CD70 antibody drug conjugates and methods of using same

Abstract

The present invention provides conjugates of CD70 antibodies and / or antigen-binding portions thereof for use in the treatment of cancer and autoimmune diseases. TIFF2026503177000370.tif67140

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2022 / 127588, filed October 26, 2022, and International Patent Application No. PCT / US23 / 74126, filed September 14, 2023, the contents of which are incorporated herein by reference. [Background technology]

[0002] background Much interest surrounds 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). Designing an antibody-drug conjugate by attaching a cytotoxic agent, immunomodulator, or other agent (collectively, "drug") to an antibody, typically 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, structural elements (if any) that provide for drug release, and structural modifications, if any, of the released free drug. If the drug is to be released into the extracellular environment, the released form of the drug must be able to reach its target. If the drug is to be released after internalization of the antibody, 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 by the 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, it was assumed that higher drug loading was superior to lower drug loading (e.g., 4 loading vs. 8 loading). The rationale was that more highly loaded conjugates would deliver more drug (e.g., cytotoxic agents) to target cells. This rationale was supported by the observation that conjugates with higher drug loading were more active against cell lines in vitro. However, a subsequent study revealed that this assumption was not confirmed in animal models. Conjugates of certain auristatins with drug loads of 4 or 8 were observed to have similar activity in mouse models. See, e.g., Hamblett et al., Clinical Cancer Res. 10:7063-70 (2004) (Non-Patent Document 1). Hamblett et al. further reported that more highly loaded ADCs were cleared from the circulation more rapidly in animal models. This faster clearance suggested PK trends for more highly loaded species compared to less loaded species. See Hamblett et al. In addition, more highly loaded conjugates had lower maximum tolerated doses (MTDs) in mice and, as a result, narrower reported therapeutic indices. In contrast, ADCs with a drug loading of 2 at engineered sites in monoclonal antibodies have been reported to have the same or better PK and therapeutic indices compared to certain 4-loaded ADCs. See, for example, Junutula et al., Clinical Cancer Res. 16:4769 (2010). Therefore, the recent trend is to develop ADCs with low drug loading.

[0004] An attractive target for cancer therapy using ADCs is CD70. CD70 is a member of the tumor necrosis factor (TNF) family of membrane-bound and secreted molecules expressed by a variety of normal and malignant cell types. CD70 is a type II transmembrane protein with its carboxyl terminus exposed to the extracellular space and its amino terminus found on the cytosolic side of the plasma membrane (Bowman et al., 1994, J. Immunol. 152:1756-61; Goodwin et al., 1993, Cell 73:447-56). Human CD70 contains a 20-amino acid cytoplasmic domain, an 18-amino acid transmembrane domain, and a 155-amino acid extracellular domain with two potential N-linked glycosylation sites (Bowman et al., supra; Goodwin et al., supra). Based on its homology with TNF-α and TNF-β, a trimeric structure has been predicted for CD70 (Petsch et al, 1995, MoI. Immunol. 32:761-72).

[0005] CD70 has limited expression in normal tissues in humans, making it an attractive target for cancer therapy. CD70 expression has been identified in several cancers, including renal cell carcinoma, colon cancer, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, some types of non-Hodgkin's lymphoma, and multiple myeloma. Although CD70 is present in a wide variety of cancers, clinical trials using CD70 antibodies and CD70 antibody-drug conjugates have had limited success to date.

[0006] Thus, there is a need for CD70 antibody-drug conjugates in general, and CD70 antibody-drug conjugates in particular that are capable of higher drug loading while maintaining other properties of lower-loaded conjugates, such as favorable PK properties. Embodiments of the present invention address these and related needs. [Prior art documents] [Non-patent literature]

[0007] [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) [Non-patent document 3] Bowman et al., 1994, J. Immunol. 152:1756-61 [Non-patent document 4] Goodwin et al., 1993, Cell 73:447-56 [Non-Patent Document 5] Petsch et al, 1995, MoI. Immunol. 32:761-72 Summary of the Invention

[0008] Provided herein are CD70 antibody-drug conjugates (ADCs) and methods of using the same. The CD70 antibody-drug conjugates comprise a binding unit comprising one or more CD70 antibodies or antigen-binding portions thereof, a linker, and one or more drug units. Additionally, provided herein are CD70 ADCs conjugated to one or more drug units via a linker, which have hydrophilic characteristics that maintain the inherent properties of the CD70 antibody. In particular, the linker helps maintain the hydrophilicity of the CD70 antibody at higher drug loadings and / or when conjugated to hydrophobic drugs and other agents. Methods of using such conjugates for the treatment of cancer and other diseases are also provided. The inventions disclosed herein are based, in part, on CD70 ADCs that specifically bind to CD70 and exhibit improved properties. CD70 is an important and advantageous therapeutic target for the treatment of certain cancers and autoimmune diseases. The CD70 ADCs provide compositions and methods based on the use of such conjugates in the treatment of CD70+ cancers and other diseases.

[0009] In some embodiments, a conjugate comprising a binding unit attached to one or more drug units by one or more linkers, (1) The binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH CDRs and VL CDRs are set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; and SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. NO:26; having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:18, respectively; and SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively; (2) each linker has the following formula (I): TIFF2026503177000002.tif11128 or a salt thereof; During the ceremony, L1 is a stretcher unit covalently linked to the binding unit, where the wavy line (~) indicates the binding site for the binding 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 binding sites for Drug units, where TIFF2026503177000003.tif4128 shows the binding site for the drug unit; wherein 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 sugar unit, a PEG unit, a carboxyl unit, and a combination thereof; (3) each drug unit is TIFF2026503177000004.tif4128, which is covalently linked to the linker subunit. A composite is provided.

[0010] In some embodiments, conjugates are provided wherein the VH and VL regions of the binding units have amino acid sequences selected from the pair of amino acid sequences set forth in the group consisting of SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10; and SEQ ID NO:11 and SEQ ID NO:12. In some embodiments, conjugates are provided wherein the VH and VL regions of the binding units have amino acid sequences selected from the pair of amino acid sequences set forth in the group consisting of SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10; and SEQ ID NO:11 and SEQ ID NO:12, respectively, and the heavy and light chain framework regions are optionally modified by 1 to 8 amino acid substitutions, deletions, or insertions within the framework regions. In some embodiments, complexes are provided in which the HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 of the binding units have the amino acid sequences shown in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:15, and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively.

[0011] In some embodiments, conjugates are provided wherein the framework regions of the binding units are human framework regions. In some embodiments, conjugates are provided wherein the binding units are antibodies or antigen-binding portions thereof. In some embodiments, the binding units are monoclonal antibodies, Fabs, Fab's, F(ab's), Fvs, disulfide-linked Fc's, scFvs, single-domain antibodies, diabodies, bispecific antibodies, or multispecific antibodies.

[0012] In some embodiments, conjugates are provided wherein the binding units have a heavy chain variable region and further comprise a heavy chain constant region. In some embodiments, conjugates are provided wherein the heavy chain constant region of the binding units is an IgG isotype. In some embodiments, conjugates are provided wherein the heavy chain constant region of the binding units is an IgG1 constant region. In some embodiments, conjugates are provided wherein the heavy chain constant region of the binding units is an IgG4 constant region. In some embodiments, conjugates are provided wherein the IgG1 constant region of the binding units has the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, conjugates are provided wherein the binding units have a light chain variable region and further comprise a light chain constant region. In some embodiments, conjugates are provided wherein the light chain constant region of the binding units is a κ isotype. In some embodiments, conjugates are provided wherein the light chain constant region of the binding units has the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, conjugates are provided wherein the heavy chain constant region of the binding units further comprises at least one amino acid modification that reduces binding affinity to a human Fc receptor (such as FcγRIII). In some embodiments, conjugates are provided wherein the heavy chain constant region of the binding unit further comprises at least one amino acid modification that reduces binding affinity to human FcγRIII.

[0013] In some aspects, conjugates are provided wherein the binding units are monospecific, in some aspects, conjugates are provided wherein the binding units are bivalent, in some aspects, conjugates are provided wherein the binding units are bispecific.

[0014] In some aspects, a pharmaceutical composition is provided comprising any of the conjugates described herein and a pharmaceutically acceptable carrier.

[0015] In some embodiments, the sugar unit of the linker has the following formula: TIFF2026503177000005.tif51128 or a salt thereof, During the ceremony, each X is independently selected from NH or O; each R is independently selected from hydrogen, acetyl, monosaccharide, disaccharide, and polysaccharide; 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; 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1); A composite is provided.

[0016] In some embodiments, the PEG unit of the linker is (a) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid Unit, a portion of a Stretcher Unit and / or a Linker Subunit L2; R 21 and R 22 are each independently any C1-C3 alkylene; R 24 and R 25 is H; polyhydroxyl group; substituted polyhydroxyl group; -C(O)-polyhydroxyl group; substituted -C(O)-polyhydroxyl group; optionally substituted C3-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; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 ; or together -NR from C3-C8 heterocycle 24 R 25 , independently selected from; 24 and R 25 provided that neither of the is H; The wavy line (~) is R 20 indicates the binding site for; n20 is 1–26; or (b) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid Unit, a portion of a Stretcher Unit and / or a Linker Subunit L2; R 21 and R 22 are each independently any 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 C3-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; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 , selected from; R24 and R 25 the other is polyethylene glycol optionally having 1 to 24 ethylene glycol subunits; The wavy line (~) is R 20 indicates the binding site for; n20 is 1–26; or (c) ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25 (XXI) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid Unit, a portion of a Stretcher Unit and / or a Linker Subunit L2; R 26 and R 27 are arbitrary, and C1 to C 12 Alkylene, -NH-C1~C 12 Alkylene, -C1~C 12 Alkylene-NH-, -C(O)-C1-C 12 Alkylene, -C1~C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)- and -C(O)-C1-C 12 independently selected from 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 C3-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; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 , selected from; 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 C3-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; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 and polyethylene glycol optionally having 1 to 24 ethylene glycol subunits; or together -NR from a C3 to C8 heterocycle. 24 R 25 , where R 24 and R 25 provided that neither of the is H; Each R 29 is optional and independently selected from —C(O)—, —NH—, —C(O)—C1-C6 alkenylene-, —NH—C1-C6 alkenylene-, —C1-C6 alkenylene-NH—, —C1-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 1–4; A conjugate is provided having a formula selected from:

[0017] In some embodiments, R 24 and R 25are each independently selected from H and a polyhydroxyl group, with the proviso that R 24 and R 25 and (b) are not H.

[0018] In some embodiments, conjugates are 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.

[0019] In some embodiments, 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 urosonic acid; or The amino sugar is selected from glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine; A composite is provided.

[0020] In some embodiments, R 24 and R 25 is provided, wherein one of the sugars is a linear monosaccharide and the other is a cyclic monosaccharide.

[0021] In some embodiments, R 24 and R 25 are independently selected from cyclic monosaccharides, disaccharides and polysaccharides.

[0022] In some embodiments, R 24 and R 25 are independently selected from linear monosaccharides and substituted linear monosaccharides, and the substituted linear monosaccharides are replaced with monosaccharides, disaccharides, or polysaccharides.

[0023] In some embodiments, R 24 and R 25are independently selected from linear monosaccharides and substituted monosaccharides, where the substituted linear monosaccharides are substituted with one or more substituents selected from alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide, and may be further substituted with a monosaccharide, disaccharide, or polysaccharide.

[0024] In some embodiments, 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 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.

[0025] In some embodiments, R 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 R 24 and R 25 and (b) are not H.

[0026] In some embodiments, R 24 and R 25 is selected from H, substituted —C(O)—C1-C8 alkyl, substituted —C(O)—C1-C4 alkyl, and substituted —C(O)—C1-C3 alkyl; and R 24 and R 25the 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 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; with the proviso that R 24 and R 25 and (b) are not H.

[0027] In some embodiments, each monosaccharide is a C5 or C6 sugar selected from glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, gulose, idose, talose, aldose, ketose, glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine; a sugar acid selected from gluconic acid, aldonic acid, uronic acid, and urosonic acid; or an amino sugar selected from glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine More independently selected conjugates are provided.

[0028] 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.

[0029] 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 a protected form thereof.

[0030] In some embodiments, the PEG unit is ~R 40 -(R 43 -R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XL) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid Unit, a portion of the Stretcher Unit and / or the Linker Subunit L2; R 41 and R 42 are absent or each independently a C1-C6 alkylene; Each R 43 are independently absent or C1 to C 12 Alkylene, -NH-C1~C 12 Alkylene, -C1~C 12 Alkylene-NH-, -C(O)-C1-C 12 Alkylene, -C1~C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)-, -C(O)-C1-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1~C 12 Alkylene, -C(O)-NH-C 12Alkylene, heteroarylene, heteroaryl-C1-C 12 Alkylene, heteroaryl-C1-C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 where 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, where the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; 44 and R 45 provided that neither of the is H; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6, A composite is provided.

[0031] In some embodiments, the PEG unit is ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLI) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid Unit, a portion of the Stretcher Unit and / or the Linker Subunit L2; R 41and R 42 are absent or each independently a 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)-C1-C 12 Alkylene, -C1~C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)-, -C(O)-C1-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 where 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, where the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; 44 and R 45 provided that neither of the is H; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6, A composite is provided.

[0032] In some embodiments, the PEG unit is ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLII) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid Unit, a portion of the Stretcher Unit and / or the Linker Subunit L2; R 41 and R 42 are absent or each independently a 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 12 Alkylene, -heteroarylene, heteroaryl-C1-C6 alkylene, heteroaryl-C1-C6 alkylene-C(O)-, or -C(O)NR 46 R 47 where 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 45are each independently H, a polyhydroxyl group, a substituted polyhydroxyl group, a —C(O)-polyhydroxyl group, or a substituted —C(O)-polyhydroxyl group, where the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; 44 and R 45 provided that neither of the is H; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1 to 4; n42 is 1 to 4, A composite is provided.

[0033] 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 a protected form thereof.

[0034] In some embodiments, R 20 or R 40 But the following structure: TIFF2026503177000006.tif179162 or a stereoisomer thereof, During the ceremony, R=H or C 1~6 is alkyl, n=0 to 12, (*) indicates a subunit of an amino acid unit, a stretcher unit and / or a portion of the linker subunit L2, 20 or R 40 indicates the binding site of TIFF2026503177000007.tif4128 is the R to the rest of the PEG unit 20 Or R 40indicates the binding site of A composite is provided.

[0035] In some embodiments, R 20 or R 40 But the following structure: TIFF2026503177000008.tif180162 or a stereoisomer thereof, During the ceremony, n=0 to 12, (*) indicates a subunit of an amino acid unit, a stretcher unit and / or a portion of the linker subunit L2, 20 or R 40 indicates the binding site of TIFF2026503177000009.tif4128 shows the R 20 Or R 40 indicates the binding site of A composite is provided.

[0036] In some embodiments, NR 43 If there is R 43 -(NR 44 R 45 ) n41 But the following structure: TIFF2026503177000010.tif75154 or a stereoisomer thereof, During the ceremony, R=H, C 1~6 alkyl, polyhydroxyl, or substituted polyhydroxyl; TIFF2026503177000011.tif4128 shows the R 43 indicates the binding site of A composite is provided.

[0037] In some embodiments, NR 43 If there is R 43 -(NR 44 R 45 ) n41 But the following structure: TIFF2026503177000012.tif75154 or a stereoisomer thereof, wherein: TIFF2026503177000013.tif4128 is the R to the rest of the PEG unit 43 A complex is provided that shows the binding site of

[0038] In some embodiments, -NR 44 R 45 But the following structure: TIFF2026503177000014.tif204144 or a stereoisomer thereof, wherein: TIFF2026503177000015.tif4128 -NR to the rest of the PEG units 44 R 45 A complex is provided that shows the binding site of

[0039] In some embodiments, ~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) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid Unit, a portion of the Stretcher Unit and / or the Linker Subunit L2; R 41 and R 42 are absent or each independently a C1-C6 alkylene; Each R 43 are independently absent or C1 to C 12 Alkylene, -NH-C1~C 12Alkylene, -C1~C 12 Alkylene-NH-, -C(O)-C1-C 12 Alkylene, -C1~C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)-, -C(O)-C1-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1~C 12 Alkylene, -C(O)-NH-C 12 Alkylene, heteroarylene, heteroaryl-C1-C 12 Alkylene, heteroaryl-C1-C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 where 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, where the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; 44 and R 45 provided that neither of the is H; 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, A composite is provided.

[0040] In some embodiments, TIFF2026503177000016.tif238163 or a stereoisomer or salt thereof, During the ceremony, Each Y is independently R 76 or TIFF2026503177000017.tif19128; 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; Each * indicates a binding site for an amino acid unit (AA) subunit, a linker subunit L2, or a stretcher unit (L1); A composite is provided.

[0041] In some embodiments, the PEG unit is TIFF2026503177000018.tif239164 or a stereoisomer or salt thereof, During the ceremony, 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; Each * indicates a binding site for an amino acid unit (AA) subunit, a linker subunit L2, or a stretcher unit (L1); A composite is provided.

[0042] In some embodiments, the PEG unit is TIFF2026503177000019.tif244159 or a stereoisomer or salt thereof, During the ceremony, 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 an amino acid unit (AA) subunit, a linker subunit L2, or a stretcher unit (L1); A composite is provided.

[0043] In some embodiments, Y is R 76 A complex is provided, wherein

[0044] In some embodiments, Y is A composite is provided, which is TIFF2026503177000020.tif19128.

[0045] In some embodiments, each R a and R b are independently H.

[0046] In some embodiments, R a and R b are combined with the carbon to which they are attached to form an oxo group.

[0047] In some embodiments, conjugates are provided wherein q is 10-20.

[0048] In some embodiments, conjugates are provided wherein q is 12.

[0049] In some embodiments, the compound of the formula: TIFF2026503177000021.tif43128 or a salt thereof, During the ceremony, (a) L 70 is selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, —C(O)—C1-C8 alkylene-, and —C(O)—C1-C8 alkylene-C(O)-; R 70 Ha~NR 71 (R 72 -R 73 ), where R 71 H, C1~C 12 Alkyl, substituted C1-C 12 alkyl, or polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits); R 72 is selected from absent or 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1); each of p1 and o1 is independently selected from 0 to 2; or (b) L 70is selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, —C(O)—C1-C8 alkylene-, and —C(O)—C1-C8 alkylene-C(O)-; R 70 Ha~NR 71 (R 75 - (R 73 )2), where R 71 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, an optionally substituted ether, an optionally substituted thioether, an optionally substituted ketone, an optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), an optionally substituted carbocycle, an optionally substituted aryl or an optionally substituted heteroaryl, and each R 73 are 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1); each of p1 and o1 is independently selected from 0 to 2; or (c) L 70 is selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, —C(O)—C1-C8 alkylene-, and —C(O)—C1-C8 alkylene-C(O)-; R 70 HA~N(R 74 -R 73 )(R72 - 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 aryl, or optionally substituted heteroaryl; and each R 73 are 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1); Each of p1 and o1 is independently selected from 0 to 2; A composite is provided.

[0050] In some embodiments, conjugates are provided that include at least one sugar unit.

[0051] In some embodiments, conjugates are provided that include at least one PEG unit.

[0052] In some embodiments, a conjugate is provided that includes at least one carboxyl unit.

[0053] In some embodiments, conjugates are provided that include at least two polar units, each polar unit selected from a sugar unit, a PEG unit, and a carboxyl unit.

[0054] In some embodiments, conjugates are provided that include at least one sugar unit and one PEG unit or one carboxyl unit.

[0055] In some embodiments, a conjugate is provided that comprises at least one carboxyl unit and one PEG unit.

[0056] In some embodiments, conjugates are provided in which the amino acid unit (AA) is present (s=1).

[0057] In some embodiments, a conjugate is provided wherein the amino acid unit comprises at least one polar unit.

[0058] In some embodiments, L2 or AA-L2 has the following structure: TIFF2026503177000022.tif156157 or a stereoisomer thereof, wherein the wavy line on the amino group indicates the attachment site for the Stretcher unit or Amino Acid unit, and the Drug unit is attached to the benzyl alcohol. A composite is provided.

[0059] In some embodiments, the linker is one of the following: ~AA-L2~ having a formula selected from TIFF2026503177000023.tif31128; In the formula, square brackets indicate amino acid units, each aa is any 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 bound to an amino acid subunit of AA, SU is a sugar unit bound to a subunit of AA or L2, and CU is a carboxyl unit bound to a subunit of AA or L2; double wavy line TIFF2026503177000024.tif4128 shows the binding site for the Drug unit, wherein aa and aa1 are independently selected from α, β and γ amino acids, and derivatives thereof. A composite is provided.

[0060] In some embodiments, the linker is one of the following: ~AA-L2~ having a formula selected from TIFF2026503177000025.tif70128; In the formula, the square brackets represent amino acid units, each aa is an amino acid subunit of AA, L2 is a linker subunit bonded to the side chain of aa, the wavy line (~) represents the binding site for the stretcher unit; aa1 (PEG) is a PEG unit bonded to aa, SU is a sugar unit bonded to aa, CU is a carboxyl unit bonded to aa, and the double wavy line TIFF2026503177000026.tif4128 shows the binding site for the Drug unit; aa and aa1 are independently selected from α, β and γ amino acids and derivatives thereof. A composite is provided.

[0061] In some embodiments, a conjugate is provided wherein the amino acid unit comprises at least two polar units.

[0062] In some embodiments, the linker is one of the following: AA-L2 having a formula selected from TIFF2026503177000027.tif31128; In the formula, the square brackets represent amino acid units, aa is any subunit of AA, L2 is a linker subunit, the wavy line (~) represents a binding site for a Stretcher unit; each of aa1(PEG) and aa2(PEG) is a PEG unit linked to aa or the other PEG unit; each SU is a sugar unit linked to aa or the other sugar unit, each CU is a carboxyl unit linked to aa or the other carboxyl unit, and the double wavy line TIFF2026503177000028.tif4128 shows the binding site for the Drug unit; aa, aa1 and aa2 are independently selected from α, β and γ amino acids and derivatives thereof. A composite is provided.

[0063] In some embodiments, the linker is one of the following: ~AA-L2~ having a formula selected from TIFF2026503177000029.tif70128; In the formula, square brackets indicate amino acid units, aa is an amino acid subunit of AA, L2 is a linker subunit bonded to the side chain of aa, and each wavy line (~) indicates a binding site for a stretcher unit; each of aa1(PEG) and aa2(PEG) is a PEG unit bonded to aa, each SU is a sugar unit bonded to aa; each CU is a carboxyl unit bonded to aa; and a double wavy line TIFF2026503177000030.tif4128 shows the binding site for the Drug unit; each of aa, aa1 and aa2 is independently selected from α, β and γ amino acids, and derivatives thereof. A composite is provided.

[0064] In some embodiments, conjugates are provided wherein the linker subunit L2 is a cleavable linker unit.

[0065] In some embodiments, a conjugate is provided wherein the linker subunit L2 comprises a peptide that is cleavable by an intracellular protease.

[0066] In some embodiments, conjugates are provided 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.

[0067] In some embodiments, a conjugate is provided wherein the linker subunit L2 comprises at least one polar unit.

[0068] In some embodiments, conjugates are provided wherein the polar unit is a sugar unit (SU).

[0069] In some embodiments, conjugates are provided 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.

[0070] In some embodiments, a conjugate is provided wherein the polar unit is a carboxyl unit (CU).

[0071] In some embodiments, conjugates are provided 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.

[0072] In some embodiments, conjugates are provided wherein the polar unit is a PEG unit (PEG).

[0073] In some embodiments, conjugates are provided 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 linked to lysine or citrulline residues, respectively.

[0074] In some embodiments, conjugates are provided in which the cleavable peptide is linked to a para-aminobenzyl alcohol self immolative group (PABA).

[0075] In some embodiments, conjugates are provided in which L2 is attached to a side chain of an AA subunit.

[0076] In some embodiments, conjugates are provided in which the amino acid unit is linked to the linker subunit L2 by a non-peptidic linking group.

[0077] In some embodiments, the non-peptidic linking group is a C1-C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Conjugates are provided in which the alkyl group is selected from alkynylene, or polyethylene glycol.

[0078] In some aspects, a conjugate is provided wherein the linker further comprises a stretcher unit.

[0079] In some embodiments, the stretcher unit comprises: Selected from TIFF2026503177000031.tif131146, In the formula, R 17 is -C1~C 10Alkylene-, -C1~C 10 Heteroalkylene-, -C3-C8 carbocyclo-, -O-(C1-C8 alkylene)-, -(CH2-O-CH2) b -C1-C8 alkylene- (where b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -(wherein b is 1 to 26), -C1 to C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene- (where 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)- (where b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-C(=O)- (where 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 to C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene -C(=O)-, -C1 to 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- (where b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-NH- (where b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C(=O)- (wherein b is 1 to 26), -C1 to 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 to C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -C1-C8 alkylene-NH- (where 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 10Heteroalkylene-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 Alkyene C(O)-, maleimide (C1-C 10 Alkyne) (CH2OCH2) p2 C(O)- or their open ring forms, wherein p2 is 1 to 26; A composite is provided.

[0080] In some embodiments, the stretcher unit comprises: Selected from TIFF2026503177000032.tif138143, In the formula, wavy line TIFF2026503177000033.tif2128 shows the attachment site of the Stretcher unit to the amino acid unit or linker subunit L2, and the attachment site to the Linker unit is on the maleimide, primary amine, or alkyne functional group. A composite is provided.

[0081] In some embodiments, conjugates are provided that include any of the conjugation units described herein, at least one linker attached to the conjugation unit, and at least one drug unit attached to each linker. In some embodiments, conjugates are provided in which each drug unit is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, a radioisotope, and a chelating ligand. In some embodiments, conjugates are provided in which each linker is attached to a conjugation unit via an interchain disulfide residue, a lysine residue, an engineered cysteine ​​residue, a glycan, a modified glycan, the N-terminal residue of the conjugation unit, or a polyhistidine residue attached to the conjugation unit. In some embodiments, the average drug loading of the conjugate 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. In some embodiments, the average drug loading of the complex is about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, or about 16. In some embodiments, the average drug loading of the complex is about 1 to about 8, about 2 to about 8, or about 4 to about 8. In some embodiments, the average drug loading of the complex is about 1 to about 12, about 2 to about 12, about 4 to about 12, about 6 to about 12, or about 8 to about 12. In some embodiments, the average drug loading of the complex is about 1 to about 16, about 2 to about 16, about 4 to about 16, about 6 to about 16, about 8 to about 16, about 10 to about 16, or about 12 to about 16.

[0082] In some embodiments, the average drug-to-antibody ratio (DAR) 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. In some embodiments, the DAR is about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, or about 16. In some embodiments, the DAR is about 1 to about 8, about 2 to about 8, or about 4 to about 8. In some embodiments, the DAR is about 1 to about 12, about 2 to about 12, about 4 to about 12, about 6 to about 12, or about 8 to about 12. In some embodiments, the DAR is about 1 to about 16, about 2 to about 16, about 4 to about 16, about 6 to about 16, about 8 to about 16, about 10 to about 16, or about 12 to about 16.

[0083] In some embodiments, conjugates are provided wherein the Drug Unit is a cytotoxic agent. In some embodiments, conjugates are provided wherein the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid, a camptothecin, a duocarmycin, or a calicheamicin. In some embodiments, conjugates are provided wherein the cytotoxic agent is an auristatin. In some embodiments, conjugates are provided wherein the cytotoxic agent is MMAE or MMAF. In some embodiments, conjugates are provided wherein the cytotoxic agent is a camptothecin. In some embodiments, conjugates are provided wherein the cytotoxic agent is exatecan. In some embodiments, conjugates are provided wherein the cytotoxic agent is a diastereomer of exatecan. In some embodiments, conjugates are provided wherein the cytotoxic agent is SN-38. In some embodiments, conjugates are provided wherein the cytotoxic agent is calicheamicin. In some embodiments, conjugates are provided wherein the cytotoxic agent is a maytansinoid. In some embodiments, conjugates are provided wherein the maytansinoid is maytansine, maytansinol, or maytansine analogs such as DM1, DM3 and DM4, and ansamatocin-2.

[0084] In some embodiments, conjugates are provided wherein the linker is a cleavable linker. In some embodiments, conjugates are provided wherein the linker comprises mc-VC-PAB, CL2, CL2A, or (succinimide-3-yl-N)-(CH)C(=O)-Gly-Gly-Phe-Gly-NH-CH-O-CH-(C=O)-(SEQ ID NO: 34), where n=1-5. In some embodiments, conjugates are provided wherein the linker comprises mc-VC-PAB. In some embodiments, conjugates are provided wherein the linker comprises CL2A. In some embodiments, conjugates are provided wherein the linker comprises CL2. In some embodiments, conjugates are provided wherein the linker comprises (succinimide-3-yl-N)-(CH)C(=O)-Gly-Gly-Phe-Gly-NH-CH-O-CH-(C=O)-(SEQ ID NO: 34). In some embodiments, conjugates are provided in which a linker is attached to at least one molecule of exatecan.

[0085] In some embodiments, the conjugate is provided wherein the drug unit is an immunomodulator. In some embodiments, the conjugate is provided wherein the immunomodulator is selected from the group consisting of a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist. In some embodiments, the conjugate is provided wherein the immunomodulator is a TLR7 agonist. In some embodiments, the conjugate is provided wherein 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 azide-2,2-dioxide, benzonaphthyridine, guanosine analog, adenosine analog, thymidine homopolymer, ssRNA, CpG-A, polyG10, or polyG3. In some embodiments, the conjugate is provided wherein the immunomodulator is a TLR8 agonist. In some embodiments, the conjugate is provided 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. In some embodiments, the conjugate is provided wherein the immunomodulator is a STING agonist. In some embodiments, the conjugate is provided wherein the immunomodulator is a RIG-I agonist. In some embodiments, the conjugate is provided wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, and KIN2000.

[0086] In some embodiments, the conjugate is provided, wherein 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); 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, samarium-153, and lead-212.

[0087] In some aspects, a pharmaceutical composition is provided comprising any of the conjugates described herein and a pharmaceutically acceptable carrier.

[0088] In some embodiments, methods of treating a CD70+ cancer are provided, comprising administering to a subject in need thereof a therapeutically effective amount of any of the conjugates described herein or any of the pharmaceutical compositions described herein. In some embodiments, the CD70+ cancer is a solid tumor or a hematological malignancy. In some embodiments, the CD70+ cancer is selected from hepatocellular carcinoma, colorectal cancer, pancreatic cancer, ovarian cancer, indolent non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the B-cell lineage, multiple myeloma, renal cell carcinoma, nasopharyngeal carcinoma, thymic carcinoma, head and neck cancer, and glioma. In some embodiments, the CD70+ cancer is a hematological malignancy. In some embodiments, the CD70+ cancer is a non-Hodgkin's lymphoma. In some embodiments, the CD70+ cancer is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the CD70+ cancer is a solid tumor. In some embodiments, the CD70+ cancer is renal cell carcinoma. In some embodiments, the CD70+ cancer is renal clear cell carcinoma (ccRCC). In some embodiments, the CD70+ cancer is head and neck cancer. In some embodiments, the CD70+ cancer is squamous cell carcinoma. In some embodiments, the CD70+ cancer is head and neck squamous cell carcinoma (HNSCC).

[0089] In some embodiments, the method further comprises administering immunotherapy to the subject. In some embodiments, the immunotherapy comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4. In some embodiments, the checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab. In some embodiments, the method further comprises administering chemotherapy to the subject.

[0090] In some embodiments, a method for treating cancer comprises administering any of the conjugates described herein or any of the pharmaceutical compositions described herein. In some embodiments, the conjugate or pharmaceutical composition is administered intravenously. In some embodiments, the conjugate or pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 12 mg / kg.

[0091] In some embodiments, the subject's treatment outcome is improved. In some embodiments, the improved treatment outcome is an objective response selected from stable disease, partial response, or complete response. In some embodiments, the improved treatment outcome is a reduction in tumor burden. In some embodiments, the improved treatment outcome is progression-free survival or disease-free survival.

[0092] In some embodiments, there is provided a use of any of the conjugates described herein or any of the pharmaceutical compositions described herein for the treatment of a CD70+ cancer in a subject.

[0093] In some aspects, provided herein are methods for treating an autoimmune disease, comprising administering a therapeutically effective amount of any of the conjugates described herein or any of the pharmaceutical compositions described herein to a subject in need thereof. In some aspects, the autoimmune disease is rheumatoid arthritis, multiple sclerosis, or systemic lupus erythematosus. In some aspects, the method further comprises administering immunosuppressive therapy to the subject. In some aspects, the method comprises administering any of the conjugates described herein or any of the pharmaceutical compositions described herein.

[0094] In some embodiments, the conjugate or pharmaceutical composition is administered intravenously. In some embodiments, the conjugate or pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 12 mg / kg. In some embodiments, the subject's treatment outcome is improved. In some embodiments, the improved treatment outcome is a reduction in disease progression or a reduction in disease severity.

[0095] In some aspects, there is provided a use of any of the conjugates described herein or any of the pharmaceutical compositions described herein for the treatment of an autoimmune disease in a subject.

[0096] These and other aspects of the present invention may be more fully understood with reference to the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings. [Brief explanation of the drawings]

[0097] [Figure 1] Binding assay of 2E7 and 2E7-LD038 to Caki-1. [Figure 2] Binding assay of 2E7 and 2E7-LD038 to 786-O. [Figure 3] Binding assay of 2E7 and 2E7-LD038 to Raji. [Figure 4] Binding assay of 2E7 and 2E7-LD038 to MCF-7. [Figure 5] In vitro blockade of CD27 binding to Caki-1 cells by 2E7 or 2E7-LD038. [Figure 6] In vitro blockade of CD27 binding to 786-O cells by 2E7 or 2E7-LD038. [Figure 7] In vitro blocking of CD27 binding to Raji cells by 2E7 or 2E7-LD038. [Figure 8] Internalization of 2E7 into tumor cells. [Figure 9] Internalization of 2E7-LD038 into tumor cells. [Figure 10] PK of 2E7 and 2E7-LD038 in rats. [Figure 11] In vitro cytotoxicity of 2E7 conjugates against 786-O. [Figure 12] In vitro cytotoxicity of 2E7 conjugates against Raji. [Figure 13] In vitro cytotoxicity of 2E7 conjugates against Caki-1. [Figure 14] In vitro cytotoxicity of 2E7 conjugates against A498. [Figure 15] In vitro cytotoxicity of 2E7 and 2E7-LD038 against Caki-1. [Figure 16] In vitro cytotoxicity of 2E7 and 2E7-LD038 against 786-O. [Figure 17] In vitro cytotoxicity of 2E7 and 2E7-LD038 against Raji. [Figure 18] Multiple-dose study of the antitumor activity of the 2E7 conjugate using Caki-1. [Figure 19] Single-dose study of the antitumor activity of the 2E7 conjugate using Caki-1. [Figure 20] Multiple-dose study of the antitumor activity of the 2E7 conjugate using Raji. [Figure 21] Single-dose study of the antitumor activity of the 2E7 conjugate using Raji. [Figure 22] Single-dose study of the antitumor activity of the 2E7 conjugate using HONE-1. [Figure 23] Single- and multiple-dose studies of the antitumor activity of the 2E7-LD038 conjugate against Caki-1. [Figure 24] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate against 786-O. [Figure 25] Single- and multiple-dose studies of the antitumor activity of the 2E7-LD038 conjugate against Raji. [Figure 26] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate against Raji. [Figure 27] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate in a patient-derived xenograft model of DLBCL. [Figure 28] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate in a patient-derived xenograft model of DLBCL. [Figure 29] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate in a patient-derived xenograft model of DLBCL. [Figure 30] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate in a patient-derived xenograft model of DLBCL. [Figure 31] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate in a patient-derived xenograft model of DLBCL. [Figure 32] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate in a patient-derived xenograft model of ccRCC. [Figure 33] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate in a patient-derived xenograft model of ccRCC. [Figure 34] Single-dose study of the antitumor activity of the 2E7-LD038 conjugate in a patient-derived xenograft model of head and neck cancer. DETAILED DESCRIPTION OF THE INVENTION

[0098] definition For convenience, certain terms in the specification, examples, and claims are defined here. Unless otherwise stated or implicit 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.

[0099] As used herein, and unless otherwise indicated, the terms "a" and "an" are used to mean "one," "at least one," or "one or more." Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0100] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprises," "including," and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is, in the sense of "including, but not limited to."

[0101] The terms "reduced," "reduce," "reduced," "reduction," "reduce," and "inhibit" are all used herein to generally mean a decrease by a statistically significant amount compared to a reference.

[0102] The terms "increased," "increase," "enhance," or "activate" all refer to In all cases, the term "measured value" is used herein to generally mean an increase by a statistically significant amount compared to a baseline.

[0103] 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 α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" also refer to polymers 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.

[0104] CD70 is a cell surface antigen on activated T and B lymphocytes, but not on resting T and B lymphocytes. It is also referred to as CD27L, tumor necrosis factor (ligand) superfamily, member 7, TNFSF7, surface antigen CD70, and Ki-24 antigen. As further described herein, it has been reported to be overexpressed in certain cancers. Human CD70 polypeptides include, but are not limited to, those having the amino acid sequences set forth in UniProt identifiers P32970-1 and P32970-2, and RefSeq NP_001243.1 and NP_001317261.1; these sequences are incorporated herein by reference.

[0105] As used herein, "epitope" refers to amino acids conventionally bound by an immunoglobulin VH / VL pair, such as an antibody or antigen-binding portion thereof. Epitopes can be formed on a polypeptide from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated 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 for an antibody or antigen-binding portion thereof and thus represents the target of specificity of the antibody or antigen-binding portion thereof. In the case of a single-domain antibody, an epitope represents the structural unit bound by an isolated variable domain.

[0106] As used herein, "specifically binds" means that a binding unit (e.g., an antibody or antigen-binding portion thereof) described herein specifically binds to an antigen of interest. -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10-11 M, 10 -12 Specific binding refers to the ability to bind to a target, such as human CD70, with a KD of M or less. Specific binding can be influenced by the affinity and avidity of the binding unit and the concentration of the target polypeptide. One skilled in the art can determine appropriate conditions under which a binding unit described herein selectively binds to CD70 using any appropriate method, such as titrating an antibody or antigen-binding portion thereof in an appropriate cell binding assay. A binding unit that specifically binds to CD70 is not displaced by a dissimilar competitor. In certain embodiments, a binding unit is said to specifically bind to CD70 if it preferentially recognizes its target antigen, CD70, in a complex mixture of proteins and / or macromolecules.

[0107] In some embodiments, the binding units described herein are 10 -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or lower dissociation constant (KD or K D In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide at about 10 -5 M~10 -6 In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of about 10 -6 M~10 -7 In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of about 10 -7 M~10 -8 In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of about 10 -8 M~10 -9In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of about 10 -9 M~10 -10 In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of about 10 -10 M~10 -11 In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of about 10 -11 M~10 -12 In some embodiments, the binding units described herein bind specifically to a CD70 polypeptide with a dissociation constant (KD) of 10 -12 It binds specifically with a dissociation constant (KD) less than M.

[0108] Unless otherwise indicated, the term "alkyl," by itself or as part of another term, refers to a substituted or unsubstituted straight or branched chain saturated hydrocarbon having the indicated number of carbon atoms (e.g., "-C1-C5 alkyl," "-C1-C8 alkyl," or "-C1-C 10"Alkyl refers to an alkyl group 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(CH3) 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 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 These include 4-methyl-2-pentyl (-CH(CH)CHCH(CH)), 4-methyl-2-pentyl (-CH(CH)CHCH(CH)), 3-methyl-3-pentyl (-C(CH)(CHCH)), 2-methyl-3-pentyl (-CH(CHCH)CH(CH)), 2,3-dimethyl-2-butyl (-C(CH)CH(CH)), and 3,3-dimethyl-2-butyl (-CH(CH)C(CH)).

[0109] 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 2refers to a C2-C8 substituted or unsubstituted straight or branched 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).

[0110] Unless otherwise indicated, "alkynyl," by itself or as part of another term, refers to a C2-C8 substituted or unsubstituted straight or branched chain 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.

[0111] Unless otherwise indicated, "alkylene" refers to a saturated, branched, or straight-chain hydrocarbon group 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 (-CH-), 1,2-ethyl (-CHCH-), 1,3-propyl (-CHCHCH-), 1,4-butyl (-CHCHCHCH-), and the like.

[0112] Unless otherwise indicated, "alkenylene" refers to an unsaturated, branched, or straight-chain hydrocarbon group 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 groups include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0113] Unless otherwise indicated, "alkynylene" refers to an unsaturated, branched or straight-chain or cyclic hydrocarbon group 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 groups include, but are not limited to, acetylene, propargyl, and 4-pentynyl.

[0114] 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 1 to 10, preferably 1 to 3, heteroatoms selected from the group consisting of O, N, Si, and S; the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, and S 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 heteroatom Si 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 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.

[0115] 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 may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N, and S 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 heteroatom Si 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.

[0116] 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 (described above), as exemplified by -CH2CH2SCH2CH2- and -CH2SCH2CH2NHCH2-. 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. Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.

[0117] 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 (described above). In some embodiments, a C2-C4 heteroalkenylene or heteroalkynylene has from 1 to 4 carbon atoms. For heteroalkenylene and heteroalkynylene groups, heteroatoms can also occupy either or both of the chain termini. Still further, for alkylene and heteroalkenylene and heteroalkynylene linking groups, no orientation of the linking group is implied.

[0118] 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.

[0119] Unless otherwise indicated, "C3-C8 carbocyclo," by itself or as part of another term, refers to a saturated or unsaturated 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).

[0120] Unless otherwise specified, "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 removing one hydrogen atom from a ring atom of a parent ring system. Representative -C3 to -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).

[0121] 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 indicated, the term "heterocarbocycle" is synonymous with the terms "heterocycle" or "heterocyclo" as used herein.

[0122] 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).

[0123] Unless otherwise indicated, "aryl," by itself or as part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon group 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, groups derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is a phenyl group.

[0124] 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 can be in the ortho, meta, or para orientation.

[0125] Unless otherwise indicated, "heteroaryl" and "heterocycle" refer to a ring system in which one or more ring atoms are heteroatoms, such as nitrogen, oxygen, and sulfur. Heterocycle groups contain 1 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. Heterocycles can be monocyclic rings having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or bicyclic rings having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), such as bicyclo[4,5], [5,5], [5,6], or [6,6] systems.

[0126] 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).

[0127] Unless otherwise indicated, "carboxyl" refers to COOH or COO - M +where M + is a cation.

[0128] Unless otherwise indicated, "oxo" refers to (C=O).

[0129] 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, each X is independently a halogen: -F, -Cl, -Br, or -I; each R 10are independently -H, -C1 to -C 20 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.

[0130] Unless otherwise specified, a "polyhydroxyl group" refers to an alkyl, alkylene, carbocyclic, or carbocyclo group containing two 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. Each carbon atom of a polyhydroxyl group may be 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, altrose, gulose, idose, talose, aldose, and ketose, sugar acids such as gluconic acid, aldonic acid, uronic acid, or ulosonic 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.

[0131] Unless the context dictates otherwise, "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, wherein a hydrogen atom of the substituent, moiety, or group may be 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 aspects, two consecutive sp3 carbon atoms of an alkyl substituent are replaced with an alkene functionality, provided that the radical carbon of the alkyl moiety is not replaced, such that the optionally substituted alkyl is an unsaturated alkyl substituent.

[0132] Any substituents replacing a hydrogen in 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)2NH2, -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(=S )OR", -C(=O)SR', -C(=S)SR', -C(=S)NH2, -C(=S)N(R')(R")2, -C(=NR')NH2, -C(=NR')N(R')R" and salts thereof, each X is independently selected from the group consisting of halogen: -F, -CI, -Br, and -I; each R" is selected from the group consisting of C1 to C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C6-C 24 Aryl, C3-C 24 Heterocyclyl (C5-C 24 or two R" together with the heteroatom to which they are attached define a heterocyclyl; R' is hydrogen or R" and R" is a C1-C 20 Alkyl, C6-C 24 Aryl, C3-C 24 Heterocyclyl (C5-C 24 and protecting groups.

[0133] Typically, optional substituents include -X, -OH, -OR", -SH, -SR", -NH2, -NH(R"), -NR'(R")2, -N(R")3, =NH, =NR", -CX3, -CN, -NO2, - 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, -S and a salt thereof, wherein each X is independently selected from the group consisting of -F and -Cl, and R" is typically C1-C6 alkyl, C6-C7 alkyl, C8-C9 alkyl, C10-C11 alkyl, C11-C12 alkyl, C12-C13 alkyl, C13-C14 alkyl, C14-C15 alkyl, C15-C16 alkyl, C16-C17 alkyl, C17-C18 alkyl, C18-C19 alkyl, C19-C20 alkyl, C19-C21 alkyl, C19-C22 alkyl, C19-C23 alkyl, C19-C24 alkyl, C19-C25 alkyl, C19-C26 alkyl, C19-C27 alkyl, C19-C28 alkyl, C19-C29 alkyl, C19-C29 alkyl, C19-C21 alkyl, C19-C22 alkyl, C19-C25 alkyl, C19-C26 alkyl, C19-C28 alkyl, C19-C29 alkyl, C19-C21 alkyl, C19-C21 alkyl, C19-C22 alkyl, C19-C23 alkyl, C19-C24 alkyl, C19-C25 alkyl, C19-C26 alkyl, C19-C27 alkyl, C19-C28 alkyl, C19-C29 alkyl, C19-C25 alkyl, C19-C26 alkyl, C19-C29 ... 10 Aryl, C3-C 10 Heterocyclyl (C5-C 10 R' is independently selected from the group consisting of hydrogen, C1-C6 alkyl, C6-C 10 Aryl, C3-C 10 Heterocyclyl (C5-C 10 and R" are protecting groups independently selected from -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(=NR')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 R' is selected from the group consisting of hydrogen, C1-C6 alkyl, and a protecting group independently selected from R".

[0134] 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 is therefore capable of forming acid addition salts 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, gentisinate, fumarate, gluconate, glucuronate, saccharinate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoic acid) salts). Pharmaceutically acceptable salts may involve the inclusion of another molecule such as acetate, succinate, or other counterion. Counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound.In addition, pharmaceutically acceptable salt can have more than one charged atom in its structure.If multiple charged atoms are part of pharmaceutically acceptable salt, pharmaceutically acceptable salt can have multiple counterions.Therefore, pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0135] 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, novel, or functional characteristics of that embodiment.

[0136] As used herein, the term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not recited in that description of an embodiment.

[0137] Except 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%.

[0138] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of 2 standard deviations (2SD) above or below the reference value.

[0139] Although the structures shown throughout this specification are depicted with particular stereocenters, this specification should be read to include variations at those stereocenters. For example, the structure of exatecan may be shown in the (S,S) configuration, but it is envisioned that the (R,S) diastereomer of exatecan is also found in other embodiments of the complexes described herein.

[0140] Other terms are defined herein within the description of various aspects of the invention.

[0141] Detailed Description Provided herein are CD70 antibody-drug conjugates (ADCs) that specifically bind to human CD70. The CD70 antibody-drug conjugates include a binding unit comprising one or more CD70 antibodies or antigen-binding portions thereof, a linker, and one or more drug units, such as a cytotoxic agent or an immunomodulator. In some embodiments, the CD70 ADCs specifically bind to and reduce the number of CD70+ cells in a subject. In some embodiments, the CD70 ADCs specifically bind to and reduce the number of CD70+ cancer cells in a subject. In some embodiments, the CD70 ADCs specifically bind to and reduce the number of CD70+ cells associated with a disease or condition, such as an autoimmune disease, in a subject.

[0142] In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0143] In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. The phrase "unmodified CDRs of the heavy or light chain variable regions" refers to VH and VL CDRs that have no amino acid substitutions, deletions, or insertions compared to the amino acid sequences set forth.

[0144] In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered.

[0145] In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered.

[0146] In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered.

[0147] In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered.

[0148] In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered. In some embodiments, a binding unit of a CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unaltered.

[0149] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; and the binding unit specifically binds to CD70. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; and the binding unit specifically binds to CD70 with a higher binding affinity (lower Kd) than antibody 69A7. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. As described herein, a binding unit comprises a CD70 antibody or antigen-binding portion thereof, and optionally, can include another peptide or polypeptide covalently linked to the CD70 antibody or antigen-binding portion thereof. In any of these embodiments, the binding unit specifically binds to CD70.

[0150] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; and the binding unit specifically binds to CD70. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; and the binding unit specifically binds to CD70 with a higher binding affinity (lower Kd) than antibody 69A7. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0151] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; and the binding unit specifically binds to CD70. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; and the binding unit specifically binds to CD70 with a higher binding affinity (lower Kd) than antibody 69A7. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0152] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; and the binding unit specifically binds to CD70. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; and the binding unit specifically binds to CD70 with a higher binding affinity (lower Kd) than antibody 69A7. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0153] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; and the binding unit specifically binds to CD70. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; and the binding unit specifically binds to CD70 with a higher binding affinity (lower Kd) than antibody 69A7. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0154] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; and the binding unit specifically binds to CD70. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; and the binding unit specifically binds to CD70 with a higher binding affinity (lower Kd) than antibody 69A7. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0155] In some embodiments, the binding unit 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 disposed within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within a light chain variable region framework region, and the VH and VL CDRs are (i) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; (ii) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; (iii) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:26, (iv) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:18, respectively; and (V) SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0156] In some embodiments, the binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0157] In some embodiments, the binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0158] In some embodiments, the binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0159] In some embodiments, the binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:18, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0160] In some embodiments, the binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0161] In some embodiments, the compositions and methods described herein relate to reducing CD70+ cells in a subject by CD70 ADCs in vivo (e.g., reducing the number of CD70+ cells in a cancer or tumor, or CD70+ cells associated with an autoimmune disease or disorder). In some embodiments, the compositions and methods described herein relate to treating a CD70+ cancer in a subject by administering a CD70 ADC. In some embodiments, the compositions and methods described herein relate to treating an autoimmune disorder in a subject by administering a CD70 ADC. In some embodiments, the compositions and methods described herein relate to treating a disease or disorder associated with CD70+ cells in a subject by administering a CD70 ADC. In any of these embodiments, the method further comprises reducing the number of CD70+ cells in the subject that are associated with the disease, condition, or cancer.

[0162] 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 an antigen, such as human CD70. The term generally refers to antibodies that are 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).

[0163] Each heavy chain is composed of a variable region (abbreviated as VH region) and a constant region. The heavy chain constant region may contain 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 can be further divided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). Thus, each VH and VL region consists of three CDRs and four FRs arranged from N-terminus to 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.

[0164] As used herein, an "antigen-binding portion" of a CD70 antibody refers to a portion of a CD70 antibody described herein that has the VH and VL sequences or CDRs of the CD70 antibody and specifically binds to CD70. Examples of antigen-binding portions include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, single-domain antibodies (also referred to as VHHs, VNARs, sdAbs, or nanobodies), or diabodies (see, e.g., 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, in the case of CD70 antibodies, to the following: (i) a Fab fragment, i.e., a monovalent fragment composed of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked together via a disulfide bridge at the hinge region; and (iii) an Fv fragment composed of the VL and VH domains. Although the two domains of the Fv fragment, VL and VH, are encoded by separate coding regions, they may be linked together via a synthetic linker, such as the poly-G4S amino acid sequence ("(G4S)" disclosed as SEQ ID NO: 27). 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 a monovalent molecule (known as a single-chain Fv or scFv). 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 domains are expressed on a single polypeptide chain but use a linker connecting the VH and VL domains that is too short to allow the two regions to combine on the same chain, thereby forcing the VH and VL domains to pair with complementary domains on different chains (VL and VH, respectively) and form two antigen-binding sites (see, e.g., Holliger, R, et al. (1993) Proc. Natl. Acad. Sci. USA 90:64446448; Poljak, R. J, et al. (1994) Structure 2:1121-1123).

[0165] A single domain antibody is an antibody moiety consisting of a single monomeric variable antibody domain. A single domain antibody can be derived from the variable domain of an antibody heavy chain from a camelid (e.g., a nanobody or VHH moiety). Furthermore, the term single domain antibody includes an autonomous human heavy chain variable domain (aVH) or a VNAR moiety derived from a shark (see, e.g., Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0166] 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 an antigen, 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 these primers may 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).

[0167] In some embodiments, the CD70 antibody, or antigen-binding portion thereof, is part of a bispecific or multispecific binding unit. 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 an IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv. See, 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.

[0168] VH and VL region modifications With respect to VH and VL amino acid sequences, those of skill in the art will recognize that individual substitutions, deletions, or additions (insertions) to amino acids in a nucleic acid encoding VH or VL, or a polypeptide that alters a single amino acid or a small percentage of amino acids in the encoded sequence, are "conservatively modified variants" if the alteration results in the substitution of an amino acid with a chemically similar amino acid (conservative amino acid substitution) and the altered polypeptide retains the ability to specifically bind to CD70.

[0169] In some embodiments, conservatively modified variants of CD70 antibodies or antigen-binding portions thereof (i.e., binding units) can have alterations in the framework regions (i.e., other than in the CDRs), for example, conservatively modified variants of CD70 antibodies can have alterations in the VH and VL CDR amino acid sequences (amino acid sequences SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:18; and the set of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26), with at least one conservative amino acid substitution in the framework region (FR). In some embodiments, the VH and VL amino acid sequences collectively have no more than eight, six, four, two, or one conservative amino acid substitutions in the FR compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences have from eight to one, six to one, four to one, or two to one conservative amino acid substitutions in the FR compared to the amino acid sequences of the unmodified VH and VL regions. In further aspects of any of these embodiments, conservatively modified variants of the binding unit (i.e., the binding unit) exhibit specific binding to CD70.

[0170] For conservative amino acid substitutions, a given amino acid can be replaced with a residue having similar physiochemical properties, e.g., one aliphatic residue can be substituted for another (e.g., Ile, Val, Leu, or Ala for another), or one polar residue can be substituted for another (e.g., Lys for Arg; Glu for Asp; or Gln for Asn). Other such conservative amino acid substitutions, e.g., substitutions of entire regions with similar hydrophobic properties, are well known. Polypeptides containing conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that the desired activity, e.g., the antigen-binding activity and specificity of the native or reference polypeptide, i.e., specificity for CD70, is retained.

[0171] In some embodiments, the binding units can be further optimized to, for example, reduce potential immunogenicity or optimize other functional properties while maintaining functional activity for therapeutic use in humans. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0172] In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, a binding unit may comprise a VH region having an amino acid sequence that is at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:3.In some embodiments, the binding unit may comprise a VL region having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 4.

[0173] In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, a binding unit may comprise a VH region having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:5.In some embodiments, the binding unit may comprise a VL region having an amino acid sequence that is at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:6.

[0174] In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, a binding unit may comprise a VH region having an amino acid sequence that is at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:7.In some embodiments, the binding unit may comprise a VL region having an amino acid sequence that is at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 8.

[0175] In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, a binding unit may comprise a VH region having an amino acid sequence that is at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:9.In some embodiments, the binding unit may comprise a VL region having an amino acid sequence that is at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:10.

[0176] In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, the binding units comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, a binding unit may comprise a VH region having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:11.In some embodiments, a binding unit may comprise a VL region having an amino acid sequence that is at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:12.

[0177] In any of these embodiments, the functional activity of the binding unit includes specific binding to CD70. Additional functional activities include depletion of CD70+ cells (e.g., cancer cells or autoimmune cells). Additionally, a binding unit having functional activity means that the polypeptide exhibits an activity similar to or better than the activity of a reference antibody or antigen-binding portion thereof described herein (e.g., a reference CD70-binding antibody or antigen-binding portion thereof described herein, comprising (i) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1 and (ii) a light chain variable region having the amino acid sequence set forth in SEQ ID NO:2, or a variant thereof), when measured in a particular assay, such as a biological assay, in the presence or absence of a dose dependency. If a dose dependency exists, it need not be identical to the dose dependency of the reference antibody or antigen-binding portion thereof described herein, but will be substantially similar to or better than the dose dependency for a given activity compared to the reference antibody or antigen-binding portion thereof described herein (i.e., the candidate polypeptide exhibits greater activity than the reference antibody).

[0178] For conservative substitutions, amino acids can be grouped according to similarities in the properties of their side chains (AL Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

[0179] Alternatively, for conservative substitutions, naturally occurring residues can be divided into groups based on shared side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging members of one of these classes for another.

[0180] Particular conservative substitutions include, for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln, or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0181] In some embodiments, conservatively modified variants of a binding unit are preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a reference VH or VL sequence, wherein the VH and VL CDRs are unaltered. The degree of homology (percent identity) between a reference and a variant sequence can be determined, for example, by comparing the two sequences using publicly available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings), which are freely available.

[0182] In some embodiments, the VH and VL amino acid sequences collectively have no more than 8, 6, 4, 2, or 1 conservative amino acid substitutions in the framework regions compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences collectively have no more than 8, 6, 1, 4, 1, or 2, 1 conservative amino acid substitutions in the framework regions compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences collectively have no more than 8, 6, 4, 2, or 1 amino acid substitutions, deletions, or insertions in the framework regions compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences collectively have no more than 8, 6, 4, 1, or 2, 1 conservative amino acid substitutions in the framework regions compared to the amino acid sequences of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences collectively have 8 or 6 or 4 or 2 or 1 amino acid substitutions, deletions or insertions compared to the amino acid sequences of the unmodified VH and VL regions.

[0183] Modification of a native (or reference) amino acid sequence can be accomplished by any of several techniques known to those skilled in the art. For example, mutations can be introduced at specific loci by synthesizing oligonucleotides containing the desired mutant sequence flanked by restriction sites that allow ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes a variant with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered by the desired substitution, deletion, or insertion. Techniques for making such modifications are well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entireties.

[0184] constant region In some embodiments, the binding unit has a fully human constant region. In some embodiments, the binding unit has a humanized constant region. In some embodiments, the binding unit has a non-human constant region. An immunoglobulin constant region refers to a heavy chain constant region or a light chain constant region. Human heavy chain constant region and light chain constant region amino acid sequences are known in the art. The constant region can be of any suitable type, which can be selected from the immunoglobulin classes IgA, IgD, IgE, IgG, and IgM. Some immunoglobulin classes can be further classified into isotypes, e.g., 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 either kappa (or κ) or lambda (or λ).

[0185] The constant region can have an IgG1 isotype. The constant region can have an IgG2 isotype. The constant region can have an IgG3 isotype. The constant region can have an IgG4 isotype. The Fc domain can have a hybrid isotype comprising constant regions from more than one isotype. The immunoglobulin constant region can be an IgG1 or IgG4 constant region. In some embodiments, the CD70 antibody heavy chain of the binding unit is an IgG1 isotype and has the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the CD70 antibody light chain of the binding unit is a κ isotype and has the amino acid sequence set forth in SEQ ID NO:29.

[0186] Fc domain engineering to alter effector function In some embodiments, the Fc region or Fc domain of the binding unit has substantially no binding 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 exhibits substantially no binding to any Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a), and FcyRIIIB (CD16b). As used herein, "does not substantially bind" refers to weak or no binding to one or more selected Fcγ receptors. In some embodiments, "does not substantially bind" refers to at least a 1000-fold reduction in binding affinity to an Fcγ receptor (i.e., an increase in Kd). 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 exhibits weak or no binding to any of the Fcγ receptors. In some embodiments, an Fc null domain or region exhibits at least a 1000-fold reduction in binding affinity to an Fcγ receptor (i.e., an increase in Kd).

[0187] 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 reductions in 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 cause 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.

[0188] In some embodiments, the Fc domain has reduced or no ADCC activity. As used herein, reduced or no ADCC activity refers to at least a 10, at least 20, at least 30, at least 50, at least 100, or at least 500-fold decrease in the ADCC activity of the Fc domain.

[0189] In some embodiments, the Fc domain has reduced or no CDC activity. As used herein, reduced or no CDC activity refers to at least a 10, at least a 20, at least a 30, at least a 50, at least a 100, or at least a 500-fold decrease in the CDC activity of the Fc domain.

[0190] In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted ADCC and / or CDC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks Fcγ receptor binding (and therefore potentially lacks ADCC activity). NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. 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 (see, 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, 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 (PBMC) 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).

[0191] C1q binding assay can also be performed to confirm that antibody or Fc domain or region cannot bind to C1q, and therefore lacks or has reduced CDC activity.See, for example, the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402.CDC assay can be performed to evaluate 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)).

[0192] In some embodiments, the Fc domain has reduced or no ADCP activity. As used herein, reduced or no ADCP activity refers to at least a 10-, at least a 20-, at least a 30-, at least a 50-, at least a 100-, or at least a 500-fold decrease in the ADCP activity of the Fc domain.

[0193] ADCP binding assays may also be performed to confirm that an antibody or Fc domain or region lacks or has reduced ADCP activity. See, e.g., US20190079077 and US20190048078 and the references disclosed therein.

[0194] Binding units with reduced effector function activity include those with one or more substitutions of Fc region residues, such as, for example, 238, 265, 269, 270, 297, 327, and 329, according to the EU numbering of Kabat (see, e.g., 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, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine, according to the EU numbering of Kabat (see, U.S. Pat. No. 7,332,581). Certain antibody variants with reduced binding to FcRs 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).) Binding units containing such amino acid modifications can be prepared that have reduced binding to FcRs.

[0195] In some embodiments, the binding unit comprises an Fc domain or region having one or more amino acid substitutions that reduce FcγR binding, e.g., 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 the EU numbering of Kabat. In some embodiments, the Fc domain comprises D265A and / or P329G according to the EU numbering of Kabat in an Fc region derived from a human IgG1 Fc region. In some embodiments, the substitutions are L234A, L235A, and P329G (LALA-PG) according to the EU numbering of Kabat in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831). In some embodiments, the substitutions are L234A, L235A, and D265A (LALA-DA) according to the EU numbering of Kabat in an Fc region derived from a human IgG1 Fc region.

[0196] In some embodiments, alterations are made within the Fc region that result in altered (i.e., decreased in either case) 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).

[0197] Method for making a binding unit In various embodiments, binding units can be produced in cell lines derived from humans, mice, or other animals. Recombinant DNA expression can be used to produce binding units. This allows for the production of a spectrum of CD70 antigen-binding moieties, not just CD70 antibodies, in the host species of choice. Production of binding units in bacteria, yeast, transgenic animals, and chicken eggs are also alternatives to cell-based production systems. The main advantage of transgenic animals is the potentially high yield from renewable sources.

[0198] In some embodiments, a VH polypeptide having an amino acid sequence set forth in SEQ ID NO:3, 5, 7, 9, or 11 is encoded by a nucleic acid. In some embodiments, a VH polypeptide having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO:3, 5, 7, 9, or 11 is encoded by a nucleic acid. In some embodiments, a VL polypeptide having an amino acid sequence set forth in SEQ ID NO:4, 6, 8, 10, or 12 is encoded by a nucleic acid. In some embodiments, a VL polypeptide having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NO:4, 6, 8, 10, or 12 is encoded by a nucleic acid. In some embodiments, the nucleic acid encodes a VH polypeptide having an amino acid sequence set forth in SEQ ID NO:3, 5, 7, 9, or 11.In some embodiments, the nucleic acid encodes a VH polypeptide having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NOs:3, 5, 7, 9, or 11. In some embodiments, the nucleic acid encodes a VL polypeptide having an amino acid sequence set forth in SEQ ID NO:4, 6, 8, 10, or 12. In some embodiments, the nucleic acid encodes a VL polypeptide having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NOs:4, 6, 8, 10, or 12. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO: 11.In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the nucleic acid encodes a VH polypeptide having the amino acid sequence set forth in SEQ ID NO:12.

[0199] In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs:3 and 4. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs:5 and 6. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs:7 and 8. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs:9 and 10. In some embodiments, the nucleic acid encodes VH and VL polypeptides having the amino acid sequences set forth in SEQ ID NOs:11 and 12.

[0200] 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. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one strand of a denatured double-stranded DNA. In some embodiments, a nucleic acid can be a cDNA, e.g., a nucleic acid lacking introns.

[0201] Nucleic acid molecules encoding the amino acid sequence of a binding unit can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding a CD70 antibody or antigen-binding portion thereof. In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding a CD70 antibody or antigen-binding portion thereof. Nucleic acid sequences encoding at least the CD70 antibody or antigen-binding portion thereof described herein, or polypeptides thereof, can be recombined with vector DNA by conventional techniques, such as, for example, blunt or staggered-ended termini for ligation, restriction enzymes to provide suitable ends, filling of sticky ends as needed, alkaline phosphatase treatment to avoid undesired ligation, and ligation using an appropriate ligase or other techniques known in the art. Techniques for such manipulations are disclosed, for example, by 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 CD70 antibodies or antigen-binding portions thereof, or VH or VL polypeptides thereof (i.e., binding units).

[0202] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" 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. Operable linkage is one in which the regulatory DNA sequence and the DNA sequence to be expressed (e.g., a CD70 antibody or its antigen-binding portion (i.e., binding unit)) are connected in a manner that allows gene expression of the polypeptide or antigen-binding portion in recoverable amounts. As is well known in the art, the exact nature of the regulatory regions required for gene expression can vary from organism to organism. See, e.g., Sambrook et al., 1989; Ausubel et al., 1987-1993.

[0203] Thus, expression of the CD70 antibodies or antigen-binding portions thereof described herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian, and mammalian cells, either in vivo or in situ, or 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 any other mammalian cells may 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 so produced can be processed in vivo or purified and processed in vitro to allow synthesis of the CD70 antibodies or antigen-binding portions thereof described herein with specific amino-terminal sequences. Moreover, problems associated with retention of the methionine residue from the start codon in direct yeast (or bacterial) expression may be circumvented. (See, e.g., Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989).) Recombinant CD70 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.

[0204] Production of binding units 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 skilled in the art, see Ausubel et al., 1987-1993.

[0205] In some embodiments, the introduced nucleic acid sequence (encoding the CD70 antibody or antigen-binding portion thereof, or polypeptide thereof) 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, which 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.

[0206] Exemplary prokaryotic vectors known in the art include plasmids, such as those that can replicate in E. coli. Other gene expression elements useful for expressing DNA encoding binding units include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 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). Immunoglobulin-encoding DNA genes can be expressed as described by Liu et al. (infra) and Weidle et al., 51 Gene 21 (1987) using the SV40 early promoter and 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 expression elements.

[0207] For immunoglobulin encoding nucleotide sequences, the transcription promoter can be, for example, human cytomegalovirus and the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin.

[0208] For expression of the DNA coding region in rodent cells, the transcription promoter can be a viral LTR sequence, and the transcription promoter enhancer can be one 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.

[0209] Each coding region or gene fusion is assembled or inserted into an expression vector. Recipient cells capable of expressing the CD70 variable region or antigen-binding portion thereof are then transfected with nucleotides encoding the CD70 antibody or antibody polypeptide or antigen-binding portion thereof, either alone or co-transfected with polynucleotides encoding the VH and VL chain coding regions. The transfected recipient cells are cultured under conditions that allow expression of the incorporated coding regions, and the expressed antibody chains or intact antibodies or antigen-binding portions are recovered from the culture.

[0210] Nucleic acids containing the coding regions encoding the binding units can be assembled into separate expression vectors, which 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, with the first designed for selection in a bacterial system and the second designed for selection in a eukaryotic system, each vector carrying a set of coding regions. This strategy results in vectors that initially direct the production of nucleotide sequences in a bacterial system and allow their amplification. The DNA vectors thus 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 acid (e.g., containing the heavy and light chains of a CD70 antibody). Non-limiting examples of selectable genes for use in bacterial systems are genes that confer ampicillin resistance and genes that confer chloramphenicol resistance. Selectable genes for use in eukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (called gpt) and the phosphotransferase gene from Tn5 (called neo). Alternatively, fused nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0211] For transfection of the expression vector and production of the binding units, 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 cells are recombinant Ig-producing myeloma cells SP2 / 0. SP2 / 0 cells produce only immunoglobulins encoded by the transfected genes. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, from which secreted immunoglobulins can be obtained from ascites.

[0212] Expression vectors encoding binding units 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 particle bombardment. As known to those skilled in the art, Johnston et al., 240 Science 1538 (1988).

[0213] Yeast offers certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeast carries out 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. (Montpelier, France, 1982).

[0214] Yeast gene expression systems can be routinely evaluated for the production, secretion, and stability levels of antibodies and assembled binding units. Various 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 1α 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 II DNA Cloning 45, (Glover, ed., IRL Press, 1985) and, for example, U.S. Patent Application Publication No. 2006 / 0270045A1.

[0215] Bacterial strains can also be used as hosts for the production of the antibody molecules or antigen-binding portions thereof 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 vectors contain not only replication sites but also specific genes capable of providing phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for the production of binding units in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).

[0216] 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.

[0217] In addition to the cells of lymphoid origin described above, mammalian cells that can 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.

[0218] The one or more binding units can be produced in vivo by any suitable method in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptide.

[0219] The antibody or antigen-binding portion thereof is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in 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).

[0220] 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 described 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 in the same plasmid, which is then transfected into cells, thereby directly selecting for cells expressing both chains. Alternatively, a plasmid encoding one chain, e.g., the VL chain, can be first transfected into cells, and the resulting cell line can then be transfected with a VH chain plasmid containing a second selection marker. Cell lines producing antibodies, antigen-binding portions thereof, via either route can also be transfected with plasmids encoding additional copies of peptides, VH, VL, or VH+VL chains, along with additional selectable markers, to generate cell lines with enhanced properties, e.g., higher production of assembled binding units or enhanced stability of the transfected cell line.

[0221] Additionally, 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. The binding units can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be whole plant, 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 WO 0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, for example, Biolex, NC).

[0222] For intact antibodies, the variable regions (VH and VL regions) of the CD70 antibody 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 by well-known procedures from various human cells, such as immortalized B cells (WO 87 / 02671). The CD70-binding antibody can contain both a light chain constant region and a heavy chain constant region. The heavy chain constant region can include CH1, hinge, CH2, CH3, and optionally CH4 regions. In some embodiments, the CH2 domain can be deleted or omitted.

[0223] Techniques described for the production of single-chain antibodies (see, 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 CD70. 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 is incorporated herein by reference in its entirety).

[0224] In some embodiments, the binding unit 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 and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. This 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 producing scFv molecules and designing suitable peptide linkers are described, for example, in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9: 132-137 (1991). scFv-Fc is described by Sokolowska-Wedzina et al., Mol. Cancer Res. 15(8):1040-1050, 2017.

[0225] In some embodiments, the binding unit is a single domain antibody or 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 an autonomous human heavy chain variable domain (aVH) or a VNAR portion derived from a shark (see, e.g., Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0226] 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 may 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 an antigen, 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 for the isolation of antibody fragments by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).

[0227] 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 / 089004A1); cross-linking two or more antibodies or antigen-binding portions thereof (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody portions (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994); and, for example, by preparing trispecific antibodies as described in Tutt et al. J. Immunol. 147: 60 (1991).

[0228] Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies," can also be binding units (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576A1).

[0229] In some embodiments, the binding unit (e.g., antibody or antigen-binding portion) herein also includes "dual-acting FAb" or "DAB" that contains antigen-binding sites that bind to two different antigens (see, e.g., US2008 / 0069820 and Bostrom et al., 2009, Science 323:1610-14). "Crossmab" antibodies are also included herein (see, e.g., WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, and WO 2013 / 026833).

[0230] In some embodiments, the binding unit comprises different antigen-binding sites fused to one or the other of the two subunits of the Fc domain; thus, 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 is advantageous to introduce modifications to the Fc domain of the binding unit that promote the association of the desired polypeptides.

[0231] Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domains with charged amino acid residues, such that homodimer formation is electrostatically disfavored, but heterodimerization is electrostatically favored.

[0232] In some embodiments, the binding 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 comprise 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 comprises a polypeptide spacer sequence between the two scFvs. Each scFv recognizes a different epitope, which may be specific for different proteins, such that both proteins are bound by the BiTE.

[0233] Because the bispecific T cell engager is a single polypeptide, it 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 required 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 an imidazole gradient. The eluate is further purified using anion exchange chromatography, and the polypeptide is eluted with a sodium chloride gradient. Finally, the eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species. In some embodiments, the binding 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.

[0234] In some embodiments, the binding units are multispecific, such as 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.

[0235] 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. Two-in-one 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., supra. ScFv-HSA-scFv antibodies are also described in Kontermann et al. (ibid.).

[0236] Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or their antigen-binding portions (i.e., binding units) 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, NY, 1982). Substantially pure binding units of at least about 90%-95% homogeneity are advantageous, with those having 98%-99% or greater homogeneity being advantageous, particularly for pharmaceutical uses. After purification, partially or to homogeneity as desired, the intact binding units can then be used therapeutically or in developing and performing assay procedures, immunofluorescence staining, and the like. See generally, Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981).

[0237] antibody-drug conjugates In some embodiments, a CD70 antibody-drug conjugate (also referred to as a CD70 conjugate or CD70 ADC) comprises a binding unit comprising a CD70 antibody or antigen-binding portion linked to at least one linker, with at least one drug unit linked to each linker. As used herein, the term "drug unit" refers to cytotoxic agents (chemotherapeutic agents or drugs), immunomodulators, nucleic acids (including siRNAs), growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant, or animal origin, enzymatically active toxins, or fragments thereof), radioisotopes, PROTACs, and other compounds that are active against target cells when delivered to those cells.

[0238] cytotoxic substances In some embodiments, the CD70 ADC comprises at least one drug unit that is a cytotoxic agent. A "cytotoxic agent" refers to an agent that has a cytotoxic effect on a cell. A "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.

[0239] Tubulin disrupting agents include, for example, auristatins, dolastatins, tubulysins, colchicine, vinca alkaloids, taxanes, cryptophycins, maytansinoids, hemiasterins, as well as 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, for example, 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 U.S. Patent No. 9,345,785, which is incorporated herein by reference.

[0240] Tubulysins include, but are not limited to, tubulysin D, tubulysin M, tubuphenylalanine, and tubutyrosine. WO 2017 / 096311 and WO 2016 / 040684 describe tubulysin analogs, including tubulysin M.

[0241] Colchicines include, but are not limited to, colchicine and CA-4.

[0242] Vinca alkaloids include, but are not limited to, vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) and vindesine (VOS).

[0243] Taxanes include, but are not limited to, paclitaxel and docetaxel.

[0244] Cryptophycins include, but are not limited to, cryptophycin-1 and cryptophycin-52.

[0245] Maytansinoids include, but are not limited to, maytansine, maytansinol, maytansine analogs such as 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.

[0246] Maytansinoid drug moieties also 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) (U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CHOAc) (U.S. Pat. No. 4,450,254) (prepared from Nocardia); C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866) (prepared by conversion of maytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (prepared from Trewia nudiflora) nudiflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces sp.); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol).

[0247] Hemiasterins include, but are not limited to, hemiasterin and HTI-286.

[0248] Other tubulin disrupting agents include takalonolide A, takalonolide B, takalonolide AF, takalonolide AJ, takalonolide Al-epoxide, discodermolide, epothilone A, epothilone B, and laulimalide.

[0249] In some embodiments, the cytotoxic agent can 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 (SS form), diastereoisomers of exatecan, RS form, and exatecan analog DXd containing exatecan (see US20150297748)) and analogs of DXd containing the RS diastereoisomer of exatecan. Other camptothecins are disclosed in WO 1996 / 021666, WO 00 / 08033, U.S. Patent Application Publication No. 2016 / 0229862, and WO 2020 / 156189.

[0250] In some embodiments, the cytotoxic agent is a duocarmycin, including the synthetic analogs KW-2189 and CBI-TMI.

[0251] immunomodulator In some embodiments, the Drug Unit is an immunomodulator, which can be, for example, a TLR7 and / or TLR8 agonist, a STING agonist, a RIG-I agonist, or other immunomodulator.

[0252] In some embodiments, the Drug Unit is an immunomodulator, 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 US20160168164, US20150299194, US20110098248, US20100143301, and US20090047249.

[0253] 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, VTX-1463.

[0254] In some embodiments, the TLR8 agonist can be any of the compounds described in WO 2018 / 170179, WO 2020 / 056198, and WO 2020056194.

[0255] Other TLR7 and TLR8 agonists are described, for example, in WO2016142250, WO2017046112, WO2007024612, WO2011022508, WO2011022509, WO2012045090, WO2012097173, WO2012097177, WO2017079283, US20160008374, US20160194350, US20160289229, U.S. Patent No. 6,043,238, US20180086755, WO2017216054, WO2017190669, WO2017202704, WO2017202703, WO20170071944, US2014 0045849, US20140073642, WO2014056953, WO2014076221, WO2014128189, US2014035 0031, WO2014023813, US20080234251, US20080306050, US20100029585, US201100924 85, US20110118235, US20120082658, US20120219615, US20140066432, US20140088085, US20140275167, and US20130251673, WO2018198091, and US20170131421.

[0256] In some embodiments, the immunomodulatory agent is a STING agonist. Examples of STING agonists include those disclosed in, for example, WO2020059895, WO2015077354, WO2020227159, WO2020075790, WO2018200812, and WO2020074004.

[0257] In some embodiments, the immunomodulator is a RIG-I agonist. Examples of RIG-I agonists include KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, and KIN2000.

[0258] toxin In some embodiments, the Drug Unit is an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-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.

[0259] radioactive isotope In some embodiments, the Drug Unit is a radioactive atom. A variety of radioisotopes are available for producing radioconjugates. Examples include I131, I125, Y90, Re186, Re188, Sm153, Bi213, P32, Pb212, and radioisotopes of lutetium (e.g., Lu177).

[0260] PROTAC In some embodiments, the drug unit is a targeted proteolysis-inducing 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.

[0261] Linker A CD70 conjugate typically includes at least one linker, each linker having at least one drug unit attached thereto. Typically, the conjugate comprises a linker between the CD70 antibody (or antigen-binding portion thereof (i.e., Binding Unit)) and the Drug Unit. In various embodiments, the linker is a protease-cleavable linker, an acid-cleavable linker, a disulfide linker, a disulfide-containing linker, or a disulfide-containing linker 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 self-stabilizing linker (see, e.g., WO2018 / 031690 and WO2015 / 095755 and Jain et al., Pharm. Res. 32:3526-3540 (2015)), non-cleavable linkers (see, e.g., WO2007 / 008603), photolabile linkers, and / or hydrophilic linkers (see, e.g., WO2015 / 123679).

[0262] In some embodiments, the linker is a cleavable linker that is cleavable under intracellular conditions, thereby releasing the binding unit and / or the drug unit from the linker into the intracellular environment by cleavage of the linker. For example, in some embodiments, the linker is cleavable by a cleavage agent present in the intracellular environment (e.g., in a lysosome, endosome, or caveolae). The linker can include a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease (see, for example, WO2004 / 010957, US20150297748, US2008 / 0166363, US20120328564, and US20200347075). Typically, the peptidyl linker is at least 1 amino acid long or at least 2 amino acids long. Intracellular cleavage agents can include cathepsin B 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). Most typical peptidyl linkers are cleavable by enzymes present in target antigen-expressing cells. For example, peptidyl linkers (e.g., Phe-Leu or Gly-Phe-Leu-Gly linkers) that can be cleaved by cathepsin-B, a thiol-dependent protease that is highly expressed in cancerous tissues, can be used. Other such linkers are described, for example, in U.S. Patent No. 6,214,345. In certain embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a val-cit linker), or a Gly-Gly-Phe-Gly (SEQ ID NO: 35) linker (see, e.g., US2015 / 0297748).One advantage of using intracellular proteolytic release of the drug unit is that the drug is typically attenuated when conjugated and the serum stability of the conjugate is typically high. See also U.S. Patent No. 9,345,785.

[0263] As used herein, the terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction within a cell on an antibody-drug conjugate that breaks the covalent bond, e.g., linker, between the drug (e.g., cytotoxic agent) and the antibody, resulting in free drug or other metabolites of the conjugate dissociated from the antibody inside the cell. Thus, the cleaved portion of the conjugate is an intracellular metabolite.

[0264] In some embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic 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 linkers are relatively stable under neutral pH conditions, e.g., blood conditions, but are unstable at pH below 5.5 or 5.0, which is approximately the pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the drug via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).

[0265] In some embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). 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-α-methyl-α-(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. Press, 1987; see also U.S. Pat. No. 4,880,935).

[0266] In some embodiments, the linker is a malonic acid linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12). In some embodiments, the linker unit is non-cleavable, such as a maleimidocaproyl linker, and the drug is released by antibody degradation. (See U.S. Patent Application Publication No. 2005 / 0238649.)

[0267] In some embodiments, the linker is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in reference to a linker means that when the antibody-drug conjugate (ADC) 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 linkers in a sample of the ADC are cleaved. Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by independently incubating both (a) the ADC ("ADC sample") and (b) an equimolar amount of unconjugated antibody or drug ("control sample") with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unconjugated antibody or drug present in the ADC sample with the amount present in the control sample, as measured, for example, by high performance liquid chromatography.

[0268] In some embodiments, the linker promotes cellular internalization. In some embodiments, the linker promotes cellular internalization when conjugated to a drug, such as a cytotoxic agent (i.e., in the context of the linker-drug moieties of the ADCs described herein). In still other embodiments, the linker promotes cellular internalization when conjugated to both a drug and a CD70 antibody (i.e., in the context of the ADCs described herein).

[0269] In other embodiments, CD70 ADCs can be generated using a variety of bifunctional protein coupling agents, including N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (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 conjugation of radionucleotides to linking units are described, for example, in WO 94 / 11026.

[0270] Conjugates of CD70 ADCs include, but are not limited to, commercially available (e.g., from 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, as well as conjugates prepared using cross-linking reagents including, but not limited to, SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0271] In some embodiments, the linker can be attached to the end of the amino acid sequence of the antibody or antigen-binding portion thereof (i.e., the binding unit) or to a side chain modification of the antibody or antigen-binding portion thereof, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, unnatural amino acid residue, glutamine, or glutamic acid residue. The bond between the antibody or antigen-binding portion thereof and the linker or drug unit can be via any of several bonds, including, but not limited to, an amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single, double, or triple bond, a disulfide bond, or a thioether bond. Functional groups capable of forming such bonds include, for example, amino groups, carboxyl groups, aldehyde groups, azide groups, alkyne and alkene groups, ketones, carbonates, cyano and succinimidyl groups, and carbonyl functions coupled with leaving groups such as hydroxyl groups.

[0272] In some embodiments, the linker is attached to the linking unit through an interchain disulfide. In some embodiments, the linker is connected to the linking unit through a hinge cysteine ​​residue. In some embodiments, the linker is connected to the linking unit through an engineered cysteine ​​residue. In some embodiments, the linker is connected to the linking unit through a lysine residue. In some embodiments, the linker is connected to the linking unit through an engineered glutamine residue. In some embodiments, the linker is connected to the linking unit through an unnatural amino acid engineered into the heavy chain.

[0273] In some embodiments, the linker is attached to the binding unit through a sulfhydryl group. In some embodiments, the linker is attached to the binding unit through a primary amine. In some embodiments, the linker is attached through a linkage created between an unnatural amino acid on the binding unit by reacting a ketone group with an oxime bond formed by modifying an alkoxyamine on the drug.

[0274] In some embodiments, the linker is attached to the binding unit via a sortase A linker, which can be created by a sortase A enzyme that fuses an LPXTG recognition motif (SEQ ID NO:33) with an N-terminal GGG motif to regenerate a native amide bond.

[0275] In some embodiments, the linker has the following formula (I): TIFF2026503177000034.tif11128 or a salt thereof, During the ceremony, L1 is a stretcher unit that has a binding site for the binding 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 binding sites for Drug units; The wavy line (~) indicates the binding site for the binding unit, and the double wavy line TIFF2026503177000035.tif4128 shows the binding site for the drug unit; wherein at least one polar unit is present within an amino acid unit, a stretcher unit, a linker subunit, or a combination thereof, and the polar unit is selected from a sugar unit, a PEG unit, a carboxyl unit, and a combination thereof.

[0276] In some embodiments, the linker has the following formula (I): TIFF2026503177000036.tif11128 or a salt thereof, During the ceremony, L1 is a stretcher unit that has a binding site for the binding 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 binding sites for Drug units; The wavy line (~) indicates the binding site for the binding unit, and the double wavy line TIFF2026503177000037.tif4128 shows the binding site for the drug unit; wherein 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 sugar unit, a PEG unit, a carboxyl unit, and a combination thereof.

[0277] Sugar Unit In some embodiments, the linker has the following formula: TIFF2026503177000038.tif51128 or a salt thereof, During the ceremony, each X is independently selected from NH or O; each R is independently selected from hydrogen, acetyl, monosaccharide, disaccharide, and polysaccharide; 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; 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1).

[0278] In some embodiments, the linker is TIFF2026503177000039.tif123128 or a stereoisomer or salt thereof, During the ceremony, each R is independently selected from hydrogen, a monosaccharide, a disaccharide, and a polysaccharide; 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1).

[0279] PEG unit In some embodiments, the linker is (a) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid Unit, a portion of a Stretcher Unit and / or a Linker Subunit L2; R 21 and R 22 are each independently any C1-C3 alkylene; R 24 and R 25 is H; polyhydroxyl group; substituted polyhydroxyl group; -C(O)-polyhydroxyl group; substituted -C(O)-polyhydroxyl group; optionally substituted C3-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; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 ; or together -NR from C3-C8 heterocycle 24 R 25 , independently selected from; 24 and R 25 provided that neither of the is H; The wavy line (~) is R 20indicates the binding site for; n20 is 1–26; or (b) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid Unit, a portion of a Stretcher Unit and / or a Linker Subunit L2; R 21 and R 22 are each independently any 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 C3-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; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 , selected from; R 24 and R 25 the other is polyethylene glycol optionally having 1 to 24 ethylene glycol subunits; The wavy line (~) is R 20 indicates the binding site for; n20 is 1–26; or (c) ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21-R 27 -] n27 -NR 24 R 25 (XXI) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid Unit, a portion of a Stretcher Unit and / or a Linker Subunit L2; R 26 and R 27 are arbitrary, and C1 to C 12 Alkylene, -NH-C1~C 12 Alkylene, -C1~C 12 Alkylene-NH-, -C(O)-C1-C 12 Alkylene, -C1~C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)- and -C(O)-C1-C 12 independently selected from 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 C3-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; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 , selected from; 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 C3-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; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 and polyethylene glycol optionally having 1 to 24 ethylene glycol subunits; or together -NR from a C3 to C8 heterocycle. 24 R 25 , where R 24 and R 25 provided that neither of the is H; Each R 29 is optional and independently selected from —C(O)—, —NH—, —C(O)—C1-C6 alkenylene-, —NH—C1-C6 alkenylene-, —C1-C6 alkenylene-NH—, —C1-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 1–4; The PEG unit has a formula selected from:

[0280] In some embodiments, the R of the PEG unit 24 and R 25 and R are not both H. In some embodiments, 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 both H.

[0281] In some embodiments, conjugates are provided that include a linker 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, glucose, idose, talose, aldose, and ketose; the sugar acid is 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; A conjugate comprising a linker is provided.

[0282] In some embodiments, the PEG unit is: TIFF2026503177000040.tif197114 or a stereoisomer or salt thereof; In the formula, R 39 is selected from H, a linear monosaccharide, and polyethylene glycol optionally having 1 to 24 ethylene glycol subunits; the wavy line on the left indicates the site of attachment of the amino acid unit to a subunit, a portion of the stretcher unit and / or linker subunit, A conjugate comprising a linker is provided.

[0283] In some embodiments, the R of the PEG unit 24 and R 25 and the other is a cyclic monosaccharide.

[0284] In some embodiments, the PEG unit is: TIFF2026503177000041.tif107128 or a stereoisomer or salt thereof; In the formula, R 41 is a cyclic monosaccharide; the wavy line on the left indicates the site of attachment of the amino acid unit to a subunit, a stretcher unit and / or a portion of the linker subunit, A conjugate comprising a linker is provided.

[0285] In some embodiments, the R of the PEG unit 24 and R 25 is independently selected from cyclic monosaccharides, disaccharides, and polysaccharides. TIFF2026503177000042.tif189143 or a stereoisomer or salt thereof; In the formula, each R 45 is selected from H and a monosaccharide, a disaccharide, or a polysaccharide; R 46 is selected from a cyclic monosaccharide, disaccharide, or polysaccharide; the wavy line on the right indicates the site of attachment of the amino acid unit to a subunit, a portion of a stretcher unit, and / or a linker subunit, A conjugate comprising a linker is provided.

[0286] In some embodiments, the R of the PEG unit 24 and R 25 is independently selected from linear monosaccharides and substituted linear monosaccharides, and the substituted linear monosaccharides are substituted with monosaccharides, disaccharides, or polysaccharides. TIFF2026503177000043.tif165128 or a stereoisomer or salt thereof; In the formula, R 47 is a linear monosaccharide; each R 49 is selected from monosaccharides, disaccharides and polysaccharides; the dashed line on the left indicates the site of attachment of the amino acid unit to a subunit, a portion of the stretcher unit and / or linker subunit, A conjugate comprising a linker is provided.

[0287] In some embodiments, the R of the PEG unit 24 and R 25are independently selected from linear monosaccharides and substituted monosaccharides, and the substituted linear monosaccharides are substituted with one or more substituents selected from alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide, and may further be substituted with a monosaccharide, disaccharide, or polysaccharide. In some embodiments, the PEG unit is one of the following: TIFF2026503177000044.tif87128 or a stereoisomer or salt thereof; 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 dashed line on the left indicates the site of attachment of the Amino Acid Unit to a subunit, a portion of a Stretcher Unit, and / or a Linker Subunit. A conjugate comprising a linker is provided.

[0288] 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 25 and 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, the PEG unit is one of the following: TIFF2026503177000045.tif75128 or a stereoisomer or salt thereof; wherein the dashed line on the left indicates the site of attachment of the Amino Acid Unit to a subunit, a Stretcher Unit and / or a portion of the Linker Subunit. A conjugate comprising a linker is provided.

[0289] In some embodiments, the R of the PEG unit 24 and R 25 are independently selected from H, substituted -C1-C8 alkyl, substituted -C1-C4 alkyl, or substituted -C1-C3 alkyl; provided that R 24 and R 25 and wherein the substituted -C1-C8 alkyl, -C1-C4 alkyl, and -C1-C3 alkyl are substituted with hydroxyl and / or carboxyl. TIFF2026503177000046.tif236106 or a stereoisomer or salt thereof; In the formula, R 48 is selected from H, OH, CHOH, COOH, or -C1-C6 alkyl substituted with hydroxyl or carboxyl; the dashed line on the left indicates the site of attachment of the Amino Acid Unit to a portion of the subunit, Stretcher Unit and / or Linker Subunit. A conjugate comprising a linker is provided.

[0290] In some embodiments, the R of the PEG unit 24 and R 25 is selected from H, substituted —C(O)—C1-C8 alkyl, substituted —C(O)—C1-C4 alkyl, and substituted —C(O)—C1-C3 alkyl; and R 24 and R 25 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. TIFF2026503177000047.tif146128TIFF2026503177000048.tif109128 or a stereoisomer or salt thereof; wherein the dashed line on the left indicates the site of attachment of the Amino Acid Unit to a subunit, a Stretcher Unit and / or a portion of the Linker Subunit. A conjugate comprising a linker is provided.

[0291] 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 —NR 24 R 25 and the nitrogen of R by an alkylene, arylene, carbocyclo, heteroarylene, or heterocarbocyclo; provided that R 24 and R 25 and N,N'-dialkyl-substituted piperazines are provided. 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 PEG unit is selected from the following: TIFF2026503177000049.tif80128 or a stereoisomer or salt thereof; wherein the dashed line on the left indicates the site of attachment of the Amino Acid Unit to a subunit, a Stretcher Unit and / or a portion of the Linker Subunit. A conjugate comprising a linker is provided.

[0292] In some embodiments, each monosaccharide of the sugar unit or PEG unit is a C5 or C6 sugar selected from glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, gulose, idose, talose, aldose, ketose, glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine; a sugar acid selected from gluconic acid, aldonic acid, uronic acid, and urosonic acid; or an amino sugar selected from glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine Conjugates are provided that include more independently selected linkers.

[0293] 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.

[0294] 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.

[0295] In some embodiments, R 20is selected from carboxyl, amino, alkynyl, azide, hydroxyl, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, or a protected form thereof.

[0296] 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.

[0297] In some embodiments, the linker is ~R 40 -(R 43 -R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XL) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid Unit, a portion of the Stretcher Unit and / or the Linker Subunit L2; R 41 and R 42 are absent or each independently represent C1-C6 alkylene; Each R 43 are independently absent or C1 to C 12 Alkylene, -NH-C1~C 12 Alkylene, -C1~C 12 Alkylene-NH-, -C(O)-C1-C 12Alkylene, -C1~C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)-, -C(O)-C1-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1~C 12 Alkylene, -C(O)-NH-C 12 Alkylene, heteroarylene, heteroaryl-C1-C 12 Alkylene, heteroaryl-C1-C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 where R 46 and R 47 One of them 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; However, R 44 and R 45 provided that neither of the following is H; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6.

[0298] In some embodiments, the linker is ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLI) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid Unit, a portion of the Stretcher Unit and / or the Linker Subunit L2; R 41 and R 42 are absent or each independently a 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)-C1-C 12 Alkylene, -C1~C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)-, -C(O)-C1-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 where 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; However, R 44 and R 45provided that neither of the is H; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1-6; n42 is 1 to 6.

[0299] In some embodiments, the linker is ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLII) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid Unit, a portion of the Stretcher Unit and / or the Linker Subunit L2; R 41 and R 42 are absent or each independently represent 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 12 Alkylene, -heteroarylene, heteroaryl-C1-C6 alkylene, heteroaryl-C1-C6 alkylene-C(O)-, or -C(O)NR 46 R 47 where R 46 and R 47One 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; However, R 44 and R 45 provided that neither of the is H; The wavy line (~) is R 40 indicates the binding site for; n40 is 1-26; n41 is 1 to 4; n42 is 1 to 4.

[0300] 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 a protected form thereof.

[0301] In some embodiments, R 20 or R 40 But the following structure: TIFF2026503177000050.tif170152 or a stereoisomer thereof, During the ceremony, R=H or C 1~6 is alkyl, n=0 to 12, (*) indicates a subunit of an amino acid unit, a stretcher unit and / or a portion of the linker subunit L2, 20 or R 40 indicates the binding site of TIFF2026503177000051.tif4128 is the R to the rest of the PEG unit 20 or R 40 indicates the binding site of A conjugate comprising a linker is provided.

[0302] In some embodiments, R 20 or R 40 But the following structure: TIFF2026503177000052.tif171152 or a stereoisomer thereof, During the ceremony, n=0 to 12, (*) indicates a subunit of an amino acid unit, a stretcher unit and / or a portion of the linker subunit L2, 20 or R 40 indicates the binding site of TIFF2026503177000053.tif4128 is the R to the rest of the PEG unit 20 or R 40 indicates the binding site of A conjugate comprising a linker is provided.

[0303] In some embodiments, R 43 If there is R 43 -(NR 44 R 45 ) n41 But the following structure: TIFF2026503177000054.tif75155 or a stereoisomer thereof, During the ceremony, n is 0 to 6, R=H, C 1~6 alkyl, polyhydroxyl, or substituted polyhydroxyl; TIFF2026503177000055.tif4128 is the R to the rest of the PEG unit 43 indicates the binding site of A conjugate comprising a linker is provided.

[0304] In some embodiments, R 43 If there is R 43 -(NR 44 R 45 ) n41 But the following structure: TIFF2026503177000056.tif75155 or a stereoisomer thereof, wherein: TIFF2026503177000057.tif4128 is the R to the rest of the PEG unit 43 A conjugate is provided that includes a linker that exhibits a binding site for

[0305] In some embodiments, -NR 44 R 45 But the following structure: TIFF2026503177000058.tif204145 or a stereoisomer thereof, wherein: TIFF2026503177000059.tif4128 -NR to the rest of the PEG units 44 R 45 A conjugate is provided that includes a linker that exhibits a binding site for

[0306] In some embodiments, the PEG unit has the following structure prior to attachment to the Amino Acid unit, Stretcher unit and / or portion of the Linker subunit L2: TIFF2026503177000060.tif114128TIFF2026503177000061.tif232101TIFF2026503177000062.tif232115TIFF2026503177000063.tif101128 or a stereoisomer thereof, wherein R is H or alkyl, each n independently is 1 to 12, and the functional group moiety at the attachment site of the PEG unit may be selected from carboxyl, hydroxyl, aminyl, azidyl, hydrazinyl, alkynyl, formyl, or triazolyl, as described above.

[0307] In some embodiments, the linker is ~R40 -(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) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid Unit, a portion of the Stretcher Unit and / or the Linker Subunit L2; R 41 and R 42 are absent or each independently represent C1-C6 alkylene; Each R 43 are independently absent or C1 to C 12 Alkylene, -NH-C1~C 12 Alkylene, -C1~C 12 Alkylene-NH-, -C(O)-C1-C 12 Alkylene, -C1~C 12 Alkylene-C(O)-, -NH-C1-C 12 Alkylene -C(O)-, -C(O)-C1-C 12 Alkylene-NH-, -NH-C(O)-NH-, -NH-C(O)-, -NH-C(O)-C1~C 12 Alkylene, -C(O)-NH-C 12 Alkylene, heteroarylene, heteroaryl-C1-C 12 Alkylene, heteroaryl-C1-C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 where R 46 and R 47 One of the two is H or C1~C 12 alkylene, and the other is C1-C 12is 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.

[0308] In some embodiments, the PEG unit has the following structure prior to attachment to the Amino Acid unit, Stretcher unit and / or portion of the Linker subunit L2: TIFF2026503177000064.tif50170, wherein R is H or alkyl, n is 1 to 12, and the functional group moiety at the attachment site of the PEG unit may be selected from carboxyl, hydroxyl, aminyl, or azidyl. A conjugate comprising a linker is provided.

[0309] In some embodiments, the linker is TIFF2026503177000065.tif250162 or a stereoisomer or salt thereof, During the ceremony, Each Y is independently R 76 or TIFF2026503177000066.tif18128; 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; Each * indicates a binding site for an amino acid unit (AA) subunit, a linker subunit L2, or a stretcher unit (L1).

[0310] In some embodiments, the linker is TIFF2026503177000067.tif240165 or a stereoisomer or salt thereof, During the ceremony, 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; Each * indicates a binding site for an amino acid unit (AA) subunit, a linker subunit L2, or a stretcher unit (L1).

[0311] In some embodiments, the linker is TIFF2026503177000068.tif245163 or a stereoisomer or salt thereof, During the ceremony, 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 an amino acid unit (AA) subunit, a linker subunit L2, or a stretcher unit (L1).

[0312] In some embodiments, Y is R 76 A conjugate is provided that includes a linker,

[0313] In some embodiments, Y is A conjugate including a linker is provided, which is TIFF2026503177000069.tif18128.

[0314] In some embodiments, each R a and R b are independently H.

[0315] In some embodiments, R a and R b are provided, which include a linker in which, together with the carbon to which they are attached, they form an oxo group.

[0316] In some embodiments, a conjugate comprising a linker wherein q is 10 to 20 is provided.

[0317] In some embodiments, a conjugate comprising a linker wherein q is 12 is provided.

[0318] Carboxyl Unit In some embodiments, the linker has the following formula: TIFF2026503177000070.tif38128 or a salt thereof, During the ceremony, (a) L 70 is selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, —C(O)—C1-C8 alkylene-, and —C(O)—C1-C8 alkylene-C(O)-; R 70 Ha~NR 71 (R 72-R 73 ), where R 71 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, 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1); Each of p1 and o1 is independently selected from 0 to 2, or (b) L 70 is selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, —C(O)—C1-C8 alkylene-, and —C(O)—C1-C8 alkylene-C(O)-; R 70 Ha~NR 71 (R 75 - (R 73 )2), where R 71 H, C1~C 12 Alkyl, substituted C1-C 12 alkyl, or polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits); R75 is a branched optionally substituted C1-C3 alkylene, an optionally substituted ether, an optionally substituted thioether, an optionally substituted ketone, an optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), an optionally substituted carbocycle, an optionally substituted aryl or an 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, 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1); Each of p1 and o1 is independently selected from 0 to 2, or (c) L 70 is selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, —C(O)—C1-C8 alkylene-, and —C(O)—C1-C8 alkylene-C(O)-; R 70 HA~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 aryl, or optionally substituted heteroaryl; and each R 73is independently carboxyl or polycarboxyl, wherein the polycarboxyl 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, heteroalkylene, amino, and / or amido; Each wavy line (~) indicates a binding site of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1); Each of p1 and o1 is independently selected from 0 to 2.

[0319] In some embodiments, conjugates are provided that include a linker comprising at least one sugar unit. In some embodiments, conjugates are provided that include a linker comprising at least one PEG unit. In some embodiments, conjugates are provided that include a linker comprising at least one carboxyl unit. In some embodiments, conjugates are provided that include a linker comprising at least two polar units, each polar unit being selected from a sugar unit, a PEG unit, and a carboxyl unit. In some embodiments, conjugates are provided that include a linker comprising at least one sugar unit and one PEG unit or one carboxyl unit. In some embodiments, conjugates are provided that include a linker comprising at least one carboxyl unit and one PEG unit.

[0320] In some embodiments, conjugates are provided that include a linker, wherein an amino acid unit (AA) is present (s=1). In some embodiments, conjugates are provided that include a linker, wherein the amino acid unit includes at least one polar unit.

[0321] In some embodiments, L2 or AA-L2 has the following structure: TIFF2026503177000071.tif150157 or a stereoisomer thereof, wherein the wavy line on the amino group indicates the attachment site for the Stretcher unit or Amino Acid unit, and the Drug unit is attached to the benzyl alcohol.

[0322] In some embodiments, the linker is one of the following: TIFF2026503177000072.tif31128, wherein the square brackets represent amino acid units, each aa is any subunit of AA, L2 is a linker subunit, and each wavy line (~) represents 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 a double wavy line TIFF2026503177000073.tif4128 shows a binding site for a Drug unit, where aa and aa1 are independently selected from α, β, and γ amino acids and derivatives thereof. Conjugates are provided that include a linker.

[0323] In some embodiments, the linker is one of the following: TIFF2026503177000074.tif70128, wherein the square brackets represent amino acid units, each aa being an amino acid subunit of AA, L2 being a linker subunit attached to the side chain of aa, the wavy line (~) representing a site of attachment to a Stretcher unit; aa1(PEG) being a PEG unit attached to aa, SU being a sugar unit attached to aa, CU being a carboxyl unit attached to aa, and the double wavy line TIFF2026503177000075.tif4128 shows the attachment site for the Drug unit; where aa and aa1 are independently selected from α, β, and γ amino acids and derivatives thereof. Conjugates are provided that include a linker.

[0324] In some embodiments, conjugates are provided in which the amino acid unit comprises a linker comprising at least two polar units.

[0325] In some embodiments, the linker is one of the following: TIFF2026503177000076.tif31128, wherein the square brackets represent amino acid units, aa is any subunit of AA, L2 is a linker subunit, the wavy line (~) represents a site of attachment to a Stretcher unit; each of aa1(PEG) and aa2(PEG) is a PEG unit linked to aa or the other PEG unit; each SU is a sugar unit linked to aa or the other sugar unit, and each CU is a carboxyl unit linked to aa or the other carboxyl unit, and the double wavy line TIFF2026503177000077.tif4128 shows the attachment site for the Drug unit; conjugates are provided that include a linker, wherein aa, aa1, and aa2 are independently selected from α, β, and γ amino acids and derivatives thereof.

[0326] In some embodiments, the linker is one of the following: TIFF2026503177000078.tif70128, 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, and 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 a double wavy line TIFF2026503177000079.tif4128 shows the attachment site for the Drug unit; conjugates are provided that include a linker, wherein each of aa, aa1, and aa2 is independently selected from α, β, and γ amino acids and derivatives thereof.

[0327] In some embodiments, a conjugate is provided that includes a linker, wherein the linker subunit L2 is a cleavable linker unit. In some embodiments, a conjugate is provided that includes a linker, wherein the linker subunit L2 includes a peptide that can be cleaved by an intracellular protease. In some embodiments, a conjugate is provided that includes a linker, wherein the cleavable peptide includes a valine-citrulline peptide, a valine-alanine peptide, a valine-lysine peptide, a phenylalanine-lysine peptide, or a glycine-glycine-phenylalanine-glycine peptide.

[0328] In some embodiments, a conjugate is provided that includes a linker, wherein the linker subunit L2 includes at least one polar unit. In some embodiments, a conjugate is provided that includes a linker, wherein the polar unit is a sugar unit (SU). In some embodiments, a conjugate is provided that includes a linker, wherein the cleavable peptide includes 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.

[0329] In some embodiments, a conjugate is provided that includes a linker in which the polar unit is a carboxyl unit (CU). In some embodiments, a conjugate is provided that includes a linker in which the cleavable peptide includes 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 that includes a lysine residue.

[0330] In some embodiments, a conjugate is provided that includes a linker, wherein the polar unit is a PEG unit (PEG). In some embodiments, a conjugate is provided that includes a linker, wherein the cleavable peptide includes 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 a PEG unit linked to a lysine residue or a citrulline residue, respectively.

[0331] In some embodiments, conjugates are provided that include a linker in which a cleavable peptide is attached to a para-aminobenzyl alcohol self-immolative group (PABA).

[0332] In some embodiments, conjugates are provided that include a linker, wherein the amino acid unit is linked to the 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 Conjugates are provided that include a linker selected from alkynylene, or polyethylene glycol.

[0333] In some embodiments, a conjugate is provided that includes a linker further comprising a Stretcher unit. In some embodiments, the Stretcher unit is one of the following: Selected from TIFF2026503177000080.tif140146, In the formula, R 17 is -C1~C 10 Alkylene-, -C1~C 10Heteroalkylene-, -C3-C8 carbocyclo-, -O-(C1-C8 alkylene)-, -(CH2-O-CH2) b -C1-C8 alkylene- (where b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -(wherein b is 1 to 26), -C1 to C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene- (where 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)- (where b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-C(=O)- (where 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 to C 10 Alkylene-(C3-C8 carbocyclo)-C(=O)-, -(C3-C8 carbocyclo)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclo-C(=O)-, -C1-C 10Alkylene-(C3-C8 heterocyclo)-C(=O)-, -(C3-C8 heterocyclo)-C1-C 10 Alkylene -C(=O)-, -C1 to 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- (where b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-NH- (where b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C(=O)- (wherein b is 1 to 26), -C1 to 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 to C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -C1-C8 alkylene-NH- (where 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 10Heteroalkylene-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 C(O)- or an open ring form thereof, and p2 is 1 to 26; A conjugate comprising a linker is provided.

[0334] In some embodiments, the stretcher unit comprises: Selected from TIFF2026503177000081.tif138143, where wavy lines TIFF2026503177000082.tif2128 shows the attachment site of the Stretcher unit to the amino acid unit or linker subunit L2, and conjugates containing linkers are provided in which the attachment site to the Linker unit is on a maleimide, primary amine, or alkyne functional group.

[0335] In some embodiments, the following structure: TIFF2026503177000083.tif254115TIFF2026503177000084.tif191128TIFF2026503 177000085.tif220136TIFF2026503177000086.tif204149TIFF2026503177000087.t if236149TIFF2026503177000088.tif190152TIFF2026503177000089.tif202149TIF F2026503177000090.tif255149TIFF2026503177000091.tif223157TIFF2026503177 000092.tif196152TIFF2026503177000093.tif166152TIFF2026503177000094.tif2 26146TIFF2026503177000095.tif201146TIFF2026503177000096.tif233133TIFF20 26503177000097.tif206149TIFF2026503177000098.tif223120TIFF2026503177000099.tif174130TIFF2026503177000100.tif156148, or a stereoisomer thereof; where each Z is joined by *, Selected individually from TIFF2026503177000101.tif125160; The Drug unit is attached to the linker subunit L2, a terminal acid group or a benzyl alcohol, or is represented by a wavy line TIFF2026503177000102.tif4128 shows the binding site for the Drug unit; A composite is provided.

[0336] In some embodiments, the following: T-Linker-Drug Unit or a pharmaceutically acceptable salt thereof, where: T is a binding unit; The linker has the following formula (I): L1-(AA) s -L2 (I) and During the ceremony, (i) L1 is a stretcher unit coupled with a coupling unit; (ii) AA is an amino acid unit having 1 to 12 subunits; (iii) s is 0 or 1; (iv) L2 is a linker subunit attached to a Drug unit, wherein the linker subunit is a cleavable linker unit comprising a cleavable peptide; (v) the Drug Unit is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, a radioisotope, and a chelating ligand; (vi) at least one PEG unit is present within an amino acid unit, a linker subunit, or a combination thereof, wherein the at least one PEG unit has the formula: TIFF2026503177000103.tif65128, 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; Each * indicates a binding site of the linker subunit to a subunit, an amino acid unit, or both.

[0337] In some embodiments, the compound of formula (III) [L1-(AA) s -L2]-D t (III) or a salt thereof, During the ceremony, (i) L1 is the stretcher unit; (ii) AA is an amino acid unit having 1 to 12 subunits; (iii) s is 0 or 1; (iv) L2 is a linker subunit attached to a Drug unit (D), wherein the linker subunit is a cleavable linker unit comprising a cleavable peptide, and t is 1 to 4; (v) the Drug Unit is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, a radioisotope, and a chelating ligand; (vi) one or more PEG units are present within the amino acid units, linker subunits, or combinations thereof, wherein at least one of the one or more PEG units has the formula: TIFF2026503177000104.tif65128, 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; Each * indicates a binding site to a linker subunit, an amino acid unit, or both; A drug-linker is provided.

[0338] In some embodiments, for the conjugate, the binding unit is an antibody or an antigen-binding portion thereof.

[0339] In some embodiments, for the complex, (1) the binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH CDRs and VL CDRs are set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; and SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. NO:26; and SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:18, respectively; and SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively.

[0340] In some embodiments, for the conjugate, the binding units are as described elsewhere herein.

[0341] In some embodiments, for a conjugate or drug-linker, each of the one or more PEG units has the formula: I have TIFF2026503177000105.tif65128.

[0342] In some embodiments, for a conjugate or drug-linker, each of the one or more PEG units has the formula: I have TIFF2026503177000106.tif30128.

[0343] In some embodiments, for conjugates or drug-linkers, (vi) has one PEG unit. In some cases, (vi) has two PEG units.

[0344] In some embodiments, for a Conjugate or Drug-Linker, q is independently 4 to 16. In some cases, q is 12. In some cases, m is 4. In some cases, n is 1.

[0345] In some embodiments, for a Drug-Linker, the Stretcher unit can form a bond with a sulfur atom. In some cases, the Stretcher unit can be a maleimide (C1-C 10 Alkylene)-C(O)-, Maleimide (CH2OCH2) p2 (C1~C 10 Alkyne) C(O)-, Maleimide (C1-C 10 Alkyne) (CH2OCH2) p2 C(O)-, or its open ring form, and p2 is 1 to 26. In some cases, the Stretcher unit is a maleimide (C1 to C 10 In some cases, the stretcher unit comprises a maleimide (C1-C 10 In some cases, the stretcher unit is The file is TIFF2026503177000107.tif19128.

[0346] In some embodiments, s is 0 for the conjugate or drug-linker.

[0347] In some embodiments, the cleavable peptide of the conjugate or drug-linker comprises a valine-citrulline peptide, a valine-alanine peptide, a valine-lysine peptide, a phenylalanine-lysine peptide, or a glycine-glycine-phenylalanine-glycine peptide. In some cases, 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 a PEG unit linked to a lysine residue or a citrulline residue, respectively, and the PEG unit is represented by Formula (XVIb).

[0348] In some embodiments, the cleavable peptide of the conjugate or drug-linker comprises a self-immolative group. In some cases, the cleavable peptide comprises a para-aminobenzyl alcohol self-immolative group (PABA) or a p-amino-benzyloxycarbonyl self-immolative group. In some cases, the cleavable peptide comprises a p-amino-benzyloxycarbonyl self-immolative group.

[0349] In some embodiments, for conjugates or drug-linkers, the cleavable peptide is attached to the drug unit via a p-amino-benzyloxycarbonyl self-immolative group.

[0350] In some embodiments, s is 1 for the conjugate or drug-linker.

[0351] In some embodiments, for the conjugate or drug-linker, the subunits of the amino acid unit are 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, aminoalkanedionic acid, aminobenzoic acid, amino-heterocycloalkanoic acid, heterocyclo-carboxylic acid, citrulline, and diaminoalkanoic acid; and one or more PEG units are bound to one of the subunits.In some cases, the subunits of the amino acid unit are selected from alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamine, phenylalanine, lysine, leucine, serine, and citrulline. In some cases, the subunits of the amino acid unit are selected from lysine, valine, and citrulline. In some cases, each of the one or more PEG units is TIFF2026503177000108.tif30128.

[0352] In some embodiments, for a conjugate or drug-linker, L2 is a cleavable peptide. In some cases, L2 is a cleavable peptide substituted with one or more PEG units.

[0353] In some embodiments, for a Drug-Linker, the Stretcher unit is Selected from TIFF2026503177000109.tif116128, where wavy lines TIFF2026503177000110.tif2128 indicates the binding site of the Stretcher unit to the amino acid unit if s is 1, or L2 if s is 0.

[0354] In some embodiments, for the conjugate or drug-linker, the drug unit is selected from a cytotoxic agent. In some cases, the cytotoxic agent is MMAE, MMAF, exatecan or SN-38. In some cases, the cytotoxic agent is exatecan. In some cases, the cytotoxic agent is MMAE. In some cases, the cytotoxic agent is MMAF.

[0355] In some embodiments, the compound of formula (III*C) below: T-[L1-AA-L2-D] S (III*C) or a salt thereof, During the ceremony, (vii) T is a binding unit; (viii)s is p load where p load is selected from about 1 to about 16; (i) L1 is TIFF2026503177000111.tif44128, where R 17 is C1-C8 alkylene-C(O)-; (ii) AA is an amino acid unit having 1 to 5 subunits; (iii) 1 to 5 subunits of the amino acid unit are 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, aminoalkynol, aminoalkandioic acid, aminobenzoic acid, amino-heterocyclo-alkanoic acid, heterocyclo-carboxylic acid, citrulline, and diaminoalkanoic acid; (iv) L2 is a cleavable peptide covalently linked to a self-immolative group; (v) D is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, a radioisotope, and a chelating ligand, and D is covalently attached to a self-immolative group of L2; (vi) one of the 1 to 5 subunits of the amino acid unit is covalently linked to a PEG unit, the PEG unit having the formula: TIFF2026503177000112.tif65128, In the above formula, each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; * indicates the binding site of the amino acid unit to one of the 1-5 subunits; A composite is provided.

[0356] In some embodiments, for the conjugate, the binding unit is an antibody or an antigen-binding portion thereof.

[0357] In some embodiments, for the complex, (1) the binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH CDRs and VL CDRs are set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; and SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. NO:26; and SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:18, respectively; and SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively.

[0358] In some aspects, the binding units can be as described elsewhere herein.

[0359] In some embodiments, the p load is selected from about 8 to about 16. In some embodiments, p load is about 8. In some embodiments, p load is about 12. In some embodiments, p load is about 16.

[0360] In some embodiments, the compound of formula (III*) L1-AA-L2-D (III*) or a salt thereof, During the ceremony, (i) L1 is maleimide (C1-C 10 alkylene)-C(O)-; (ii) AA is an amino acid unit having 1 to 5 subunits; (iii) 1 to 5 subunits of the amino acid unit are 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, aminoalkynol, aminoalkandioic acid, aminobenzoic acid, amino-heterocyclo-alkanoic acid, heterocyclo-carboxylic acid, citrulline, and diaminoalkanoic acid; (iv) L2 is a cleavable peptide covalently linked to a self-immolative group; (v) D is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, a radioisotope, and a chelating ligand, wherein D is covalently attached to a self-immolative group of L2; (vi) one of the 1 to 5 subunits of the amino acid unit is covalently linked to a PEG unit, the PEG unit having the formula: TIFF2026503177000113.tif65128, In the above formula, each q is independently 1 to 26; each m is independently 1 to 4; each n is independently 1 to 4; * indicates the binding site of the amino acid unit to one of the 1-5 subunits; A drug-linker is provided.

[0361] In some embodiments, for the conjugate or drug-linker, D is a cytotoxic agent. In some cases, the cytotoxic agent is MMAE, MMAF, exatecan, or SN-38. In some cases, the cytotoxic agent is exatecan. In some cases, D is TIFF2026503177000114.tif39128. In some cases, D is The file is TIFF2026503177000115.tif28128.

[0362] In some embodiments, for a conjugate or drug-linker, the cleavable peptide is selected from a valine-citrulline peptide, a valine-alanine peptide, a valine-lysine peptide, a phenylalanine-lysine peptide, and a glycine-glycine-phenylalanine-glycine peptide. TIFF2026503177000116.tif37131. In some cases, the cleavable peptide is TIFF2026503177000117.tif17128. In some cases, the cleavable peptide is TIFF2026503177000118.tif36128. In some cases, the cleavable peptide is The file is TIFF2026503177000119.tif33128.

[0363] In some embodiments, for a conjugate or drug-linker, the self-immolative group is selected from a para-aminobenzyl alcohol self-immolative group (PABA) and a p-amino-benzyloxycarbonyl self-immolative group. Selected from TIFF2026503177000120.tif21128.

[0364] In some embodiments, L2 is TIFF2026503177000121.tif23128, TIFF2026503177000122.tif43145. In some cases, L2 is TIFF2026503177000123.tif23128. In some cases, L2 is TIFF2026503177000124.tif42128. In some cases, L2 is The file is TIFF2026503177000125.tif39128.

[0365] In some embodiments, for the Drug-Linker, L is The file is TIFF2026503177000126.tif22128.

[0366] In some embodiments, for a conjugate or drug-linker, one to five subunits of the amino acid unit are selected from alanine, arginine, asparagine, histidine, glycine, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, ornithine, β-alanine, and citrulline. In some cases, one to five subunits of the amino acid unit are selected from alanine, arginine, asparagine, histidine, glycine, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, ornithine, β-alanine, and citrulline. In some cases, the amino acid unit has one subunit. In some cases, the amino acid unit has two subunits. In some cases, the amino acid unit has three subunits.

[0367] In some embodiments, for a Conjugate or Drug-Linker, AA is TIFF2026503177000127.tif34128. In some cases, AA is TIFF2026503177000128.tif44128. In some cases, q is independently 1 to 16. In some cases, each m is independently 3 to 4. In some cases, each n is independently 1 to 2. In some cases, n is 1. In some cases, each m is 4. In some cases, q is selected from 4, 8, and 12. In some cases, q is 12.

[0368] In some embodiments, a conjugate is provided, comprising a Drug-Linker of Formula (III*) and a Linking Unit. In some cases, the Linking Unit comprises a reactive substituent that reacts with the maleimide of the Drug-Linker of Formula (III*) to form a new covalent bond, thus forming the conjugate.

[0369] In some embodiments, a conjugate is provided comprising a Drug-Linker of Formula (III) and a Linking Unit, wherein the Drug-Linker of Formula (III) optionally comprises a reactive substituent that reacts with the maleimide of the Drug-Linker of Formula (III) to form a new covalent bond, thus forming the conjugate.

[0370] In some embodiments, conjugates are provided that include a linker and further include at least one drug unit attached to the linker subunit L2 to form a drug-linker. In some embodiments, conjugates are provided that include a drug-linker, wherein the drug unit is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, a radioisotope, and a chelating ligand. In some embodiments, conjugates are provided that include a drug-linker, wherein the drug unit is a cytotoxic agent. In some embodiments, conjugates are provided that include a drug-linker, 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, conjugates are provided that include a drug-linker, wherein the cytotoxic agent is an auristatin. In some embodiments, conjugates are provided that include a drug-linker, wherein the cytotoxic agent is MMAE or MMAF. In some embodiments, conjugates are provided that include a drug-linker, wherein the cytotoxic agent is a camptothecin. In some embodiments, conjugates comprising a drug-linker are provided, wherein the cytotoxic agent is exatecan or SN-38. In some embodiments, conjugates comprising a drug-linker are provided, wherein the cytotoxic agent is exatecan (SS). In some embodiments, conjugates comprising a drug-linker are provided, wherein the cytotoxic agent is a diastereoisomer of exatecan, the RS form. In some embodiments, conjugates comprising a drug-linker are provided, wherein the cytotoxic agent is calicheamicin. In some embodiments, conjugates comprising a drug-linker are provided, wherein the cytotoxic agent is a maytansinoid. In some embodiments, conjugates comprising a drug-linker are provided, wherein the maytansinoid is maytansine, maytansinol, or ansamatocin-2.

[0371] In some embodiments, the Drug unit is an immunomodulator. Conjugates comprising a Drug-Linker are provided. In some embodiments, conjugates comprising a Drug-Linker are provided, wherein the immunomodulator is selected from a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist. In some embodiments, conjugates comprising a Drug-Linker are provided, wherein the immunomodulator is a TLR7 agonist. In some embodiments, a drug-linker-containing conjugate 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-containing conjugate is provided in which the immunomodulator is a TLR8 agonist. In some embodiments, a conjugate comprising a drug-linker is provided, 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. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the immunomodulator is a STING agonist. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the immunomodulator is a RIG-I agonist. In some embodiments, a drug-linker comprising conjugate is provided wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400 and KIN2000.

[0372] 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); 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, samarium-153, and lead-212.

[0373] In some embodiments, conjugates are provided that include a binding unit conjugated to any of the drug-linkers described herein. In some embodiments, conjugates are provided in which the binding unit is selected from an antibody or an antigen-binding portion thereof. In some embodiments, conjugates are provided in which the binding 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, conjugates are provided in which the binding unit is monospecific. In some embodiments, conjugates are provided in which the binding unit is bivalent. In some embodiments, conjugates are provided in which the binding unit is bispecific.

[0374] In some embodiments, the average drug loading of the complex (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.

[0375] In some embodiments, the following: TIFF2026503177000129.tif202164TIFF2026503177000130.tif162159TIFF2026503177000131.tif182146TIFF2026503177000 132.tif177154TIFF2026503177000133.tif181157TIFF2026503177000134.tif196165TIFF2026503177000135.tif250168TIFF2 026503177000136.tif212164TIFF2026503177000137.tif198164TIFF2026503177000138.tif229164TIFF2026503177000139.t if224164TIFF2026503177000140.tif219164TIFF2026503177000141.tif208167TIFF2026503177000142.tif221164TIFF202650 3177000143.tif205164TIFF2026503177000144.tif202164TIFF2026503177000145.tif221164TIFF2026503177000146.tif190 161TIFF2026503177000147.tif237164TIFF2026503177000148.tif199161TIFF2026503177000149.tif212164TIFF20265031770 00150.tif213161TIFF2026503177000151.tif253164TIFF2026503177000152.tif226164TIFF2026503177000153.tif244165TIFF2026503177000154.tif253161TIFF2026503177000155.tif227161TIFF2026503177000156.tif215163, in the above formula, each Z is connected with * and individually Selected from TIFF2026503177000157.tif155164; or TIFF2026503177000158.tif73164, in the above formula, each Z is connected with * and individually Selected from TIFF2026503177000159.tif154164; or a stereoisomer thereof, wherein Ab represents a binding unit and n is p load For example, 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.

[0376] In some embodiments, the above conjugate is provided, wherein the binding unit is antibody 2E7 and the drug-linker is LD038. In certain embodiments, the binding unit is antibody 2E7 (VH is SEQ ID NO:7 and VL is SEQ ID NO:8).

[0377] In some embodiments, the conjugate has the following structure: TIFF2026503177000160.tif85143, where Ab is 2E7 and n is p load For example, 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.

[0378] In some embodiments, the conjugate has the following structure: TIFF2026503177000161.tif86160, where Ab is 2E7 and n is p load For example, 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.

[0379] Exemplary Linker-Drug Combinations In some embodiments, conjugates are provided in which a drug unit, such as a tubulin disrupting agent, e.g., an auristatin, is attached to the linker by its C-terminal carboxyl group, which forms an amide bond with the linker subunit L2. In some embodiments, conjugates are provided in which the linker comprises at least one amino acid.

[0380] In some embodiments, conjugates are provided wherein the linker comprises, in addition to the linker subunit L2, a stretcher unit and / or an amino acid unit.

[0381] In some embodiments, the Stretcher unit can link the Binding Unit to an Amino Acid Unit or a Linker Subunit L2 via a sulfhydryl group in the Binding Unit. The sulfhydryl group can be generated, for example, by reduction of an interchain disulfide bond in the Binding Unit. For example, the Stretcher unit can be linked to the Binding Unit through a sulfur atom generated from reduction of the interchain disulfide bond in the Binding Unit. In some embodiments, the Stretcher unit is linked to the Binding Unit solely through a sulfur atom generated from reduction of the interchain disulfide bond in the Binding Unit. In some embodiments, the sulfhydryl group can be generated by reaction of an amino group of a lysine moiety in the Binding Unit with 2-iminothiolane (Traut's Reagent) or other sulfhydryl-generating reagent. In some embodiments, the Binding Unit is a recombinant antibody engineered to contain one or more additional lysines. In some embodiments, the recombinant Binding Unit is engineered to contain additional sulfhydryl groups, e.g., additional cysteines, e.g., engineered cysteines.

[0382] The synthesis and structure of MMAE are described in U.S. Patent No. 6,884,869, which is incorporated herein by reference in its entirety and for all purposes. The synthesis and structure of exemplary Stretcher units and methods for making antibody-drug conjugates are described, for example, in U.S. Patent Application Publication Nos. 2006 / 0074008 and 2009 / 0010945, each of which is incorporated herein by reference in its entirety.

[0383] Representative stretcher units are described within the brackets of formulas IIa and IIb in U.S. Pat. No. 9,211,319, incorporated herein by reference.

[0384] In some embodiments, the CD70 complex comprises monomethyl auristatin E (MMAE) and a protease-cleavable linker. In some embodiments, the CD70 complex comprises exatecan and a protease-cleavable linker. It is envisioned that the protease-cleavable linker comprises a thiol-reactive spacer and a dipeptide. In various embodiments, the protease-cleavable linker comprises a thiol-reactive maleimidocaproyl spacer, a valine-citrulline (val-cit) dipeptide, and a p-amino-benzyloxycarbonyl or PAB spacer.

[0385] The abbreviation "PAB" stands for self-immolating spacer: Points to TIFF2026503177000162.tif19128.

[0386] The abbreviation "MC" stands for stretcher maleimidocaproyl: Refers to TIFF2026503177000163.tif20128.

[0387] In some embodiments, conjugates are provided in which the Drug Unit is a camptothecin or a camptothecin (CPT) analog, such as irinotecan (also known as CPT-11), belotecan, topotecan, 10-hydroxy-CPT, exatecan, a diastereomer of exatecan, DXd, a diastereomer of DXd, or SN-38. Representative structures are shown below. TIFF2026503177000164.tif123131

[0388] Attachment of the Drug-Linker to the Binding Unit Techniques for linking a drug unit to a binding unit via a linker are well known in the art. See, for example, Alley et al., Current Opinion in Chemical Biology 2010 14:1-9; Senter, Cancer J., 2008, 14(3):154-169. In some embodiments, conjugates are provided in which a linker is first bound to a drug unit (e.g., a cytotoxic agent), and then a drug-linker is bound to a binding unit. In some embodiments, conjugates are provided in which a linker is first bound to a drug unit, and then a drug unit is bound to a linker. In the following description, the term drug-linker is used to illustrate the attachment of a linker or drug-linker to a binding unit; those skilled in the art will recognize that the selected attachment method can be determined by the linker and the drug unit (e.g., a cytotoxic agent or other drug unit). In some embodiments, conjugates are provided in which the Drug Unit is attached to the Linker Unit in a manner that reduces the activity of the Drug Unit until the Drug Unit is released from the conjugate (e.g., by hydrolysis, proteolysis, or a cleaving agent).

[0389] Generally, the conjugates can be prepared by several routes using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group of a linking unit with a bivalent linker reagent to form a linking unit-linker intermediate via a covalent bond, followed by reaction with a drug unit (e.g., a cytotoxic agent); and (2) reacting a nucleophilic group of a drug unit (e.g., a cytotoxic agent) with a bivalent linker reagent to form a drug-linker via a covalent bond, followed by reaction with a nucleophilic group of a linking unit. An exemplary method for preparing conjugates via the latter route is described in U.S. Patent No. 7,498,298, expressly incorporated herein by reference.

[0390] Nucleophilic groups on the linking unit include, but are not limited to, (i) N-terminal amine groups, (ii) side-chain amine groups, such as lysine, (iii) side-chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups on glycosylated antibodies. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyl groups, and maleimide groups. Certain linking units have reducible interchain disulfides, i.e., cysteine ​​bridges. Linking units can be made reactive for conjugation with linker reagents by treating them with a reducing agent, such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), to completely or partially reduce the linking unit. Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into a binding unit via modification of lysine residues, for example, by reacting the lysine residue with 2-iminothiolane (Traut's reagent), resulting in conversion of the amine to a thiol. Reactive thiol groups can also be introduced into a binding unit by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a binding unit containing one or more non-native cysteine ​​amino acid residues).

[0391] Conjugates can also be produced by the reaction of an electrophilic group on a linking unit, such as an aldehyde or ketone carbonyl group, with a nucleophilic group on a linker reagent or drug unit. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In one aspect, the linking unit is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or drug unit. In another aspect, the sugar of a glycosylated linking unit can be oxidized, for example, with a periodate oxidation reagent, to form an aldehyde or ketone group that can react with an amine group on a linker reagent or drug unit moiety. The resulting imine Schiff base group can form a stable bond or can be reduced, for example, with a borohydride reagent, to form a stable amine bond. In one embodiment, reaction of the carbohydrate moiety of a glycosylated linking unit with either galactose oxidase or sodium metaperiodate can result in carbonyl (aldehyde and ketone) groups in the linking unit that can react with appropriate groups on the drug unit (see, e.g., Hermanson, Bioconjugate Techniques). In another embodiment, a linking unit containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate, resulting in the production of an aldehyde in place of the initial amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; U.S. Patent No. 5,362,852). Such aldehydes can react with cytotoxic agents or linkers.

[0392] Exemplary nucleophilic groups on cell units, such as cytotoxic agents, include, but are not limited to, amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl halides and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups.

[0393] In some embodiments, conjugates are provided in which the drug-linker is attached to an interchain cysteine ​​residue of a linking unit. See, for example, WO2004 / 010957 and WO2005 / 081711. In such embodiments, the linker typically contains a maleimide group for attachment to a cysteine ​​residue of an interchain disulfide. In some embodiments, conjugates are provided in which the linker or drug-linker is attached to a cysteine ​​residue of a linking unit as described in U.S. Pat. No. 7,585,491 or U.S. Pat. No. 8,080,250.

[0394] In some embodiments, conjugates are provided wherein the linker or drug-linker is attached to a lysine or cysteine ​​residue of a binding unit as described in WO 2005 / 037992 or WO 2010 / 141566.

[0395] In some embodiments, conjugates are provided in which engineered cysteine ​​residues, polyhistidine sequences, glycoengineered tags, or transglutaminase recognition sequences can be used for site-specific attachment of linkers or drug-linkers to binding units.

[0396] In some embodiments, conjugates are provided in which the drug-linker is attached to an engineered cysteine ​​residue in an Fc residue other than the interchain disulfide. In some embodiments, the drug-linker is attached to an engineered cysteine ​​residue in an Fc residue other than the interchain disulfide, in accordance with the EU numbering of Kabat. , 292, 293, 294, 295, 296, 297, 298, 299, 300, 302, 305, 313, 318, 323, 324, 325, 327, 328, 329, 330, 331, 332, 333, 335, 336, 396, and / or 428, and / or linked to the light chain at positions 106, 108, 142 (light chain), 149 (light chain), and / or V205. An exemplary substitution for site-specific conjugation using an engineered cysteine ​​is S239C (see, e.g., US20100158909; Fc region numbering is according to the EU index).

[0397] In some embodiments, conjugates are provided in which a linker or drug-linker is attached to one or more introduced cysteine ​​residues of a drug unit described in WO2006 / 034488, WO2011 / 156328, and / or WO2016040856.

[0398] In some embodiments, conjugates are provided, wherein an exemplary substitution for site-specific conjugation using bacterial transglutaminase is N297S or N297Q in the Fc region. In some embodiments, conjugates are provided, wherein a linker or drug-linker is attached to the glycan or modified glycan of the linking unit. See, for example, WO2017 / 147542, WO2020 / 123425, WO2020 / 245229, WO2014 / 072482; WO2014 / / 065661, WO2015 / 057066 and WO2016 / 022027; the disclosures of which are incorporated herein by reference.

[0399] Drug Loading A CD70 ADC can contain one or more drug units per binding unit. The number of drug units per binding unit is referred to as the drug loading. The drug loading of a CD70 ADC is defined as the average number of drug units (drug molecules (e.g., cytotoxic agents)) per binding unit (e.g., antibody or antigen-binding moiety) in the CD70 ADC, p load For example, p load is about 4, the average drug loading considering all of the binding 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 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 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.

[0400] The average number of drug units per binding unit (e.g., antibody or antigen-binding moiety) 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 complexes with respect to p can also be determined. In some instances, the p distribution from complexes with other drug loadings can be determined. load Isolation, purification, and characterization of the homogeneous conjugate, where Λ is a certain value, can be achieved by means such as reverse phase HPLC or hydrophobic interaction chromatography (HIC) HPLC.

[0401] Pharmaceutical preparations Other aspects relate to compositions comprising the CD70 complexes described herein. In some embodiments, the compositions are pharmaceutical compositions. As used herein, the term "pharmaceutical composition" refers to an active agent combined with a pharmaceutically acceptable carrier accepted for use in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0402] The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on any particular formulation. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, although solid forms suitable for rehydration or suspension in liquid prior to use can also be prepared. Preparations can also be emulsified or presented as liposomal compositions. The CD70 complex can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts appropriate for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, pharmaceutical compositions can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance or maintain the effectiveness of the active ingredient (e.g., CD70 complex). The pharmaceutical compositions described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution containing an active ingredient (e.g., a CD70 complex) and water, which may contain a buffer such as sodium phosphate at a physiological pH value, physiological saline, or both, e.g., phosphate-buffered saline. Furthermore, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and in addition to water.Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0403] In some embodiments, pharmaceutical compositions comprising the CD70 complexes described herein can be lyophilized.

[0404] In some embodiments, a syringe containing a therapeutically effective amount of a CD70 complex or pharmaceutical composition thereof described herein is provided.

[0405] Cancer Treatment In some embodiments, the CD70 complexes described herein can be used in a method comprising administering a CD70 complex described herein to a subject in need thereof, such as a subject with cancer.

[0406] In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0407] In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; wherein the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and wherein the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified with 1 to 8, 1 to 6, 1 to 4, or 1 to 2 conservative amino acid substitutions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0408] In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in a pair of amino acid sequences selected from SEQ ID NO:3 and SEQ ID NO:4, respectively; SEQ ID NO:5 and SEQ ID NO:6, respectively; SEQ ID NO:7 and SEQ ID NO:8, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; and SEQ ID NO:11 and SEQ ID NO:12, respectively; and wherein the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively; the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively; the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively; the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:9 and SEQ ID NO:10, respectively; the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified. In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12, respectively; the heavy and light chain variable framework regions are optionally modified by 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acid substitutions, deletions, or insertions within the framework regions, and the CDRs of the heavy or light chain variable regions are unmodified.In some embodiments, methods are provided for treating cancer comprising administering a CD70 complex comprising a heavy chain variable region having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any one of SEQ ID NOs:3, 5, 7, 9, or 11. In some embodiments, methods are provided for treating cancer comprising administering a CD70 complex comprising a VL region having an amino acid sequence at least 60% identical (e.g., at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of SEQ ID NOs:4, 6, 8, 10, or 12.

[0409] In some embodiments, methods of treating cancer are provided that include administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed within the heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within the light chain variable region framework region, and the VH and VL CDRs are (i) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; (ii) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively; and (iii) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, respectively. (iv) SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:18, respectively; and (V) SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0410] In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0411] In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0412] In some embodiments, methods of treating cancer are provided that include administering a CD70 complex 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 disposed within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within a light chain variable region framework region, and the VH CDRs and VL CDRs have the amino acid sequences set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments of treating cancer, each VH and VL region comprises a human framework region.

[0413] In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 disposed within a light chain variable region framework region, and the VH and VL CDRs have the amino acid sequences set forth in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:18, respectively. In some embodiments, each VH and VL region comprises a humanized framework region. In some embodiments, each VH and VL region comprises a human framework region.

[0414] In some embodiments, methods of treating cancer are provided comprising administering a CD70 complex comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3 disposed within a heavy chain variable region framework region, and the VL region comprises LCDR...

Claims

1. A conjugate comprising a binding unit attached to one or more drug units by one or more linkers, wherein the binding unit comprises at least a portion of an anti-CD70 antibody.

2. The conjugate of claim 1 , wherein the binding unit comprises an anti-CD70 antibody.

3. A conjugate comprising a binding unit attached to one or more drug units by one or more linkers, (1) The binding unit 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 within a heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2, and LCDR3 arranged within a light chain variable region framework region, and the VH CDR and VL CDR are a. SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:13, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively; b. SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:14, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively; c. SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively; d. SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:18, respectively; and e. SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively. having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of: (2) Each linker has the following formula (I): or a salt thereof; During the ceremony, L1 is a Stretcher unit covalently linked to the Binding Unit, where the wavy line (~) indicates the binding site for the Binding 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, where indicates the binding site for the Drug unit; 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 sugar unit, a PEG unit, a carboxyl unit, and a combination thereof; (3) Each drug unit is is covalently attached to each linker subunit by The complex.

4. The VH and VL regions of the binding unit are each a. SEQ ID NO:3 and SEQ ID NO:4; b. SEQ ID NO:5 and SEQ ID NO:6; c. SEQ ID NO:7 and SEQ ID NO:8; d. SEQ ID NO:9 and SEQ ID NO:10; and e. SEQ ID NO:11 and SEQ ID NO:12 4. The conjugate of claim 3, having an amino acid sequence selected from the pair of amino acid sequences shown in the group consisting of:

5. The VH and VL regions of the binding unit are each a. SEQ ID NO:3 and SEQ ID NO:4; b. SEQ ID NO:5 and SEQ ID NO:6; c. SEQ ID NO:7 and SEQ ID NO:8; d. SEQ ID NO:9 and SEQ ID NO:10; and e. SEQ ID NO:11 and SEQ ID NO:12 and having an amino acid sequence selected from the pair of amino acid sequences shown in the group consisting of: the heavy and light chain framework regions are optionally modified by 1 to 8 amino acid substitutions, deletions, or insertions within the framework regions; The complex of claim 3.

6. The conjugate of any one of claims 3 to 5, wherein the HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 of the binding units have the amino acid sequences shown in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:15, and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, respectively.

7. The conjugate of claim 3, wherein the framework regions of the binding units are human framework regions.

8. The conjugate of any one of claims 3 to 7, wherein the binding unit is an antibody or an antigen-binding portion thereof.

9. 9. The conjugate of any one of claims 3 to 8, wherein the binding unit is a monoclonal antibody, Fab, Fab', F(ab'), Fv, disulfide-linked Fc, scFv, single domain antibody, diabody, bispecific antibody, or multispecific antibody.

10. The conjugate of any one of claims 3 to 9, wherein the binding unit comprises a heavy chain variable region and further comprises a heavy chain constant region.

11. The conjugate of claim 10, wherein the heavy chain constant region of the binding unit is an IgG isotype.

12. 12. The conjugate of claim 11, wherein the heavy chain constant region of the binding unit is an IgG1 constant region.

13. 11. The conjugate of claim 10, wherein the heavy chain constant region of the binding unit is an IgG4 constant region.

14. 13. The conjugate of claim 12, wherein the IgG1 constant region of the binding unit has the amino acid sequence shown in SEQ ID NO:

28.

15. The conjugate of any one of claims 3 to 14, wherein the binding unit comprises a light chain variable region and further comprises a light chain constant region.

16. 16. The conjugate of claim 15, wherein the light chain constant region of the binding unit is a kappa isotype.

17. 17. The conjugate of claim 16, wherein the light chain constant region of the binding unit has the amino acid sequence shown in SEQ ID NO:

29.

18. 18. The conjugate of any one of claims 10 to 17, wherein the heavy chain constant region of the binding unit further comprises at least one amino acid modification that reduces binding affinity to a human Fc receptor, such as FcγRIII.

19. The conjugate of any one of claims 3 to 18, wherein the binding unit is monospecific.

20. The conjugate of any one of claims 3 to 18, wherein the binding unit is bivalent.

21. The conjugate of any one of claims 3 to 18, wherein the binding unit is bispecific.

22. The sugar unit of the linker has the following formula: or a salt thereof, During the ceremony, 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 are independently selected from H, OH, and OR; k is 1 to 10; 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 of an amino acid unit (AA) to another subunit, a linker subunit (L2), or a stretcher unit (L1); The conjugate of any one of claims 3 to 21.

23. The PEG unit of the linker is （a） ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -NR 24 R 25 (XX) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid unit, a portion of a Stretcher unit and / or a Linker subunit L2; R 21 and R 22 are each independently any C 1 ~C 3 alkylene; R 24 and R 25 is H; a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C(O)-polyhydroxyl group; 3 ~C 10 Carbocyclic ring; optionally substituted C 1 ~C 3 Alkylene C 3 ~C 10 Carbocyclic ring; Optionally substituted heteroaryl; Optionally substituted carbocyclic ring; Substituted -C 1 ~C 8 Alkyl; Substituted -C(O)-C 1 ~C 8 Alkyl; Chelating agent; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 ; or together C 3 ~C 8 -NR from heterocycles 24 R 25 , independently selected from; 24 and R 25 provided that neither of The wavy line (~) is R 20 indicates the binding site for n20 is 1 to 26; or （b） ~R 20 -R 21 -[O-CH 2 -CH 2 ] n20 -R 22 -NR 24 R 25 (XX) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid unit, a portion of a Stretcher unit and / or a Linker subunit L2; R 21 and R 22 are each independently any C 1 ~C 3 alkylene; R 24 and R 25 one of which is a polyhydroxyl group; a substituted polyhydroxyl group; a -C(O)-polyhydroxyl group; a substituted -C(O)-polyhydroxyl group; an optionally substituted C(O)-polyhydroxyl group; 3 ~C 10 Carbocyclic ring; optionally substituted C 1 ~C 3 Alkylene C 3 ~C 10 Carbocyclic ring; Optionally substituted heteroaryl; Optionally substituted carbocyclic ring; Substituted -C 1 ~C 8 Alkyl; Substituted -C(O)-C 1 ~C 8 Alkyl; Chelating agent; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 , selected from; R 24 and R 25 the other is polyethylene glycol optionally having 1 to 24 ethylene glycol subunits; The wavy line (~) is R 20 indicates the binding site for n20 is 1 to 26; 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) or a salt thereof, wherein R 20 is a functional group for binding to a subunit of an Amino Acid unit, a portion of a Stretcher unit and / or a Linker subunit L2; R 26 and R 27 are arbitrary, 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 independently selected from 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(O)-polyhydroxyl group; 3 ~C 10 Carbocyclic ring; optionally substituted C 1 ~C 3 Alkylene C 3 ~C 10 Carbocyclic ring; Optionally substituted heteroaryl; Optionally substituted carbocyclic ring; Substituted -C 1 ~C 8 Alkyl; Substituted -C(O)-C 1 ~C 8 Alkyl; Chelating agent; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 , selected from; 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(O)-polyhydroxyl group; 3 ~C 10 Carbocyclic ring; optionally substituted C 1 ~C 3 Alkylene C 3 ~C 10 Carbocyclic ring; Optionally substituted heteroaryl; Optionally substituted carbocyclic ring; Substituted -C 1 ~C 8 Alkyl; Substituted -C(O)-C 1 ~C 8 Alkyl; Chelating agent; R 28 is a sugar unit of formula (XII) or (XIII) -C(O)-R 28 and polyethylene glycol optionally having 1 to 24 ethylene glycol subunits; or together C 3 ~C 8 -NR from heterocycles 24 R 25 , where R 24 and R 25 provided that neither of 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 indicates the binding site for n20 is 1 to 26; n21 is 1 to 4; n27 is 1–4; 23. The conjugate of any one of claims 3 to 22, having a formula selected from:

24. 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 (b) are not H.

25. 25. The conjugate of any one of claims 23 to 24, wherein the polyhydroxyl group is a linear monosaccharide optionally selected from a C6 or C5 sugar, a sugar acid, or an amino sugar.

26. 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 urosonic acid; or the amino sugar is selected from glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine; 26. The conjugate of claim 25.

27. R 24 and R 25 24. The conjugate of claim 23, wherein one of said monosaccharides is a linear monosaccharide and the other is a cyclic monosaccharide.

28. R 24 and R 25 24. The conjugate of claim 23, wherein is independently selected from cyclic monosaccharides, disaccharides, and polysaccharides.

29. R 24 and R 25 are independently selected from linear monosaccharides and substituted linear monosaccharides, and the substituted linear monosaccharides are substituted with a monosaccharide, a disaccharide, or a polysaccharide.

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

31. 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 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.

32. R 24 and R 25 But H, substituted -C 1 ~C 8 Alkyl, substituted -C 1 ~C 4 Alkyl or substituted -C 1 ~C 3 alkyl; 24 and R 25 and -C are not H; 1 ~C 8 Alkyl, -C 1 ~C 4 Alkyl and -C 1 ~C 3 alkyl is substituted with hydroxyl and / or carboxyl; provided that R 24 and R 25 and (b) are not H.

33. 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; 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 alkyl is substituted with hydroxyl and / or carboxyl; provided that R 24 and R 25 and (b) are not H.

34. Each monosaccharide is a C5 or C6 sugar selected from glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, gulose, idose, talose, aldose, ketose, glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine; a sugar acid selected from gluconic acid, aldonic acid, uronic acid, and urosonic acid; or an amino sugar selected from glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine The conjugate of any one of claims 24 to 31, independently selected from the following:

35. 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.

36. R 20 24. The conjugate of claim 23, wherein 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.

37. ~R 40 -(R 43 -R 41 -[O-CH 2 -CH 2 ] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XL) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid unit, the Stretcher unit and / 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, Heteroaryl-C 1 ~C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 where 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, where the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; 44 and R 45 provided that neither of The wavy line (~) is R 40 indicates the binding site for n40 is 1 to 26; n41 is 1 to 6; n42 is 1 to 6, The conjugate of any one of claims 3 to 22.

38. ~R 40 -(R 41 -[O-CH 2 -CH 2 ] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLI) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid unit, the Stretcher unit and / 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 where 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, where the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; 44 and R 45 provided that neither of The wavy line (~) is R 40 indicates the binding site for n40 is 1 to 26; n41 is 1 to 6; n42 is 1 to 6, The conjugate of any one of claims 3 to 22.

39. ~R 40 -(R 41 -[O-CH 2 -CH 2 ] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLII) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid unit, the Stretcher unit and / 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 where 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, where the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; 44 and R 45 provided that neither of The wavy line (~) is R 40 indicates the binding site for n40 is 1 to 26; n41 is 1 to 4; n42 is 1 to 4, The conjugate of any one of claims 3 to 22.

40. 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.

41. R 20 or R 40 but has the following structure: or a stereoisomer thereof, During the ceremony, R=H or C 1～6 is alkyl, n=0 to 12, (*) indicates the R to a subunit of the amino acid unit, the stretcher unit and / or a portion of the linker subunit L2. 20 or R 40 indicates the binding site of R to the remainder of the PEG unit 20 or R 40 indicates the binding site of The conjugate of any one of claims 37 to 40.

42. R 20 or R 40 but has the following structure: or a stereoisomer thereof, During the ceremony, n=0 to 12, (*) indicates the R to a subunit of the amino acid unit, the stretcher unit and / or a portion of the linker subunit L2. 20 or R 40 indicates the binding site of R to the remainder of the PEG unit 20 or R 40 indicates the binding site of 42. The conjugate of claim 41.

43. NR 43 If there is R 43 -(NR 44 R 45 ) n41 but has the following structure: or a stereoisomer thereof, During the ceremony, R=H, C 1～6 alkyl, polyhydroxyl, or substituted polyhydroxyl; R to the remainder of the PEG unit 43 indicates the binding site of The conjugate of any one of claims 37 to 42.

44. NR 43 If there is R 43 -(NR 44 R 45 ) n41 but has the following structure: or a stereoisomer thereof, wherein R to the remainder of the PEG unit 43 indicates the binding site of 44. The conjugate of claim 43.

45. -NR 44 R 45 but has the following structure: or a stereoisomer thereof, wherein -NR to the remainder of the PEG unit 44 R 45 indicates the binding site of The conjugate of any one of claims 37 to 44.

46. ~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) or a salt thereof, During the ceremony, R 40 is a functional group for binding to a subunit of the Amino Acid unit, the Stretcher unit and / 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, Heteroaryl-C 1 ~C 12 Alkylene -C(O)- or -C(O)NR 46 R 47 where 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, where the optional substituents are selected from sulfate, phosphate, alkyl sulfate, and alkyl phosphate; 44 and R 45 provided that neither of R 46 is 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 to 26; n41 is 1 to 6; n42 is 1 to 6, The conjugate of any one of claims 3 to 22.

47. The compound according to claim 47, comprising a PEG unit having a formula selected from the following: During the ceremony, Each Y is independently R 76 or and Each R 76 are independently H, acetyl, -P(=O)(OH) 2 , or -(CH 2 ) 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 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; Each * indicates a binding site for the amino acid unit (AA) subunit, the linker subunit L2, or the stretcher unit (L1); The conjugate of any one of claims 3 to 22.

48. The PEG unit is or a stereoisomer or salt thereof, During the ceremony, 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; Each * indicates a binding site for the amino acid unit (AA) subunit, the linker subunit L2, or the stretcher unit (L1); 48. The conjugate of claim 47.

49. The PEG unit is or a stereoisomer or salt thereof, During the ceremony, 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 the amino acid unit (AA) subunit, the linker subunit L2, or the stretcher unit (L1); 49. The conjugate of claim 47 or 48.

50. Y is R 76 48. The conjugate of claim 47, wherein

51. Y is 48. The conjugate of claim 47, wherein

52. Each R a and R b 48. The conjugate of claim 47, wherein:

53. R a and R b 48. The conjugate of claim 47, wherein together with the carbon to which they are attached form an oxo group.

54. 54. The conjugate of any one of claims 47 to 53, wherein q is 10 to 20.

55. 55. The conjugate of any one of claims 47 to 54, wherein q is 12.

56. The following formula: or a salt thereof, During the ceremony, （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 Ha~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 an optionally absent or substituted C 1 ~C 3 is 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, 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a 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 Ha~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 C is an optionally branched substituted 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 is independently carboxyl or polycarboxyl, where 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a 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 Ha~N(R 74 -R 73 )(R 72 - R 73 ), where R 72 and R 74 may be substituted C 1 ~C 3 are each independently 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 is independently carboxyl or polycarboxyl, where 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 of an amino acid unit (AA) to another subunit, a linker subunit L2, or a stretcher unit (L1); each of p1 and o1 is independently selected from 0 to 2; The conjugate of any one of claims 3 to 22.

57. 57. The conjugate of any one of claims 3 to 56, comprising at least one sugar unit.

58. 58. The conjugate of any one of claims 3 to 57, comprising at least one PEG unit.

59. 59. The conjugate of any one of claims 3 to 58, comprising at least one carboxyl unit.

60. 57. The conjugate of any one of claims 3 to 56, comprising at least two polar units, each polar unit selected from a sugar unit, a PEG unit, and a carboxyl unit.

61. 57. The conjugate of any one of claims 3 to 56, comprising at least one sugar unit and one PEG unit or one carboxyl unit.

62. 57. The conjugate of any one of claims 3 to 56, comprising at least one carboxyl unit and one PEG unit.

63. The conjugate of any one of claims 3 to 56, wherein the amino acid unit (AA) is present (s=1).

64. 64. The conjugate of claim 63, wherein the amino acid unit comprises at least one polar unit.

65. L2 or AA-L2 has the following structure: or a stereoisomer thereof, wherein the wavy line on the amino group indicates the attachment site for a Stretcher unit or an Amino Acid unit, and the Drug unit is attached to a benzyl alcohol.

65. The conjugate of any one of claims 3 to 64.

66. The linker may be: AA-L2, having a formula selected from In the formula, the square brackets represent the amino acid units, each aa is any 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; double wavy line indicates the binding site for the Drug unit, and aa and aa 1 are independently selected from α, β and γ amino acids and derivatives thereof, 64. The conjugate of any one of claims 3 to 63.

67. The linker may be: AA-L2, having a formula selected from In the formula, 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; 1 (PEG) is a PEG unit linked to aa, SU is a sugar unit linked to aa, CU is a carboxyl unit linked to aa, and the double wavy line indicates the binding site for the Drug unit; aa and aa 1 are independently selected from α, β and γ amino acids and derivatives thereof, 64. The conjugate of any one of claims 3 to 63.

68. 64. The conjugate of any one of claims 3 to 63, wherein the amino acid unit comprises at least two polar units.

69. The linker may be: AA-L2, having a formula selected from In the formula, the square brackets represent the amino acid units, aa is any subunit of AA, L2 is the linker subunit, and the wavy line (~) represents the binding site for the stretcher unit; 1 (PEG) and aa 2 Each of (PEG) is a PEG unit linked to aa or the other PEG unit; each SU is a sugar unit linked to aa or the other sugar unit, and each CU is a carboxyl unit linked to aa or the other carboxyl unit, and the double wavy line indicates the binding site for the Drug unit; aa, aa 1 and aa 2 are independently selected from α, β and γ amino acids and derivatives thereof, 69. The conjugate of claim 68.

70. The linker may be: AA-L2, having a formula selected from In the formula, the square brackets represent the amino acid units, aa represents an amino acid subunit of AA, L2 represents a linker subunit attached to the side chain of aa, and each wavy line (~) represents a binding site for a stretcher unit; 1 (PEG) and aa 2 Each of (PEG) is a PEG unit linked to aa; each SU is a sugar unit linked to aa; each CU is a carboxyl unit linked to aa; indicates the binding site for the Drug unit; aa, aa 1 and aa 2 are each independently selected from α, β and γ amino acids and derivatives thereof; 69. The conjugate of claim 68.

71. 71. The conjugate of any one of claims 3 to 70, wherein the linker subunit L2 is a cleavable linker unit.

72. 72. The conjugate of claim 71, wherein the linker subunit L2 comprises a peptide that is cleavable by an intracellular protease.

73. 73. The conjugate of claim 72, 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.

74. 74. The conjugate of any one of claims 3 to 73, wherein the linker subunit L2 comprises at least one polar unit.

75. 75. The conjugate of any one of claims 3 to 74, wherein the polar unit is a sugar unit (SU).

76. 76. The conjugate of claim 75, 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.

77. 75. The conjugate of claim 74, wherein the polar unit is a carboxyl unit (CU).

78. 78. The conjugate of claim 77, 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.

79. 75. The conjugate of claim 74, wherein the polar unit is a PEG unit (PEG).

80. 80. The conjugate of claim 79, 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 a PEG unit linked to a lysine or citrulline residue, respectively.

81. 81. The conjugate of any one of claims 71-80, wherein the cleavable peptide is conjugated to a para-aminobenzyl alcohol self immolative group (PABA).

82. 82. The conjugate of any one of claims 71 to 81, wherein L2 is attached to a side chain of an AA subunit.

83. 83. The conjugate of any one of claims 3 to 82, wherein the amino acid unit is linked to the linker subunit L2 by a non-peptide linking group.

84. The non-peptidic linking group is C 1 ~C 10 Alkylene, C 2 ~C 10 Alkenylene, C 2 ~C 10 84. The conjugate of claim 83, wherein the alkyl group is selected from alkynylene, or polyethylene glycol.

85. 85. The conjugate of any one of claims 3 to 84, wherein the linker further comprises a stretcher unit.

86. The stretcher unit consists of: wherein R 17 -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- (where b is 1 to 26), -C 1 ~C 8 Alkylene-(CH 2 -O-CH 2 ) b - (where b is 1 to 26), -C 1 ~C 8 Alkylene-(CH 2 -O-CH 2 ) b -C 1 ~C 8 alkylene- (where 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- (where b is 1 to 26), -C 1 ~C 8 Alkylene-(CH 2 -O-CH 2 ) b -C 1 ~C 8 alkylene-NH- (where 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- (where 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 made of maleimide (C 1 ~C 10 Alkylene-C(O)-, maleimide (CH 2 OCH 2 ) p2 (C 1 ~C 10 Alkyene (C(O)-), maleimide (C 1 ~C 10 Alkyen) (CH 2 OCH 2 ) p2 C(O)- or their open ring forms, wherein p2 is 1 to 26; 86. The conjugate of claim 85.

87. The stretcher unit comprises: Selected from In the formula, wavy line indicates the attachment site of the Stretcher unit to an Amino Acid unit or Linker subunit L2, and the attachment site to the Linking unit is on a maleimide, a primary amine, or an alkyne functional group; 86. The conjugate of claim 85.

88. 88. The conjugate of any one of claims 3-87, wherein each Drug unit is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitor, a PROTAC, a toxin, a radioisotope, and a chelating ligand.

89. 89. The conjugate of any one of claims 3-88, wherein each linker is attached to the linking unit via an interchain disulfide residue, a lysine residue, an engineered cysteine ​​residue, a glycan, a modified glycan, an N-terminal residue of the linking unit, or a polyhistidine peptide attached to the linking unit.

90. The average drug loading of the complex (p load 90. The conjugate of any one of claims 3-89, 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.

91. 91. The conjugate of any one of claims 3 to 90, wherein the drug is a cytotoxic agent.

92. 92. The conjugate of claim 91, wherein the cytotoxic agent is selected from the group consisting of an auristatin, a maytansinoid, a camptothecin, a duocarmycin, or a calicheamicin.

93. 93. The conjugate of claim 92, wherein the cytotoxic agent is an auristatin.

94. 94. The conjugate of claim 93, wherein the cytotoxic agent is MMAE or MMAF.

95. 93. The conjugate of claim 92, wherein the cytotoxic agent is camptothecin.

96. 96. The conjugate of claim 95, wherein the cytotoxic agent is exatecan.

97. 96. The conjugate of claim 95, wherein the cytotoxic agent is SN-38.

98. 93. The conjugate of claim 92, wherein the cytotoxic agent is calicheamicin.

99. 93. The conjugate of claim 92, wherein the cytotoxic agent is a maytansinoid.

100. 100. The conjugate of claim 99, wherein the maytansinoid is maytansine, maytansinol, or maytansine analogs such as DM1, DM3 and DM4, and ansamatocin-2.

101. 89. The conjugate of claim 88, wherein the Drug unit is an immunomodulator.

102. 102. The conjugate of claim 101, wherein the immunomodulatory agent is selected from a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist.

103. 103. The conjugate of claim 102, wherein the immunomodulator is a TLR7 agonist.

104. 104. The conjugate of claim 103, wherein 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, heteroarothiadiazide-2,2-dioxide, benzonaphthyridine, guanosine analog, adenosine analog, thymidine homopolymer, ssRNA, CpG-A, polyG10, or polyG3.

105. 103. The conjugate of claim 102, wherein the immunomodulator is a TLR8 agonist.

106. 106. The conjugate of claim 105, wherein the TLR8 agonist is selected from 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.

107. 103. The conjugate of claim 102, wherein the immunomodulator is a STING agonist.

108. 103. The conjugate of claim 102, wherein the immunomodulator is a RIG-I agonist.

109. The conjugate of claim 108, wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400 and KIN2000.

110. 89. The conjugate of claim 88, wherein the Drug unit is a chelating ligand.

111. 111. The complex of claim 110, 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 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, samarium-153, and lead-212.

112. The linker is mc-VC-PAB, CL2, CL2A, or (succinimide-3-yl-N)-(CH 2 ) n -C(=O)-Gly-Gly-Phe-Gly-NH-CH 2 -O-CH 2 72. The conjugate of claim 71, comprising -(C=O)-, where n=1 to 5.

113. 113. The conjugate of claim 112, wherein the linker comprises mc-VC-PAB.

114. 113. The conjugate of claim 112, wherein the linker comprises CL2A.

115. 113. The conjugate of claim 112, wherein the linker comprises CL2.

116. The linker is (succinimide-3-yl-N)-(CH 2 ) n -C(=O)-Gly-Gly-Phe-Gly-NH-CH 2 -O-CH 2 113. The conjugate of claim 112, comprising -(C=O)-.

117. 117. The conjugate of any one of claims 3-92, 95-96, or 112-116, wherein said linker is attached to at least one molecule of exatecan.

118. below: In the above formula, each Z is bonded with * and individually Selected from: or In the above formula, each Z is bonded with * and individually Selected from: or a stereoisomer thereof, Ab represents the binding unit, and n is p load wherein pload 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; The conjugate of any one of claims 3 to 21.

119. The following structure: wherein Ab is 2E7 and n is p load 4. The conjugate of claim 3, wherein pload 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.

120. The following structure: wherein Ab is 2E7 and n is p load 4. The conjugate of claim 3, wherein pload 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.

121. 121. A pharmaceutical composition comprising the conjugate of any one of claims 3 to 120 and a pharmaceutically acceptable carrier.

122. 122. A method of treating CD70+ cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the conjugate of any one of claims 3-120 or the pharmaceutical composition of claim 121.

123. 123. The method of claim 122, wherein the CD70+ cancer is a solid tumor or a hematological malignancy.

124. 124. The method of claim 123, wherein the CD70+ cancer is selected from hepatocellular carcinoma, colorectal cancer, pancreatic cancer, ovarian cancer, indolent non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the B-cell lineage, multiple myeloma, renal cell carcinoma, nasopharyngeal carcinoma, thymic carcinoma, head and neck cancer, and glioma.

125. 124. The method of claim 123, wherein the CD70+ cancer is a hematological malignancy.

126. 124. The method of claim 123, wherein the CD70+ cancer is non-Hodgkin's lymphoma.

127. 126. The method of claim 125, wherein the CD70+ cancer is diffuse large B-cell lymphoma (DLBCL).

128. 124. The method of claim 123, wherein the CD70+ cancer is a solid tumor.

129. 124. The method of claim 123, wherein the CD70+ cancer is renal cell carcinoma.

130. 124. The method of claim 123, wherein the CD70+ cancer is clear cell renal cell carcinoma (ccRCC).

131. 124. The method of claim 123, wherein the CD70+ cancer is head and neck cancer.

132. 124. The method of claim 123, wherein the CD70+ cancer is squamous cell carcinoma.

133. 124. The method of claim 123, wherein the CD70+ cancer is head and neck squamous cell carcinoma (HNSCC).

134. 134. The method of any one of claims 122-133, further comprising administering immunotherapy to said subject.

135. 135. The method of claim 134, wherein said immunotherapy comprises a checkpoint inhibitor.

136. 136. The method of claim 135, wherein said checkpoint inhibitor is selected from an antibody that specifically binds to human PD-1, human PD-L1, or human CTLA4.

137. 137. The method of claim 136, wherein said checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or ipilimumab.

138. 138. The method of any one of claims 122-137, further comprising administering chemotherapy to said subject.

139. 140. The method of any one of claims 122-139, wherein the conjugate or pharmaceutical composition is administered intravenously.

140. 140. The method of claim 139, wherein said conjugate or pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 12 mg / kg.

141. The method of any one of claims 122-140, wherein the subject's treatment outcome is improved.

142. 142. The method of claim 141, wherein said improved treatment outcome is an objective response selected from stable disease, a partial response, or a complete response.

143. 142. The method of claim 141, wherein said improved treatment outcome is a reduction in tumor burden.

144. 142. The method of claim 141, wherein said improved treatment outcome is progression-free survival or disease-free survival.

145. 122. Use of the conjugate of any one of claims 3 to 120 or the pharmaceutical composition of claim 121 for the treatment of CD70+ cancer in a subject.

146. 122. A method of treating an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of the conjugate of any one of claims 3 to 120 or the pharmaceutical composition of claim 121.

147. 147. The method of claim 146, wherein the autoimmune disease is rheumatoid arthritis, multiple sclerosis, or systemic lupus erythematosus.

148. 148. The method of any one of claims 146-147, further comprising administering to said subject an immunosuppressive therapy.

149. 149. The method of any one of claims 146-148, wherein said conjugate or pharmaceutical composition is administered intravenously.

150. 150. The method of claim 149, wherein said conjugate or pharmaceutical composition is administered at a dose of about 0.1 mg / kg to about 12 mg / kg.

151. 151. The method of any one of claims 146-150, wherein the subject's treatment outcome is improved.

152. 152. The method of claim 151, wherein said improved treatment outcome is a reduction in disease progression or a decrease in disease severity.

153. Use of the conjugate of any one of claims 3 to 120 or the pharmaceutical composition of claim 121 for the treatment of an autoimmune disease in a subject.

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