CD70 antibody drug conjugates and methods of use thereof

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.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to International Patent Application No. PCT / CN2022 / 127588 filed on October 26, 2022 and International Patent Application No. PCT / US23 / 74126 filed on September 14, 2023, the contents of which are incorporated herein by reference. Background Art

[0003] There has been significant interest in the use of monoclonal antibodies (mAbs) to target cytotoxic agents to disease-associated cells (e.g., cancer cells and other cells) in the form of antibody-drug conjugates (ADCs). The design of antibody-drug conjugates, which involves attaching cytotoxic agents, immunomodulators, or other drugs (collectively referred to as "drugs") to antibodies (usually via linkers), requires consideration of a variety of factors. These factors include the identity and position of the chemical groups used to attach the drug, the mechanism of drug release, the structural elements that provide for drug release (if any), and the structural modifications of the released free drug (if any). If the drug is released in the extracellular environment, the drug must be released in a form that is capable of reaching 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.

[0004] Another important factor in the design of antibody drug conjugates is the amount of drug that can be delivered per targeting agent (i.e., the number of drugs attached to each targeting agent (e.g., antibody), referred to as drug load or drug loading). Historically, higher drug loads have been considered superior to lower drug loads (e.g., 8 loadings versus 4 loadings). The rationale is that conjugates with higher loadings deliver more drug (e.g., cytotoxic agent) to target cells. This rationale is supported by the observation that conjugates with higher drug loadings are more active against cell lines in vitro. However, some subsequent studies have shown that this view was not confirmed in animal models. In mouse models, conjugates with loadings of 4 or 8 specific auristatins were observed to have similar activity. See, for example, Hamblett et al., Clinical Cancer Res. 10:7063-70 (2004). Hamblett et al. further reported that in animal models, ADCs with higher loadings were cleared faster from the circulation. This faster clearance suggests that species with higher drug loads are more susceptible to pharmacokinetic (PK) changes compared to species with lower drug loads. See Hamblett et al. In addition, conjugates with higher drug loads have lower maximum tolerated doses (MTDs) in mice, and therefore have narrower reported therapeutic indexes. Ibid. In contrast, it has been reported that ADCs with a drug load of 2 at an engineered site in a monoclonal antibody have the same or better pharmacokinetics and therapeutic index than certain ADCs with a drug load of 4. For example, see Junutula et al., Clinical Cancer Res. 16: 4769 (2010). Therefore, the recent trend is to develop ADCs with lower drug loads.

[0005] CD70 is an attractive target for cancer therapy using ADCs. CD70 is a member of the tumor necrosis factor (TNF) family, which are cell 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 outside of the cell and its amino terminus located on the cytoplasmic side of the plasma membrane (Bowman et al., 1994, J. Immunol. 152:1756-61; Goodwin et al., 1993, Cell 73:447-56). Human CD70 comprises 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 to TNF-α and TNF-β, CD70 is predicted to have a trimeric structure (Petsch et al., 1995, Mol. Immunol. 32:761-72).

[0006] CD70 expression is limited in normal human tissues. This makes it an attractive target for cancer therapy. CD70 has been identified and expressed in a variety of cancers, including renal cell carcinoma, colon cancer, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, certain types of non-Hodgkin's lymphoma, and multiple myeloma. Despite the presence of CD70 in various cancer types, clinical trials targeting CD70 antibodies and CD70 antibody-drug conjugates have had limited success to date.

[0007] Therefore, there is a general need for CD70 antibody-drug conjugates, and in particular, a need for CD70 antibody-drug conjugates that can achieve higher drug loadings while retaining other properties of lower-load conjugates (e.g., good pharmacokinetic properties). Embodiments of the present invention are intended to meet these and related needs. 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, a linker, and one or more drug units, wherein the binding unit comprises one or more CD70 antibodies or antigen-binding portions thereof. In addition, provided herein are CD70 ADCs having hydrophilic properties that maintain the inherent properties of the CD70 antibodies conjugated to the one or more drug units via the linker. Specifically, the linker helps maintain the hydrophilicity of the CD70 antibodies when conjugated with higher drug loads and / or with hydrophobic drugs and other agents. Also provided herein are methods of using such conjugates to treat cancer and other diseases. The invention disclosed herein is 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. CD70 ADCs provide compositions and methods based on the use of such conjugates to treat CD70+ cancers and other diseases.

[0009] In some embodiments, a conjugate is provided comprising a Binding unit bound to one or more Drug units via one or more Linkers, wherein:

[0010] (1) The binding unit comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3 positioned in the heavy chain variable region framework region and the VL region comprising LCDR1, LCDR2 and LCDR3 positioned in the light chain variable region framework region, the VH and VL CDRs each having an amino acid sequence selected from the group consisting of 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: 15, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26; NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:18; 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;

[0011] (2) Each joint has the following formula (I):

[0012] ~L1–(AA) s –L2≈

[0013] (I)

[0014] or a salt thereof, wherein:

[0015] L1 is a stretcher unit covalently bound to the binding unit, wherein the wavy (~) line indicates the attachment site to the binding unit;

[0016] AA is an amino acid unit having 1 to 12 subunits;

[0017] s is 0 or 1;

[0018] L2 is a linker subunit having 1 to 4 attachment sites for a Drug unit, wherein a double wavy (≈) line indicates an attachment site to a Drug unit; and

[0019] wherein at least one polar unit is present in an amino acid unit, a linker subunit, a stretcher unit, or a combination thereof, and wherein the polar unit is selected from a sugar unit, a PEG unit, a carboxyl unit, and a combination thereof; and

[0020] (3) Each Drug unit is covalently linked to a Linker subunit at (≈).

[0021] In some embodiments, a conjugate is provided, wherein the VH and VL regions of the binding unit each have an amino acid sequence selected from the group consisting of an amino acid sequence pair as shown in the following group: 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, a conjugate is provided, wherein the VH and VL regions of the binding unit each have an amino acid sequence selected from the group consisting of an amino acid sequence pair as shown in the following group: 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, wherein the heavy and light chain framework regions are optionally modified with 1 to 8 amino acid substitutions, deletions, or insertions in the framework regions. In some embodiments, a conjugate is provided wherein the HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 of the binding unit 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.

[0022] In some embodiments, conjugates are provided wherein the framework regions of the binding unit are human framework regions. In some embodiments, conjugates are provided wherein the binding unit is an antibody or an antigen-binding portion thereof. In some embodiments, 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.

[0023] In some embodiments, conjugates are provided wherein the heavy chain variable region of the binding unit further comprises a heavy chain constant region. In some embodiments, conjugates are provided wherein the heavy chain constant region of the binding unit is of the IgG isotype. In some embodiments, conjugates are provided wherein the heavy chain constant region of the binding unit is an IgG1 constant region. In some embodiments, conjugates are provided wherein the heavy chain constant region of the binding unit is an IgG4 constant region. In some embodiments, conjugates are provided wherein the IgG1 constant region of the binding unit has the amino acid sequence of SEQ ID NO: 28. In some embodiments, conjugates are provided wherein the light chain variable region of the binding unit further comprises a light chain constant region. In some embodiments, conjugates are provided wherein the light chain constant region of the binding unit is of the kappa isotype. In some embodiments, conjugates are provided wherein the light chain constant region of the binding unit has the amino acid sequence of SEQ ID NO: 29. 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 for human Fc receptors (such as FcγRIII). In some embodiments, a conjugate is 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.

[0024] In some embodiments, conjugates are provided wherein the binding unit is monospecific. In some embodiments, conjugates are provided wherein the binding unit is divalent. In some embodiments, conjugates are provided wherein the binding unit is bispecific.

[0025] In some embodiments, a pharmaceutical composition is provided, comprising any one of the conjugates described herein and a pharmaceutically acceptable carrier.

[0026] In some embodiments, a conjugate is provided wherein the saccharide unit of the linker has the formula:

[0027]

[0028] or a salt thereof, wherein:

[0029] Each X is independently selected from NH or O;

[0030] each R is independently selected from hydrogen, acetyl, monosaccharide, disaccharide, and polysaccharide;

[0031] Each X1 is independently selected from CH2 and C(O);

[0032] Each X2 is independently selected from H, OH and OR;

[0033] k is 1 to 10;

[0034] L3a is selected from C1-C 10 Alkylene and polyethylene glycol having 1 to 24 ethylene glycol subunits;

[0035] p and o are independently 0 to 2; and

[0036] Each * and each # indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1).

[0037] In some embodiments, a conjugate is provided wherein the PEG unit of the linker has a formula selected from:

[0038] (a)

[0039] ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25

[0040] (XX)

[0041] or a salt thereof, wherein:

[0042] R 20 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0043] R 21 and R 22 Each independently is, optionally C1-C3 alkylene;

[0044] R 24 and R 25 Each independently selected from H; polyhydroxy group; substituted polyhydroxy group; -C(O)-polyhydroxy group; substituted -C(O)-polyhydroxy 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; -C(O)-R 28 , where R 28 is a sugar unit of formula (XII) or (XIII); or -NR 24 R 25 together to form a C3-C8 heterocycle; provided that R 24 and R 25 Not all are H;

[0045] The wavy line (~) indicates the same as R 20Attachment site; and

[0046] n20 is 1 to 26;

[0047] or

[0048] (b)

[0049] ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25

[0050] (XX)

[0051] or a salt thereof, wherein:

[0052] R 20 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0053] R 21 and R 22 Each independently is, optionally C1-C3 alkylene;

[0054] R 24 and R 25 One selected from H; polyhydroxy group; substituted polyhydroxy group; -C (O) - polyhydroxy group; substituted -C (O) - polyhydroxy 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; -C(O)-R 28 , where R 28 is a sugar unit of formula (XII) or (XIII); and R 24 and R 25 The other is polyethylene glycol, optionally having 1 to 24 ethylene glycol subunits;

[0055] The wavy line (~) indicates the same as R 20 Attachment site; and

[0056] n20 is 1 to 26;

[0057] or

[0058] (c)

[0059] ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-]n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25

[0060] (XXI)

[0061] or a salt thereof, wherein:

[0062] R 20 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0063] R 26 and R 27 Each is optional and independently selected from 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)- and -C(O)-C1-C 12 Alkylene-NH-;

[0064] R 24 and R 25 One selected from H; polyhydroxy group; substituted polyhydroxy group; -C (O) - polyhydroxy group; substituted -C (O) - polyhydroxy 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; -C(O)-R 28 , where R 28 is a sugar unit of formula (XII) or (XIII); and R 24 and R 25 The other is selected from H; polyhydroxy group; substituted polyhydroxy group; -C (O) -polyhydroxy group; substituted -C (O) -polyhydroxy 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; -C(O)-R 28 , where R 28is a saccharide unit of formula (XII) or (XIII); and polyethylene glycol, optionally having 1 to 24 ethylene glycol subunits; or -NR 24 R 25 together to form a C3-C8 heterocycle; provided that R 24 and R 25 Not all are H;

[0065] 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;

[0066] The wavy line (~) indicates the same as R 20 The attachment site;

[0067] n20 is 1 to 26;

[0068] n21 is 1 to 4; and

[0069] n27 is 1 to 4.

[0070] In some embodiments, a conjugate is provided wherein R 24 and R 25 are each independently selected from H and polyhydroxy groups, provided that R 24 and R 25 Not all are H.

[0071] In some embodiments, conjugates are provided wherein the polyhydroxy group is a linear monosaccharide, optionally selected from a C6 or C5 sugar, a sugar acid, or an amino sugar.

[0072] In some embodiments, a conjugate is provided wherein:

[0073] a C6 or C5 sugar selected from the group consisting of glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, gulose, idose, talose, aldose, and ketose;

[0074] The sugar acid is selected from the group consisting of gluconic acid, aldonic acid, uronic acid and ketonic acid; or

[0075] The amino sugar is selected from the group consisting of glucosamine, N-acetylglucosamine, galactosamine and N-acetylgalactosamine.

[0076] In some embodiments, a conjugate is provided wherein R 24 and R 25 One of the two is a linear monosaccharide and the other is a cyclic monosaccharide.

[0077] In some embodiments, a conjugate is provided wherein R 24 and R 25 Independently selected from cyclic monosaccharides, disaccharides and polysaccharides.

[0078] In some embodiments, a conjugate is provided wherein R 24 and R 25 Independently selected from linear monosaccharides and substituted linear monosaccharides, wherein the substituted linear monosaccharides are substituted with monosaccharides, disaccharides or polysaccharides.

[0079] In some embodiments, a conjugate is provided wherein R 24 and R 25 Independently selected from linear monosaccharides and substituted monosaccharides, wherein the substituted linear monosaccharides are substituted with one or more substituents selected from the group consisting of alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide, and are optionally further substituted with monosaccharides, disaccharides, or polysaccharides.

[0080] In some embodiments, a conjugate is provided wherein R 24 and R 25 One of them is a -C(O)-polyhydroxy group or a substituted -C(O)-polyhydroxy group, and R 24 and R 25 The other is H, -C(O)-polyhydroxy group, substituted -C(O)-polyhydroxy group, polyhydroxy group or substituted polyhydroxy group; wherein the substituted -C(O)-polyhydroxy group and polyhydroxy group are substituted with monosaccharide, disaccharide, polysaccharide, alkyl, -O-alkyl, aryl, carboxyl, ester, or amide.

[0081] In some embodiments, a conjugate is provided wherein R 24 and R 25 independently selected from H, substituted-C1-C8 alkyl, substituted-C1-C4 alkyl or substituted-C1-C3 alkyl; provided that R 24 and R 25 Not all are H; wherein the substituted -C1-C8 alkyl, -C1-C4 alkyl and -C1-C3 alkyl are substituted with hydroxyl and / or carboxyl; provided that R 24 and R 25 Not all are H.

[0082] In some embodiments, a conjugate is provided wherein R 24 and R 25 One of the following 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 of the group 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 groups; provided that R 24 and R 25 Not all are H.

[0083] In some embodiments, conjugates are provided wherein each monosaccharide is independently selected from:

[0084] C5 or C6 sugars selected from the group consisting of 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;

[0085] a sugar acid selected from the group consisting of gluconic acid, aldonic acid, uronic acid, and ketonic acid; or

[0086] An amino sugar selected from the group consisting of glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine.

[0087] In some embodiments, a conjugate is provided wherein R 20 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

[0088] In some embodiments, a conjugate is provided wherein R 20 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

[0089] In some embodiments, a conjugate is provided wherein the PEG unit has a formula selected from:

[0090] ~R 40 -(R 43 -R 41 -[O-CH2-CH2] n40 -R 42-R 43 -(NR 44 R 45 ) n41 ) n42

[0091] (XL)

[0092] or a salt thereof, wherein:

[0093] R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0094] R 41 and R 42 Not present or each independently represents a C1-C6 alkylene group;

[0095] Each R 43 Independently absent or selected from 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 them is H or C1-C 12 Alkylene and the other is C1-C 12 alkylene;

[0096] R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates;

[0097] The condition is R 44 and R 45 Not all are H;

[0098] The wavy line (~) indicates the same as R 40 The attachment site;

[0099] n40 is 1 to 26;

[0100] n41 is 1 to 6; and

[0101] n42 is 1 to 6.

[0102] In some embodiments, a conjugate is provided wherein the PEG unit has a formula selected from:

[0103] ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42

[0104] (XLI)

[0105] or a salt thereof, wherein:

[0106] R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0107] R 41 and R 42 Not present or each independently represents a C1-C6 alkylene group;

[0108] R 43 Not present or selected from 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 46R 47 , where R 46 and R 47 One of them is H or C1-C 12 Alkylene and the other is C1-C 12 alkylene;

[0109] R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates;

[0110] The condition is R 44 and R 45 Not all are H;

[0111] The wavy line (~) indicates the same as R 40 The attachment site;

[0112] n40 is 1 to 26;

[0113] n41 is 1 to 6; and

[0114] n42 is 1 to 6.

[0115] In some embodiments, a conjugate is provided wherein the PEG unit has a formula selected from:

[0116] ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42

[0117] (XLII)

[0118] or a salt thereof, wherein:

[0119] R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0120] R 41 and R 42 Not present or each independently represents a C1-C3 alkylene group;

[0121] R 43 Not present or selected from C1-C6 alkylene, -NH-C1-C 12Alkylene, -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 them is H or C1-C6 alkylene and the other is C1-C 12 alkylene;

[0122] R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates;

[0123] The condition is R 44 and R 45 Not all are H;

[0124] The wavy line (~) indicates the same as R 40 The attachment site;

[0125] n40 is 1 to 26;

[0126] n41 is 1 to 4; and

[0127] n42 is 1 to 4.

[0128] In some embodiments, a conjugate is provided wherein R 40 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

[0129] In some embodiments, a conjugate is provided wherein R 20 or R 40 Has one of the following structures:

[0130]

[0131]

[0132] Where R=H or C 1-6 alkyl; and

[0133] n=0 to 12;

[0134] or its stereoisomers, wherein (*) indicates R 20 or R 40 A site of attachment to a subunit of an Amino Acid unit, a Stretcher unit and / or a portion of a Linker subunit L2 and Indicator R 20 or R 40 The site of attachment to the remainder of the PEG unit.

[0135] In some embodiments, a conjugate is provided wherein R 20 or R 40 Has one of the following structures:

[0136]

[0137]

[0138] wherein n=0 to 12;

[0139] or its stereoisomers, wherein (*) indicates R 20 or R 40 A site of attachment to a subunit of an Amino Acid unit, a Stretcher unit and / or a portion of a Linker subunit L2 and Indicator R 20 or R 40 The site of attachment to the remainder of the PEG unit.

[0140] In some embodiments, a conjugate is provided wherein R 43 -(NR 44 R 45 ) n41 , when NR 43 When present, has one of the following structures:

[0141]

[0142] Where R=H、C 1-6 Alkyl, polyhydroxy or substituted polyhydroxy;

[0143] or a stereoisomer thereof, wherein Indicator R 43 The site of attachment to the remainder of the PEG unit.

[0144] In some embodiments, a conjugate is provided wherein R 43 -(NR 44 R 45 ) n41, when NR 43 When present, has one of the following structures:

[0145]

[0146] or a stereoisomer thereof, wherein Indicator R 43 The site of attachment to the remainder of the PEG unit.

[0147] In some embodiments, a conjugate is provided wherein -NR 44 R 45 Has one of the following structures:

[0148]

[0149]

[0150] or a stereoisomer thereof, wherein Instructions-NR 44 R 45 The site of attachment to the remainder of the PEG unit.

[0151] In some embodiments, a conjugate is provided comprising a PEG unit having a formula selected from the group consisting of:

[0152] ~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

[0153] (XLIII)

[0154] or a salt thereof, wherein:

[0155] R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0156] R 41 and R 42 Not present or each independently represents a C1-C6 alkylene group;

[0157] Each R 43 Independently absent or selected from C1-C 12 Alkylene, -NH-C1-C12 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 them is H or C1-C 12 Alkylene and the other is C1-C 12 alkylene;

[0158] R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates; provided that R 44 and R 45 Not all are H;

[0159] R 46 Selected from amino, amino-alkyl-amino or -NH-C(O)-NH-S(O)2-NH-;

[0160] The wavy line (~) indicates the same as R 40 The attachment site;

[0161] n40 is 1 to 26;

[0162] n41 is 1 to 6; and

[0163] n42 is 1 to 6.

[0164] In some embodiments, a conjugate is provided comprising a PEG unit having a formula selected from:

[0165]

[0166]

[0167] or a stereoisomer or salt thereof, wherein:

[0168] Each Y is independently R 76 or

[0169] Each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2) v -OS(=O)2(OH);

[0170] Each R a and R b are independently H or R a and R b Together with the carbon to which they are attached, they form an oxo group;

[0171] Each q is independently 1-26;

[0172] Each m is independently 1 to 4;

[0173] each n is independently 1 to 4;

[0174] each v is independently 1 to 6; and

[0175] Each * indicates a site of attachment to a subunit of the Amino Acid Unit (AA), a Linker Subunit L2, or a Stretcher Unit (L1).

[0176] In some embodiments, a conjugate is provided wherein the PEG unit has a formula selected from:

[0177]

[0178]

[0179] or a stereoisomer or salt thereof, wherein:

[0180] Each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2) v S(=O)2(OH);

[0181] Each q is independently 1-26;

[0182] Each m is independently 1 to 4;

[0183] each n is independently 1 to 4;

[0184] each v is independently 1 to 6; and

[0185] Each * indicates a site of attachment to a subunit of the Amino Acid Unit (AA), a Linker Subunit L2, or a Stretcher Unit (L1).

[0186] In some embodiments, a conjugate is provided wherein the PEG unit has a formula selected from:

[0187]

[0188] or a stereoisomer or salt thereof, wherein:

[0189] Each q is independently 1-26;

[0190] Each m is independently 1 to 4;

[0191] each n is independently 1 to 4; and

[0192] Each * indicates a site of attachment to a subunit of the Amino Acid Unit (AA), a Linker Subunit L2, or a Stretcher Unit (L1).

[0193] In some embodiments, a conjugate is provided wherein Y is R 76 .

[0194] In some embodiments, a conjugate is provided wherein Y is

[0195] In some embodiments, a conjugate is provided wherein each R a and R b are independently H.

[0196] In some embodiments, a conjugate is provided wherein R a and R b Together with the carbon to which they are attached they form an oxo group.

[0197] In some embodiments, a conjugate is provided wherein q is 10-20.

[0198] In some embodiments, a conjugate is provided wherein q is 12.

[0199] In some embodiments, a conjugate is provided that comprises a carboxyl unit having the formula:

[0200]

[0201] or a salt thereof, wherein:

[0202] (a)

[0203] L 70 Selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, -C(O)-C1-C8 alkylene- and -C(O)-C1-C8 alkylene-C(O)-;

[0204] R 70 for NR71 (R 72 -R 73 ), where R 71 Selected from 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, and R 73 is carboxyl or polycarboxyl, wherein the polycarboxyl group comprises 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are linked to each other through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino and / or amide groups;

[0205] Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and

[0206] p1 and o1 are each independently selected from 0 to 2;

[0207] or

[0208] (b)

[0209] L 70 Selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, -C(O)-C1-C8 alkylene- and -C(O)-C1-C8 alkylene-C(O)-;

[0210] R 70 for NR 71 (R 75 -(R 73 )2), where R 71 Selected from 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, a 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 73independently carboxyl or polycarboxyl, wherein the polycarboxyl group comprises 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amide groups;

[0211] Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and

[0212] p1 and o1 are each independently selected from 0 to 2;

[0213] or

[0214] (c)

[0215] L 70 Selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, -C(O)-C1-C8 alkylene- and -C(O)-C1-C8 alkylene-C(O)-;

[0216] R 70 is ~N(R 74 -R 73 )(R 72 -R 73 ), where R 72 and R 74 are each independently selected from optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl or optionally substituted heteroaryl, and each R 73 is independently carboxyl or polycarboxyl, comprising 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amide groups;

[0217] Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and

[0218] p1 and o1 are each independently selected from 0 to 2.

[0219] In some embodiments, conjugates are provided that comprise at least one saccharide unit.

[0220] In some embodiments, conjugates are provided that comprise at least one PEG unit.

[0221] In some embodiments, conjugates are provided that comprise at least one carboxyl unit.

[0222] In some embodiments, a conjugate is provided that comprises at least two polar units, each polar unit being selected from a sugar unit, a PEG unit, and a carboxyl unit.

[0223] In some embodiments, conjugates are provided that comprise at least one saccharide unit and a PEG unit or a carboxyl unit.

[0224] In some embodiments, conjugates are provided that comprise at least one carboxyl unit and a PEG unit.

[0225] In some embodiments, conjugates are provided wherein an amino acid unit (AA) is present (s=1).

[0226] In some embodiments, conjugates are provided wherein the amino acid unit comprises at least one polar unit.

[0227] In some embodiments, a conjugate is provided wherein L2 or AA-L2 has one of the following structures, or a stereoisomer thereof:

[0228]

[0229]

[0230] The wavy line above the amino group indicates the site of attachment to the Stretcher unit or the Amino Acid unit, and the Drug unit is attached to the benzyl alcohol.

[0231] In some embodiments, conjugates are provided wherein the linker comprises AA-L2-, which has a formula selected from the group consisting of:

[0232] ~[SU–aa]-L2≈,

[0233] ~[aa1(PEG)–aa]-L2≈, or

[0234] ~[CU–aa]-L2≈

[0235] wherein square brackets indicate an amino acid unit, each aa is an optional subunit of AA, L2 is a linker subunit, each wavy line (~) indicates a site of attachment to 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 indicates a site of attachment to a drug unit, wherein aa and aa1 are independently selected from α, β, and γ amino acids and derivatives thereof.

[0236] In some embodiments, conjugates are provided wherein the linker comprises AA-L2-, which has a formula selected from the group consisting of:

[0237]

[0238]

[0239] wherein square brackets indicate amino acid units, each aa is an amino acid subunit of AA, L2 is a linker subunit connected to the side chain of aa, a wavy line (~) indicates the site of attachment to the stretcher unit; aa1(PEG) is a PEG unit connected to aa, SU is a sugar unit connected to aa, CU is a carboxyl unit connected to aa, and a double wavy (≈) line indicates the site of attachment to the drug unit; wherein aa and aa1 are independently selected from α, β and γ amino acids and derivatives thereof.

[0240] In some embodiments, conjugates are provided wherein the Amino Acid unit comprises at least two polar units.

[0241] In some embodiments, conjugates are provided wherein the linker comprises AA-L2-, which has a formula selected from the group consisting of:

[0242] ~[SU–aa–SU]–L2≈,

[0243] ~[aa1(PEG)–aa–aa2(PEG)]–L2≈, or

[0244] ~[CU–aa–CU]–L2≈

[0245] wherein square brackets indicate an amino acid unit, aa is an optional subunit of AA, L2 is a linker subunit, and a wavy line (-) indicates the site of attachment to the stretcher unit; aa1(PEG) and aa2(PEG) are each a PEG unit connected to aa or another PEG unit; each SU is a sugar unit connected to aa or another sugar unit, each CU is a carboxyl unit connected to aa or another carboxyl unit, and a double wavy (≈) line indicates the site of attachment to the drug unit; wherein aa, aa1, and aa2 are independently selected from α, β, and γ amino acids and derivatives thereof.

[0246] In some embodiments, conjugates are provided wherein the linker comprises AA-L2-, which has a formula selected from the group consisting of:

[0247]

[0248]

[0249] wherein square brackets indicate amino acid units, aa is an amino acid subunit of AA, L2 is a linker subunit connected to the side chain of aa, each wavy line (~) indicates a site of attachment to a stretcher unit; aa1(PEG) and aa2(PEG) are each a PEG unit connected to aa, each SU is a sugar unit connected to aa; each CU is a carboxyl unit connected to aa; and a double wavy (≈) line indicates a site of attachment to a drug unit; wherein aa, aa1, and aa2 are each independently selected from α, β, and γ amino acids and derivatives thereof.

[0250] In some embodiments, a conjugate is provided wherein the linker subunit L2 is a cleavable linker unit.

[0251] In some embodiments, conjugates are provided wherein the linker subunit L2 comprises a peptide cleavable by an intracellular protease.

[0252] 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.

[0253] In some embodiments, conjugates are provided wherein the linker subunit L2 comprises at least one polar unit.

[0254] In some embodiments, a conjugate is provided wherein the polar unit is a saccharide unit (SU).

[0255] In some embodiments, conjugates are provided wherein the cleavable peptide comprises a SU-valine-citrulline peptide, a SU-valine-lysine peptide, a SU-valine-alanine peptide, a SU-phenylalanine-lysine peptide, or a SU-glycine-glycine-phenylalanine-glycine peptide.

[0256] In some embodiments, a conjugate is provided wherein the polar unit is a carboxyl unit (CU).

[0257] 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.

[0258] In some embodiments, a conjugate is provided wherein the polar unit is a PEG unit (PEG).

[0259] 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 a PEG unit attached to a lysine residue or a citrulline residue, respectively.

[0260] In some embodiments, conjugates are provided wherein the cleavable peptide is linked to a p-aminobenzyl alcohol self-immolative group (PABA).

[0261] In some embodiments, conjugates are provided wherein L2 is linked to the side chain of a subunit of AA.

[0262] In some embodiments, conjugates are provided wherein the Amino Acid unit is bound to the Linker subunit L2 via a non-peptide linker.

[0263] In some embodiments, a conjugate is provided wherein the non-peptide linker is selected from the group consisting of C1-C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylidene or polyethylene glycol.

[0264] In some embodiments, conjugates are provided wherein the linker further comprises a Stretcher unit.

[0265] In some embodiments, a conjugate is provided wherein the Stretcher unit is selected from the group consisting of:

[0266]

[0267] where R 17 -C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-, -C3-C8 carbocyclyl-, -O-(C1-C8 alkylene)-, -(CH2-O-CH2) b -C1-C8 alkylene-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-(wherein b is 1 to 26), -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C10 Alkylene-(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-, -C3-C8 heterocycle-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C1-C8alkylene-(CH2-O-CH2) b -C(=O)-(wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C3-C8 carbocyclyl-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-C(=O)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocycle-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-C(=O)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C1-C8alkylene-(CH2-O-CH2) b -NH- (wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-NH-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-NH-(wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b-C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -NH-(wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C3-C8 carbocyclyl-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 carbocyclyl)-NH-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-NH-, -C3-C8 heterocycle-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-NH-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclyl-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 carbocyclyl)-S-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-S-, -C3-C8 heterocycle-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-S- or -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-S-; or

[0268] The extension unit includes a maleimide group (C1-C 10 Alkylene-C(O)-, maleimide (CH2OCH2) p2 (C1-C 10 Alkylene) C(O)-, maleimide (C1-C 10 Alkylene)(CH2OCH2) p2 C(O)-, or a ring-opened form thereof, wherein p2 is 1 to 26.

[0269] In some embodiments, a conjugate is provided wherein the Stretcher unit is selected from the group consisting of:

[0270]

[0271] The wavy line The attachment site of the Stretcher unit to the Amino Acid unit or Linker subunit L2 is indicated, and the attachment site to the Binding unit is at a maleimide, primary amine, or alkyne functional group.

[0272] In some embodiments, a conjugate is provided, which comprises any binding unit described herein, at least one linker connected to the binding unit, and at least one drug unit connected to each linker. In some embodiments, a conjugate is provided, 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. In some embodiments, a conjugate is provided, wherein each linker is connected to the binding unit through an interchain disulfide residue, a lysine residue, an engineered cysteine residue, a glycan, a modified glycan, the N-terminal residue of the binding unit, or a polyhistidine residue connected to the binding unit. In some embodiments, the average drug load 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 load of the conjugate is about 1, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16. In some embodiments, the average drug loading of the conjugate is from about 1 to about 8, from about 2 to about 8, from about 4 to about 8. In some embodiments, the average drug loading of the conjugate is from about 1 to about 12, from about 2 to about 12, from about 4 to about 12, from about 6 to about 12, from about 8 to about 12, and in some embodiments, the average drug loading of the conjugate is from about 1 to about 16, from about 2 to about 16, from about 4 to about 16, from about 6 to about 16, from about 8 to about 16, from about 10 to about 16, and from about 12 to about 16.

[0273] 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, about 16. In some embodiments, the DAR is about 1 to about 8, about 2 to about 8, 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, about 8 to about 12, and 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, and about 12 to about 16.

[0274] In some embodiments, a conjugate is provided wherein the drug unit is a cytotoxic agent. In some embodiments, a conjugate is provided wherein the cytotoxic agent is selected from auristatin, maytansinoid, camptothecin, duocarmycin or calicheamicin. In some embodiments, a conjugate is provided wherein the cytotoxic agent is auristatin. In some embodiments, a conjugate is provided wherein the cytotoxic agent is MMAE or MMAF. In some embodiments, a conjugate is provided wherein the cytotoxic agent is camptothecin. In some embodiments, a conjugate is provided wherein the cytotoxic agent is exatecan. In some embodiments, a conjugate is provided wherein the cytotoxic agent is a diastereomer of exatecan. In some embodiments, a conjugate is provided wherein the cytotoxic agent is SN-38. In some embodiments, a conjugate is provided wherein the cytotoxic agent is calicheamicin. In some embodiments, a conjugate is provided wherein the cytotoxic agent is maytansinoid. In some embodiments, a conjugate is provided wherein the maytansine is maytansine, maytansinol, or a maytansine analog in the form of DM1, DM3, and DM4, and ansamatocin-2.

[0275] 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 (succinimidyl-3-yl-N)-(CH2)nC(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-(SEQ ID NO: 34), wherein n=1 to 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 (succinimidyl-3-yl-N)-(CH2)nC(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-(SEQ ID NO: 34). In some embodiments, a conjugate is provided wherein the linker is attached to at least one molecule of exatecan.

[0276] In some embodiments, a conjugate is provided wherein the drug unit is an immunomodulator. In some embodiments, a conjugate is provided wherein the immunomodulator is selected from a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist. In some embodiments, a conjugate is provided wherein the immunomodulator is a TLR7 agonist. In some embodiments, a conjugate is provided wherein the TLR7 agonist is an imidazoquinoline, an imidazoquinolineamine, a thiazoquinoline, an aminoquinoline, an aminoquinazoline, a pyrido[3,2-d]pyrimidine-2,4-diamine, a pyrimidine-2,4-diamine, a 2-aminoimidazole, a 1-alkyl-1H-benzimidazol-2-amine, a tetrahydropyridopyrimidine, a heteroaromatic thiadiazine-2,2-dioxide, a benzonaphthyridine, a guanosine analog, an adenosine analog, a thymidine homopolymer, ssRNA, CpG-A, PolyG10, and PolyG3. In some embodiments, a conjugate is provided wherein the immunomodulator is a TLR8 agonist. In some embodiments, a conjugate is provided wherein the TLR8 agonist is selected from an imidazoquinoline, a thiazoquinoline, an aminoquinoline, an aminoquinazoline, a pyrido[3,2-d]pyrimidine-2,4-diamine, a pyrimidine-2,4-diamine, a 2-aminoimidazole, a 1-alkyl-1H-benzimidazol-2-amine, a tetrahydropyridopyrimidine, or an ssRNA. In some embodiments, a conjugate is provided wherein the immunomodulator is a STING agonist. In some embodiments, a conjugate is provided wherein the immunomodulator is a RIG-I agonist. In some embodiments, a conjugate is provided wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, and KIN2000.

[0277] In some embodiments, a conjugate is provided wherein the drug unit is a chelating ligand. In some embodiments, a conjugate is provided 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); radioactive isotopes 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.

[0278] In some embodiments, a pharmaceutical composition is provided, comprising any one of the conjugates described herein and a pharmaceutically acceptable carrier.

[0279] In some embodiments, a method for treating CD70+ cancer is provided, comprising administering to a subject in need thereof a therapeutically effective amount of any conjugate described herein or any pharmaceutical composition 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, B-cell lineage cancer, multiple myeloma, renal cell cancer, nasopharyngeal carcinoma, thymic carcinoma, head and neck cancer, and glioma. In some embodiments, the CD70+ cancer is a hematologic 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 clear cell renal 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).

[0280] In some embodiments, the method further comprises administering immunotherapy to the subject. In some embodiments, immunotherapy includes checkpoint inhibitors. In some embodiments, the checkpoint inhibitors are selected from antibodies that specifically bind to human PD-1, human PD-L1, or human CTLA4. In some embodiments, the checkpoint inhibitors are pembrolizumab, nivolumab, cemiplimab, or ipilimumab. In some embodiments, the method further comprises administering chemotherapy to the subject.

[0281] In some embodiments, the method of 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.

[0282] In some embodiments, the subject has an improved treatment outcome. 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 decrease in tumor burden. In some embodiments, the improved treatment outcome is progression-free survival or disease-free survival.

[0283] In some embodiments, provided is a use of any conjugate described herein or any pharmaceutical composition described herein for treating a CD70+ cancer in a subject.

[0284] In some embodiments, provided herein is a method for treating an autoimmune disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of any conjugate described herein or any pharmaceutical composition described herein. In some embodiments, the autoimmune disease is rheumatoid arthritis, multiple sclerosis, or systemic lupus erythematosus. In some embodiments, the method further comprises administering immunosuppressive therapy to the subject. In some embodiments, the method comprises administering any conjugate described herein or any pharmaceutical composition described herein.

[0285] 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 therapeutic outcome is improved. In some embodiments, improved therapeutic outcome refers to a slowing of disease progression or a reduction in disease severity.

[0286] In some embodiments, provided is a use of any conjugate described herein or any pharmaceutical composition described herein for treating an autoimmune disease in a subject.

[0287] These and other aspects of the present invention will become more fully understood by reference to the following detailed description, non-limiting examples of specific embodiments, and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0288] Figure 1 Binding assay of 2E7 and 2E7-LD038 to Caki-1.

[0289] Figure 2 Binding test of 2E7 and 2E7-LD038 to 786-O.

[0290] Figure 3 .Binding assay of 2E7 and 2E7-LD038 to Raji.

[0291] Figure 4 Binding assay of 2E7 and 2E7-LD038 to MCF-7.

[0292] Figure 5 .2E7 or 2E7-LD038 blocked the binding of CD27 to Caki-1 cells in vitro.

[0293] Figure 6 .2E7 or 2E7-LD038 blocked the binding of CD27 to 786-O cells in vitro.

[0294] Figure 7 .2E7 or 2E7-LD038 blocked the binding of CD27 to Raji cells in vitro.

[0295] Figure 8 .2E7 internalization in tumor cells.

[0296] Figure 9 .Internalization of 2E7-LD038 in tumor cells.

[0297] Figure 10 PK of 2E7 and 2E7-LD038 in rats.

[0298] Figure 11 In vitro cytotoxicity of 2E7-conjugates against 786-O.

[0299] Figure 12 . In vitro cytotoxicity of 2E7-conjugates against Raji.

[0300] Figure 13 In vitro cytotoxicity of 2E7-conjugates against Caki-1.

[0301] Figure 14 In vitro cytotoxicity of 2E7-conjugates against A498 cells.

[0302] Figure 15 .In vitro cytotoxicity of 2E7 and 2E7-LD038 against Caki-1.

[0303] Figure 16 .In vitro cytotoxicity of 2E7 and 2E7-LD038 against 786-O.

[0304] Figure 17 .In vitro cytotoxicity of 2E7 and 2E7-LD038 against Raji cells.

[0305] Figure 18 .Multiple-dose antitumor activity studies using 2E7-conjugates of Caki-1.

[0306] Figure 19 Single-dose antitumor activity studies using 2E7-conjugates of Caki-1.

[0307] Figure 20 .Multiple-dose antitumor activity studies using Raji's 2E7-conjugate.

[0308] Figure 21 Single-dose antitumor activity studies using Raji's 2E7-conjugate.

[0309] Figure 22Single-dose antitumor activity study of 2E7-conjugate using HONE-1.

[0310] Figure 23 .Study on the single-dose and multiple-dose antitumor activity of 2E7-LD038 conjugate against Caki-1.

[0311] Figure 24 .Study on the single-dose antitumor activity of 2E7-LD038 conjugate against 786-O.

[0312] Figure 25 .Study on the single-dose and multiple-dose antitumor activity of 2E7-LD038 conjugate against Raji.

[0313] Figure 26 .Study on the single-dose antitumor activity of 2E7-LD038 conjugate against Raji.

[0314] Figure 27 Single-dose antitumor activity study of the 2E7-LD038 conjugate in a patient-derived DLBCL xenograft model.

[0315] Figure 28 Single-dose antitumor activity study of the 2E7-LD038 conjugate in a patient-derived DLBCL xenograft model.

[0316] Figure 29 Single-dose antitumor activity study of the 2E7-LD038 conjugate in a patient-derived DLBCL xenograft model.

[0317] Figure 30 Single-dose antitumor activity study of the 2E7-LD038 conjugate in a patient-derived DLBCL xenograft model.

[0318] Figure 31 Single-dose antitumor activity study of the 2E7-LD038 conjugate in a patient-derived DLBCL xenograft model.

[0319] Figure 32 Single-dose antitumor activity study of the 2E7-LD038 conjugate in a patient-derived ccRCC xenograft model.

[0320] Figure 33 Single-dose antitumor activity study of the 2E7-LD038 conjugate in a patient-derived ccRCC xenograft model.

[0321] Figure 34 Single-dose antitumor activity study of the 2E7-LD038 conjugate in a patient-derived head and neck cancer xenograft model.

[0322] definition

[0323] For convenience, certain terms in this specification, examples, and claims are defined here. Unless otherwise specified or implicit from the context, the following terms and phrases have the following meanings. These definitions are intended to aid in describing specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited solely 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.

[0324] Unless otherwise specified, the terms "a" and "an" used herein should be understood to mean "one," "at least one," or "one or more." Unless otherwise required by context, as used herein, singular terms shall include pluralities and plural terms shall include the singular.

[0325] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprises," "comprising," and the like should be construed in an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0326] The terms "reduce," "lower," "decreased," "reduction," and "inhibit" all refer herein generally to a decrease by a statistically significant amount relative to a reference value.

[0327] The terms "increase," "increase," "enhance," or "activate" as used herein generally refer to an increase by a statistically significant amount relative to a reference value.

[0328] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues, each of which is interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" also refer to amino acid polymers, including modified amino acids (e.g., phosphorylation, saccharification, glycosylation, etc.) and amino acid analogs, regardless of their size or function. "Protein" and "polypeptide" are generally used to refer to relatively large polypeptides, while the term "peptide" is generally used to refer to small polypeptides, but the usage of these terms in the art overlaps. When referring to encoded gene products and fragments thereof, "protein" and "polypeptide" are used interchangeably herein. Therefore, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments and analogs of the aforementioned substances.

[0329] CD70 is a cell surface antigen of activated T cells and B lymphocytes (but not resting T cells and B lymphocytes). It is also known as CD27L, tumor necrosis factor (ligand) superfamily member 7, TNFSF7, surface antigen CD70 and Ki-24 antigen. It is reported to be overexpressed in certain cancers, which will be further described herein. Human CD70 polypeptides include, but are not limited to, polypeptides having the amino acid sequences shown in UniProt identifiers P32970-1 and P32970-2 and RefSeq NP_001243.1 and NP_001317261.1; these sequences are incorporated herein by reference.

[0330] As used herein, "epitope" refers to the amino acids that are typically bound by an immunoglobulin VH / VL pair (e.g., an antibody or its antigen-binding portion). An epitope can be formed by continuous amino acids on a polypeptide or by non-continuous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by continuous 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 3 amino acids, more typically at least 5, about 9, or about 8-10 amino acids, and has a unique spatial conformation. An epitope defines the minimum binding site of an antibody or its antigen-binding portion and therefore represents a target that is specific for an antibody or its antigen-binding portion. For single-domain antibodies, an epitope represents a structural unit bound by a single variable domain.

[0331] As used herein, "specific binding" refers to the binding of a binding unit (e.g., an antibody or antigen binding portion thereof) described herein to a specific binding site. -5 M (10000nM) or less, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 The ability to bind to a target (e.g., human CD70) with a KD of M or less. Specific binding can be affected by, for example, the affinity and avidity of the binding unit and the concentration of the target polypeptide. One of ordinary skill in the art can determine appropriate conditions for the binding units described herein to selectively bind to CD70 using any suitable method (e.g., titration of the antibody or antigen-binding portion thereof in a suitable cell binding assay). A binding unit that specifically binds to CD70 is not displaced by non-similar competitors. In certain embodiments, a binding unit is said to specifically bind to CD70 when it preferentially recognizes its target antigen, CD70, in a complex mixture of proteins and / or macromolecules.

[0332] In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD or K D ) is 10 -5 M (10000nM) or less, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of about 10 -5 M to 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 to 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 to 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 to 10 -9 In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of about 10 -9 M to 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 to 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 to 10 -12 In some embodiments, the binding units described herein specifically bind to a CD70 polypeptide with a dissociation constant (KD) of less than 10 - 12 M.

[0333] 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, isopropyl, -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, tert-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(C H3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl- 2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2) and 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3.

[0334] Unless otherwise indicated, "alkenyl" alone or as part of another term refers to a C2-C8 substituted or unsubstituted straight or branched chain hydrocarbon group having at least one site of unsaturation (i.e., carbon-carbon sp 2 Double bond). Examples include, but are not limited to, ethylene or vinyl (-CH=CH2), allyl (-CH2CH=CH2), cyclopentenyl (-C5H7), and 5-hexenyl (-CH2CH2CH2CH2CH=CH2).

[0335] Unless otherwise indicated, "alkynyl" alone or as part of another term refers to a C2-C8 substituted or unsubstituted straight or branched chain hydrocarbon group having at least one site of unsaturation (i.e., a carbon-carbon sp triple bond). Examples include, but are not limited to, acetylenic and propargyl.

[0336] Unless otherwise indicated, "alkylene" refers to a saturated, branched or straight-chain hydrocarbon radical having 1 to 8 carbon atoms and having two monovalent radical centers formed by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene groups include, but are not limited to, methylene (-CH2-), 1,2-ethyl (-CH2CH2-), 1,3-propyl (-CH2CH2CH2-), 1,4-butyl (-CH2CH2CH2CH2-), and the like.

[0337] Unless otherwise indicated, "alkenylene" refers to an unsaturated, branched or straight-chain hydrocarbon radical having 2 to 8 carbon atoms, having two monovalent radical centers derived from the same or two different carbon atoms of a parent olefin by removing two hydrogen atoms. Typical alkenylene radicals include, but are not limited to, 1,2-ethylene (-CH=CH-).

[0338] Unless otherwise indicated, "alkynylene" refers to an unsaturated, branched, straight-chain, or cyclic hydrocarbon radical having 2 to 8 carbon atoms, which has two monovalent radical centers derived from the parent alkyne by removing two hydrogen atoms from the same or two different carbon atoms. Typical alkynylene radicals include, but are not limited to, ethynyl, propargyl, and 4-pentynyl.

[0339] Unless otherwise indicated, the term "heteroalkyl", alone or in combination with other terms, refers to a substituted or unsubstituted stable straight or branched chain hydrocarbon or combinations thereof, which is saturated and contains from 1 to 10 (preferably from 1 to 3) heteroatoms selected from O, N, Si, and S, wherein 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 located 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 located at any position in the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Examples of heteroalkyl groups include: -CH2CH2OCH3, -CH2CH2NHCH3, -CH2CH2N(CH3)CH3, -CH2SCH2CH3, CH2CH2S(O)CH3, -CH2CH2S(O)2CH3, and -Si(CH3)3. Up to two heteroatoms may be consecutive, for example, -CH2NHOCH3 and CH2OSi(CH3)3. In some embodiments, the C1 to C4 heteroalkyl group has 1 to 4 carbon atoms and 1 or 2 heteroatoms, while the C1 to C3 heteroalkyl group has 1 to 3 carbon atoms and 1 or 2 heteroatoms.

[0340] Unless otherwise indicated, the terms "heteroalkenyl" and "heteroalkynyl," alone or in combination with other terms, refer to a substituted or unsubstituted stable straight or branched chain alkenyl or alkynyl group having from one to ten, preferably from one to three, heteroatoms selected from 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 located 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 located 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.

[0341] Unless otherwise indicated, the term "heteroalkylene" by itself or as part of another substituent refers to a substituted or unsubstituted divalent radical derived from heteroalkyl (as described above), for example, -CH2CH2SCH2CH2- and -CH2SCH2CH2NHCH2-. In some embodiments, C1 to C4 heteroalkylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms, while C1 to C3 heteroalkylene has 1 to 3 carbon atoms and 1 or 2 heteroatoms. For heteroalkylene, heteroatoms may also occupy either or both ends of the chain. In addition, for alkylene and heteroalkylene linking groups, no direction of the linking group is implied.

[0342] Unless otherwise indicated, the terms "heteroalkenylene" and "heteroalkynylene" by themselves or as part of another substituent refer to a substituted or unsubstituted divalent radical derived from a heteroalkenyl or heteroalkynyl group (as described above). In some embodiments, the C2 to C4 heteroalkenylene or heteroalkynylene group has 1 to 4 carbon atoms. For heteroalkenylene and heteroalkynylene groups, heteroatoms may also occupy either or both of the chain termini. In addition, for alkylene, heteroalkenylene, and heteroalkynylene linking groups, no direction of the linking group is implied.

[0343] 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 from the removal of one hydrogen atom from a ring atom of the 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.

[0344] Unless otherwise indicated, "C3-C8 carbocycle", alone or as part of another term, means a substituted or unsubstituted C3-C8 carbocycle group as defined above, wherein another hydrogen atom of the carbocycle group is replaced by a bond (i.e., it is divalent).

[0345] Unless otherwise stated, "C3-C 10 "Carbocycle", alone or as part of another term, means 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 carbon ring derived by removing one hydrogen atom from a ring atom of the parent ring system. Representative -C3-C 10 Carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl, and cyclooctadienyl. 10 The carbocycle may also include a fused cyclooctyne carbocycle, such as the fused cyclooctyne compounds disclosed in International Publication No. WO 2011 / 136645 (the disclosure of which is incorporated herein by reference), including BCN (bicyclo[6.1.0]nonyne) and DBCO (dibenzocyclooctyne).

[0346] Unless otherwise indicated, "C3-C8 heterocycle" by itself or as part of another term refers to a substituted or unsubstituted monovalent 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, and is 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 may be oxidized. Rings containing heteroatoms may be aromatic or non-aromatic. Unless otherwise indicated, the heterocycle and its pendant groups are attached at any heteroatom or carbon atom so as to form 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, thienyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl and isoxazolyl. Unless otherwise indicated, "heterocarbocycle" has the same meaning as "heterocycle" or "heterocyclyl" as described herein.

[0347] Unless otherwise stated, "C3-C8 heterocyclyl" by itself or as part of another term refers to a substituted or unsubstituted C3-C8 heterocyclyl as defined above wherein one of the hydrogen atoms of the heterocyclyl is replaced by a bond (ie, it is divalent).

[0348] Unless otherwise indicated, "aryl" by itself or as part of another term refers to a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of 6 to 20 carbon atoms (preferably 6 to 14 carbon atoms) derived from a single carbon atom of a parent aromatic ring system by removing a hydrogen atom. Some aryl groups are represented as "Ar" in the exemplary structures. Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzenes, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is phenyl.

[0349] Unless otherwise indicated, "arylene" by itself or as part of another term means an unsubstituted or substituted aryl group as defined above, wherein one of the aryl group's hydrogen atoms is replaced by a bond (ie, it is divalent) and can be in the ortho, meta, or para position.

[0350] Unless otherwise indicated, "heteroaryl" and "heterocycle" refer to ring systems in which one or more of the ring atoms is a heteroatom (e.g., nitrogen, oxygen, and sulfur). A heterocyclic group contains 1 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. A heterocycle can be a monocyclic ring with 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or a bicyclic ring with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example, a bicyclic [4,5], [5,5], [5,6], or [6,6] system.

[0351] Unless otherwise stated, "heteroarylene" by itself or as part of another term is an unsubstituted or substituted heteroaryl as defined above, wherein one of the heteroaryl's hydrogen atoms is replaced by a bond (ie, it is divalent).

[0352] Unless otherwise indicated, "carboxyl" refers to COOH or COO - M + , where M + It is a cation.

[0353] Unless otherwise indicated, "oxo" means (C=O).

[0354] Unless otherwise indicated, "substituted alkyl" and "substituted aryl" refer to alkyl and aryl groups, respectively, in which one or more hydrogen atoms are independently replaced by a substituent. Typical substituents include, but are not limited to, -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, wherein each X is independently a halogen: -F, -Cl, -Br, or -I; and each R 10 are independently -H, -C1-C 20 Alkyl, -C6-C 20 Aryl, -C3-C 14 Heterocycle, protecting group or prodrug moiety. Typical substituents also include (=O). The above-mentioned alkylene, carbocycle, carbocyclyl, arylene, heteroalkyl, heteroalkylene, heterocycle and heterocyclyl groups can also be substituted similarly.

[0355] Unless otherwise indicated, "polyhydroxy" refers to an alkyl, alkylene, carbocycle or carbocyclic group in which the hydrogen on the carbon atom of the carbon chain is replaced by two or more, three or more hydroxy groups. In some embodiments, the polyhydroxy group comprises at least three hydroxy groups. In some embodiments, the polyhydroxy group comprises a carbon atom in which each carbon atom contains only one hydroxy group. The polyhydroxy group may comprise one or more carbon atoms not replaced by hydroxy groups. Each carbon atom of the polyhydroxy group may be replaced by a hydroxy group. Examples of polyhydroxy groups include monosaccharides in linear (non-cyclic) or cyclic forms, 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, aldoses and ketoses, sugar acids such as gluconic acid, aldonic acid, uronic acid or ketonic acid and amino sugars such as glucosamine, N-acetylglucosamine, galactosamine and N-acetylgalactosamine. In some embodiments, the polyols include disaccharides and polysaccharides in linear or cyclic form.

[0356] Unless the context indicates otherwise, "optionally substituted" refers to an alkyl, alkenyl, alkynyl, alkylaryl, aralkylheterocycle, aryl, heteroaryl, alkylheteroaryl, heteroaralkyl, or other substituent, moiety, or group as defined or disclosed herein, wherein a hydrogen atom of the substituent, moiety, or group is optionally replaced with a different moiety or group, or an alicyclic carbon chain comprising one of these substituents, moieties, or groups is interrupted by replacing a carbon atom in the chain with a different moiety or group. In some aspects, an alkene functional group replaces two adjacent sp3 carbon atoms of an alkyl substituent, provided that the radical carbon of the alkyl moiety is not replaced, such that the optionally substituted alkyl group is an unsaturated alkyl substituent.

[0357] The optional substituents replacing hydrogen in the aforementioned substituents, moieties or groups are independently selected from aryl, heteroaryl, hydroxy, alkoxy, aryloxy, cyano, halogen, nitro, fluoroalkoxy and amino, including mono-, di- and tri-substituted amino groups, and protected derivatives thereof, or from -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, wherein each X is independently selected from the halogen: -F, -CI, -Br and -I; and wherein each R" is independently selected from the C1-C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C6-C 24 Aryl, C3-C 24 Heterocyclic groups (including C5-C 24 heteroaryl), protecting groups and prodrug moieties, or two R" together with the heteroatom to which they are attached define a heterocyclic group; and R' is hydrogen or R", wherein R" is selected from C1-C 20 Alkyl, C6-C 24 Aryl, C3-C 24 Heterocyclic groups (including C5-C 24 heteroaryl) and protecting groups.

[0358] Typically, the optional substituents are selected from -X, -OH, -OR", -SH, -SR", -NH2, -NH(R"), -NR'(R"), -N(R"), =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(=O)2N(R')R", -S(=O)2NH2, -S(=O)2N(R')(R"), -S(=O)2OR', -S(=O)R", -C(=S)R", -C(=S)NH2, -C(=S)N(R')R", -C(=NR')N(R")2, and salts thereof, wherein each X is independently selected from -F and -Cl, and R" is typically selected from C1-C6 alkyl, C6-C 10 Aryl, C3-C 10 Heterocyclic groups (including C5-C 10 Heteroaryl) and a protecting group; and R' is independently hydrogen, C1-C6 alkyl, C6-C 10 Aryl, C3-C 10 Heterocyclic groups (including C5-C 10 More typically, the substituents are selected from -X, -R", -OH, -OR", -NH2, -NH(R"), -N(R"), -N(R"), -CX3, -NO2, -NHC(=O)H, -NHC(=O)R", -C(=O)NH2, -C(=O)NHR", -C(=O)N(R"), -CO2H, -CO2R", -C(=O)H, -C(=O)R", -C(=O)NH2, -C(=O)NH(R"), -C(=O)N(R"), -C(=NR')NH2, -C(=NR')NH(R"), -C(=NR')N(R"), protecting groups and salts thereof, wherein each X is -F and R" is independently selected from C1-C6 alkyl, C6-C6 alkyl, C1-C6 alkyl, C6-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, C1-C6 alkyl, 10 Aryl, C5-C 10 heteroaryl and a protecting group; and R' is selected from hydrogen, C1-C6 alkyl and a protecting group, independently selected from R".

[0359] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic salt or inorganic salt of a compound (e.g., a joint, a drug linker, or a conjugate). The compound typically contains at least one amino group and therefore can form an acid addition salt with the amino group. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, linoleate, gentisate, fumarate, gluconate, glucuronate, sucrose, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). A pharmaceutically acceptable salt may contain another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge of the parent compound. Additionally, a pharmaceutically acceptable salt may contain multiple charged atoms in its structure. If a pharmaceutically acceptable salt contains multiple charged atoms, multiple counterions may be present. Thus, a pharmaceutically acceptable salt may contain one or more charged atoms and / or one or more counterions.

[0360] As used herein, the term "consisting essentially of refers to the elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic, novel, or functional characteristics of the embodiment.

[0361] As used herein, the term "consisting of refers to the compositions, methods, and individual components as described herein, excluding any elements not recited in the description of the embodiment.

[0362] It is to be understood that, except in the embodiments or where otherwise indicated, all numbers used herein expressing quantities of ingredients or reaction conditions are modified in all instances by the term “about.” The term “about” when used in conjunction with a percentage can mean + / - 1%.

[0363] The term "statistically significant" or "significant" refers to statistical significance and generally refers to a difference of two standard deviations (2SD) above or below a reference value.

[0364] Although the structures shown in this specification are described with specific stereocenters, this specification should be understood to include variations of these stereocenters. For example, the structure of exatecan may be shown in the (S, S) configuration, but the (R, S) diastereomer of exatecan is also contemplated to exist in separate embodiments of the conjugates described herein.

[0365] Other terms are defined herein in the description of various aspects of the invention. Detailed Description of the Invention

[0367] Provided herein are CD70 antibody drug conjugates (ADCs) that specifically bind to human CD70. 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, such as a cytotoxic agent or an immunomodulatory agent. In some embodiments, the CD70 ADC specifically binds to CD70+ cells in a subject and reduces their number. In some embodiments, the CD70 ADC specifically binds to CD70+ cancer cells in a subject and reduces their number. In some embodiments, the CD70 ADC specifically binds to CD70+ cancer cells in a subject and reduces their number. In some embodiments, the CD70 ADC specifically binds to CD70+ cancer cells in a subject and reduces their number. In some embodiments, the CD70 ADC specifically binds to CD70+ cells associated with a disease or condition (e.g., an autoimmune disease) in a subject and reduces their number.

[0368] In some embodiments, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions each having an amino acid sequence selected from 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, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions each having an amino acid sequence selected from 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, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions each having an amino acid sequence selected from the group consisting of: SEQ ID NO: 3 and SEQ ID NO: 4. In some embodiments, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions each having an amino acid sequence selected from the group consisting of: SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions have the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In some embodiments, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions have the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. In some embodiments, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions have the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0369] In some embodiments, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions respectively have an amino acid sequence set forth in an amino acid sequence pair selected from 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; 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 in the framework regions, and wherein the CDRs of the heavy or light chain variable regions are not modified. In some embodiments, the binding unit of the CD70 ADC comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have an amino acid sequence selected from 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are unmodified. The phrase "wherein the CDRs of the heavy chain or light chain variable region are unmodified" refers to VH and VL CDRs that do not have amino acid substitutions, deletions, or insertions compared to the amino acid sequence.

[0370] In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0371] In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0372] In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0373] In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are unmodified. In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are unmodified.

[0374] In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are unmodified. In some embodiments, the binding unit of the 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are unmodified.

[0375] 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 each have an amino acid sequence set forth in an amino acid sequence pair selected from 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; wherein 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 respectively have an amino acid sequence set forth in the following amino acid sequence pairs selected from 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; wherein 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 respectively have an amino acid sequence set forth in the following amino acid sequence pairs selected from: 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; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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 respectively have an amino acid sequence set forth in the following amino acid sequence pairs: 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; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified.As described herein, the binding unit comprises a CD70 antibody, or an antigen binding portion thereof, and may optionally comprise an additional peptide or polypeptide covalently linked to the CD70 antibody, or an antigen binding portion thereof. In any of these embodiments, the binding unit specifically binds to CD70.

[0376] 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; wherein 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; wherein 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 SEQ ID NO: 3 and SEQ ID NO: 4, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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 shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0377] 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; wherein 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; wherein 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 SEQ ID NO: 5 and SEQ ID NO: 6, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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 shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0378] 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; wherein 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; wherein 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 SEQ ID NO: 7 and SEQ ID NO: 8, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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 shown in SEQ ID NO:7 and SEQ ID NO:8, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0379] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; wherein 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), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; wherein 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), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0380] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein 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), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein 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), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable regions are not modified. 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 shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0381] In some embodiments, the binding unit comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region comprising complementarity determining regions HCDR1, HCDR2, and HCDR3 positioned in the heavy chain variable region framework region and the VL region comprising LCDR1, LCDR2, and LCDR3 positioned in the light chain variable region framework region, the VH and VL CDRs each having an amino acid sequence selected from the group consisting of: (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; (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; (iii) 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; (iv) 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. NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 18; 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. In some embodiments, each of the VH and VL regions comprises a humanized framework region. In some embodiments, each of the VH and VL regions comprises a human framework region.

[0382] 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 complementary determining regions HCDR1, HCDR2, and HCDR3 positioned in a heavy chain variable region framework region and the VL region comprises LCDR1, LCDR2, and LCDR3 positioned in a light chain variable region framework region, wherein the VH and VL CDRs have the amino acid sequences shown 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.

[0383] 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 complementary determining regions HCDR1, HCDR2, and HCDR3 positioned in a heavy chain variable region framework region and the VL region comprises LCDR1, LCDR2, and LCDR3 positioned in a light chain variable region framework region, wherein the VH and VL CDRs have the amino acid sequences shown 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.

[0384] 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 complementary determining regions HCDR1, HCDR2, and HCDR3 positioned in a heavy chain variable region framework region and the VL region comprises LCDR1, LCDR2, and LCDR3 positioned in a light chain variable region framework region, wherein the VH and VL CDRs have the amino acid sequences shown 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.

[0385] 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 complementary determining regions HCDR1, HCDR2, and HCDR3 positioned in a heavy chain variable region framework region and the VL region comprises LCDR1, LCDR2, and LCDR3 positioned in a light chain variable region framework region, wherein the VH and VL CDRs have the amino acid sequences shown 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.

[0386] 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 complementary determining regions HCDR1, HCDR2, and HCDR3 positioned in a heavy chain variable region framework region and the VL region comprises LCDR1, LCDR2, and LCDR3 positioned in a light chain variable region framework region, wherein the VH and VL CDRs have the amino acid sequences shown 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.

[0387] In some embodiments, the compositions and methods described herein relate to reducing CD70+ cells in a subject in vivo by CD70 ADCs (e.g., reducing the number of CD70+ cells in a cancer or tumor, or reducing the number of 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 disease 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 associated with a disease, disorder, or cancer in the subject.

[0388] 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 (e.g., human CD70). The term generally refers to antibodies comprising two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including full-length antibodies (having heavy and light chain constant regions).

[0389] Each heavy chain is composed of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may include three domains, CH1, CH2, and CH3, and an optional fourth domain, CH4. Each light chain is composed of a variable region (abbreviated as VL) and a constant region. The light chain constant region is the 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). Each VH and VL region is therefore composed 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.

[0390] As used herein, the "antigen-binding portion" of a CD70 antibody refers to a portion of a CD70 antibody as described herein that has the VH and VL sequences of a CD70 antibody or the CDRs of a 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 known as VHH, VNAR, sdAb or nanobodies), or bifunctional antibodies (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, in each case of CD70 antibodies, the terms Fab, F(ab')2, and Fv refer to: (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked to each other via a disulfide bridge in the hinge region; and (iii) an Fv fragment consisting of the VL and VH domains. Although the two domains of the Fv fragment (i.e., VL and VH) are encoded by separate coding regions, they can further use a synthetic linker, such as a poly G4S amino acid sequence ("(G4S) n ", as shown in SEQ ID NO: 27, wherein n = 1 to 5) are linked to each other so that it is possible to prepare it as a single protein chain in which the VL and VH regions combine to form a monovalent molecule (called single-chain Fv or scFv). The term "antigen-binding portion" of an antibody also includes 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 are connected by a linker that is too short for the two domains to be combined on the same chain, thereby forcing the VH and VL domains to pair with complementary domains (VL and VH, respectively) of different chains and form two antigen-binding sites (see, e.g., Holliger, R, et al. (1993) Proc. Natl. Acad. Sci. USA 90: 64446448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123).

[0391] Single-domain antibodies are antibody portions consisting of a single monomeric variable antibody domain. Single-domain antibodies can be derived from the variable domains of the antibody heavy chains of camelids (e.g., nanobodies or VHH portions). In addition, the term single-domain antibody includes autonomous human heavy chain variable domains (aVH) or VNAR portions derived from sharks (see, e.g., Hasler et al., Mol. Immunol. 75: 28-37, 2016).

[0392] Techniques for producing single-domain antibodies (e.g., DABs or VHHs) are known in the art, as disclosed in, for example, 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, for example, 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 capacity and can interact with novel antigenic determinants that are inaccessible to conventional VH-VL pairs (see, for example, Muyldermans et al., 2001). Alpaca serum IgG contains only approximately 50% camelid heavy chain IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens, and VHHs that bind to and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries that can be used to isolate antibody fragments by standard biopanning techniques known in the art (see, e.g., Maass et al., 2007).

[0393] 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: scFv1-ScFv2, ScFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, and scFv-HSA-scFv. In some embodiments, the IgG-scFv is IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, or IgG-2scFv. For example, see 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.

[0394] Modification of VH and VL regions

[0395] With respect to VH and VL amino acid sequences, as recognized by those skilled in the art, altering a single amino acid or a small percentage of amino acids in the encoding sequence, or making individual substitutions, deletions, or additions (insertions) to the nucleic acid encoding the VH or VL or amino acids in the polypeptide, results in "conservatively modified variants," wherein the alteration results in the substitution of an amino acid with a chemically similar amino acid (conservative amino acid substitution) and the altered polypeptide is still capable of specifically binding to CD70.

[0396] In some embodiments, conservatively modified variants of a CD70 antibody, or an antigen-binding portion thereof (i.e., a binding unit), may have alterations in the framework regions (i.e., other than in the CDRs). For example, conservatively modified variants of a CD70 antibody have the amino acid sequences of the VH and VL CDRs set forth in the following amino acid sequence groups: 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: 18; and SEQ ID NO: NO: 16, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26) and have at least one conservative amino acid substitution in the framework region (FR). In some embodiments, the VH and VL amino acid sequences have no more than 8, or 6, or 4, or 2, or 1 conservative amino acid substitutions in the FR compared to the amino acid sequence of the unmodified VH and VL regions. In some embodiments, the VH and VL amino acid sequences have 8 to 1, 6 to 1, 4 to 1, or 2 to 1 conservative amino acid substitutions in the FR compared to the amino acid sequence of the unmodified VH and VL regions. In other aspects of any of these embodiments, the conservatively modified variants of the binding unit (i.e., binding unit) exhibit specific binding to CD70.

[0397] For conservative amino acid substitutions, a given amino acid can be substituted with a residue having similar physiochemical properties, for example, replacing one aliphatic residue with another (e.g., Ile, Val, Leu, or Ala for each other), or replacing one polar residue with another (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn). Other such conservative amino acid substitutions, such as substitutions of entire regions with similar hydrophobic properties, are well known in the art. Polypeptides comprising conservative amino acid substitutions can be tested in any of the assays described herein to confirm that they retain the desired activity of a native or reference polypeptide (i.e., for CD70), such as antigen binding activity and specificity.

[0398] In some embodiments, the binding unit can be further optimized to reduce potential immunogenicity or optimize other functional properties while maintaining functional activity in human therapy. 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 respectively have an amino acid sequence selected from the following amino acid sequence pairs: 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; wherein the heavy chain 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 in the framework region, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions respectively have an amino acid sequence set forth in the following amino acid sequence pairs: 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified.

[0399] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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, 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:4.

[0400] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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:5. 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:6.

[0401] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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, 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:8.

[0402] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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, 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: 10.

[0403] In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, the binding unit comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein the heavy chain 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 in the framework regions and wherein the CDRs of the heavy chain or light chain variable region are not modified. 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: 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.

[0404] In any of these embodiments, the functional activity of the binding unit includes specific binding to CD70. Other functional activities include depletion of CD70+ cells (e.g., cancer cells or autoimmune cells). In addition, the 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 as described herein (e.g., a reference CD70-binding antibody or antigen-binding portion thereof comprising (i) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1, and (ii) a light chain variable region having the amino acid sequence shown in SEQ ID NO: 2, or a variant thereof, as described herein), as measured by a specific assay (e.g., a bioassay), regardless of whether there is dose dependence. In the event that dose dependence does exist, it need not be consistent with the dose dependence of the reference antibody or antigen-binding portion thereof, but rather is substantially similar to or better than the dose dependence of the reference antibody or antigen-binding portion thereof as described herein for a given activity (i.e., the candidate polypeptide will exhibit greater activity relative to the reference antibody).

[0405] For conservative substitutions, amino acids can be grouped according to the similarity of their side chain properties (in ALLehninger, in Biochemistry, 2nd 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).

[0406] Alternatively, for conservative substitutions, naturally occurring residues can be grouped based on common 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will necessarily involve exchanging a member of one or the other of these categories.

[0407] Specific conservative substitutions include, for example: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0408] In some embodiments, conservatively modified variants of binding units preferably have 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 higher identity to a reference VH or VL sequence, wherein the VH and VL CDRs are not modified. The degree of homology (percent identity) between a reference sequence and a modified sequence can be determined by comparing the two sequences using computer programs commonly available for this purpose on the Internet (e.g., BLASTp or BLASTn with preset settings).

[0409] In some embodiments, the VH and VL amino acid sequences 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 have a total of 8 to 1, or 6 to 1, or 4 to 1, or 2 to 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 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 have 8 to 1, 6 to 1, 4 to 1, or 2 to 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 have no more than 8, 6, 4, 2, or 1 amino acid substitutions, deletions, or insertions in total compared to the amino acid sequences of the unmodified VH and VL regions.

[0410] Modification of the native (or reference) amino acid sequence can be achieved by any of a variety of techniques known to those skilled in the art. For example, mutations are introduced at a specific locus by synthesizing oligonucleotides containing the desired mutant sequence, flanked by restriction sites capable of ligating to fragments of the native sequence. After 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 according to the desired substitution, deletion, or insertion. The techniques for making such changes are well established and include those disclosed by, among others, 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.

[0411] constant region

[0412] 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. Immunoglobulin constant region refers to a heavy chain or light chain constant region. The amino acid sequences of human heavy chain and light chain constant regions are known in the art. The constant region can be of any suitable type, which can be selected from the classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM. Several immunoglobulin classes can be further divided into isotypes, such as IgG1, IgG2, IgG3, IgG4, or IgA1 and IgA2. The heavy chain constant region (Fc) corresponding to different classes of immunoglobulins can be α, δ, ε, γ, and μ, respectively. The light chain can be one of kappa (or κ) and lambda (or λ).

[0413] The constant region may be of the IgG1 isotype. In some embodiments, the constant region may be of the IgG2 isotype. The constant region may be of the IgG3 isotype. The constant region may be of the IgG4 isotype. The Fc domain may have a hybrid isotype comprising constant regions of two or more isotypes. The immunoglobulin constant region may be an IgG1 or IgG4 constant region. In some embodiments, the CD70 antibody heavy chain of the binding unit is of the 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 of the kappa isotype and has the amino acid sequence set forth in SEQ ID NO: 29.

[0414] Fc domain modifications to alter effector function

[0415] In some embodiments, the Fc region or Fc domain of the binding unit does not substantially bind to at least one Fc receptor selected from FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). In some embodiments, the Fc region or domain exhibits substantially no binding to any Fc receptor selected from FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). As used herein, "substantially no binding" means that the binding to one or more selected Fcγ receptors is weak to non-existent. In some embodiments, "substantially no binding" means that the binding affinity to the Fcγ receptor is reduced (i.e., Kd is increased) by at least 1000 times. In some embodiments, the Fc domain or region is an Fc null region (Fc null). As used herein, an "Fc null region" refers to an Fc region or Fc domain that exhibits weaker to no binding to any of the Fcγ receptors. In some embodiments, the Fc null domain or region exhibits at least a 1000-fold decrease in binding affinity (i.e., an increase in Kd) to an Fcγ receptor.

[0416] In some embodiments, the Fc domains have reduced effector function activity 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, compared to wild-type Fc domains, Fc domains exhibit reduced ADCC, ADCP or CDC activity. In some embodiments, compared to wild-type Fc domains, Fc domains exhibit ADCC, ADCP and CDC reduction. In some embodiments, Fc domains exhibit substantially no effector function (i.e., the ability to stimulate or affect ADCC, ADCP or CDC). As used herein, compared to wild-type or reference Fc domains, "substantially no effector function" refers to an effector function activity reduction of at least 1000 times.

[0417] In some embodiments, the Fc domain has reduced ADCC activity or no ADCC activity. As used herein, reduced ADCC activity or no ADCC activity refers to an Fc domain whose ADCC activity is reduced by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500-fold.

[0418] In some embodiments, the Fc domain has reduced CDC activity or no CDC activity. As used herein, reduced CDC activity or no CDC activity refers to that the CDC activity of the Fc domain is reduced by at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times or at least 500 times.

[0419] In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of ADCC and / or CDC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks Fcγ receptor binding (and therefore may lack ADCC activity). NK cells, the primary cells used to mediate ADCC, express only FcγRIII, while 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 for assessing ADCC activity of molecules 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 assays can be used (see, e.g., the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc. Mountain View, Calif.); and the CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.). Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the assay can be performed, for example, in an animal model, e.g., Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).

[0420] A C1q binding assay can also be performed to confirm that the antibody or Fc domain or region is unable to bind to C1q and therefore lacks or has reduced CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (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)).

[0421] In some embodiments, the Fc domain has reduced ADCP activity or no ADCP activity. As used herein, reduced ADCP activity or no ADCP activity means that the ADCP activity of the Fc domain is reduced by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500 times.

[0422] ADCP binding assays can also be performed to confirm that the antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity. See, for example, US20190079077 and US20190048078 and references disclosed therein.

[0423] Binding units with reduced effector function activity include those with substitutions of one or more Fc region residues (e.g., 238, 265, 269, 270, 297, 327, and 329), according to EU numbering as in Kabat (see, e.g., U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, according to EU numbering as in Kabat, including so-called (DANA) Fc mutants in which residues 265 and 297 are substituted with alanine (see, e.g., U.S. Pat. No. 7,332,581). Certain antibody variants with reduced FcR binding 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 with reduced FcR binding can be prepared containing such amino acid modifications.

[0424] In some embodiments, the binding unit comprises an Fc domain or region having one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region. In some embodiments, the substitutions are L234A and L235A (LALA) according to EU numbering as in Kabat. In some embodiments, the Fc domain comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region according to EU numbering as in Kabat. In some embodiments, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region according to EU numbering as in Kabat. (See, e.g., WO 2012 / 130831). In some embodiments, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region according to EU numbering as in Kabat.

[0425] In some embodiments, the alteration occurs in the Fc region, resulting in altered (ie, reduced) CIq binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0426] Method for preparing binding unit

[0427] In various embodiments, binding units can be produced in cell lines of human, murine, or other animal origin. Recombinant DNA expression can be used to produce binding units. This allows for the production of CD70 antibodies and a range of CD70 antigen-binding portions in the host species of choice. Production of binding units in bacteria, yeast, transgenic animals, and eggs are also alternatives to cell-based production systems. The main advantage of transgenic animals is the potential for high yields from renewable sources.

[0428] In some embodiments, a VH polypeptide having an amino acid sequence as 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 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 as set forth in SEQ ID NO: 4, 6, 8, 10, or 12 is encoded by a nucleic acid. In some embodiments, a nucleic acid encodes a VL polypeptide 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: 4, 6, 8, 10, or 12. In some embodiments, a 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 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, 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 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 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.

[0429] 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.

[0430] As used herein, the term "nucleic acid" or "nucleic acid sequence" or "polynucleotide sequence" or "nucleotide" refers to a polymeric molecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid can be single-stranded or double-stranded. A single-stranded nucleic acid can be one strand of denatured double-stranded DNA. In some embodiments, the nucleic acid can be a cDNA, e.g., a nucleic acid lacking introns.

[0431] Nucleic acid molecules encoding the amino acid sequence of the binding unit can be prepared by various methods known in the art. These methods include, but are not limited to, preparing synthetic nucleotide sequences encoding CD70 antibodies, or antigen-binding portions thereof. In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding CD70 antibodies, or antigen-binding portions thereof. At least the nucleic acid sequence encoding the CD70 antibodies, or antigen-binding portions thereof, or polypeptides thereof as described herein can be recombined with vector DNA according to conventional techniques, such as blunt or staggered ends for ligation, restriction enzyme digestion to provide appropriate ends, filling in sticky ends as needed, alkaline phosphatase treatment to avoid undesirable ligation, and ligation with 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).

[0432] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences containing transcriptional and translational regulatory information, and such sequences are "operably linked" to a nucleotide sequence encoding the polypeptide. Operably linked means that the regulatory DNA sequence and the DNA sequence to be expressed (e.g., a CD70 antibody or antigen-binding portion thereof (i.e., a binding unit)) are linked in a manner that permits gene expression of the polypeptide or antigen-binding portion in reproducible amounts. The exact nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in the art. See, e.g., Sambrook et al., 1989; Ausubel et al., 1987-1993.

[0433] Thus, expression of the CD70 antibodies, or antigen-binding portions thereof, as described herein can occur in prokaryotic or eukaryotic cells. Suitable hosts include bacteria or eukaryotic hosts, including yeast, insect, fungal, avian, and mammalian cells, or host cells of mammalian, insect, avian, or yeast origin, either in vivo or in situ. Mammalian cells or tissues can be derived from humans, primates, hamsters, rabbits, rodents, cows, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells can be used. In addition, by using, for example, the yeast ubiquitin hydrolase system, in vivo synthesis of ubiquitin transmembrane polypeptide fusion proteins can be achieved. The fusion proteins thus produced can be manipulated in vivo or purified and manipulated in vitro, thereby allowing the synthesis of CD70 antibodies, or antigen-binding portions thereof, as described herein, having a specified amino-terminal sequence. Furthermore, problems associated with start codon-derived methionine residues in direct yeast (or bacterial) expression can be avoided. (See, e.g., Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989). Recombinant CD70 antibodies, or antigen-binding portions thereof, can be obtained using any of a number of yeast gene expression systems in which the promoter and terminator elements are derived from actively expressed genes encoding glycolytic enzymes that are produced in large quantities 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 used.

[0434] Binding units can be achieved, for example, by infecting an insect host with a baculovirus engineered to express the polypeptide, as is known to those skilled in the art. See Ausubel et al., 1987-1993.

[0435] In some embodiments, the introduced nucleic acid sequence (encoding a CD70 antibody, or antigen-binding portion thereof, or polypeptide thereof) is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host cell. Any of a variety of vectors can be used for this purpose and are known and available to those of ordinary skill 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 identified and selected from recipient cells that do not contain the vector; the desired copy number of the vector in a particular host; and the need to be able to "shuttle" the vector between host cells of different species.

[0436] Exemplary prokaryotic vectors known in the art include plasmids, such as plasmids capable of replication in E. coli. Other gene expression elements that can be used to express DNA encoding the binding unit include, but are not limited to, (a) viral transcriptional promoters and enhancer elements thereof, 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 (Okayarea et al., 1983), and (c) polyadenylation sites such as those in SV40 (Okayama et al., 1983). Immunoglobulin-encoding DNA genes can be expressed using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S-globulin insert, immunoglobulin and rabbit S-globulin polyadenylation sites, and the SV40 polyadenylation element as expression elements as described by Liu et al. (see below) and Weidle et al., 51 Gene 21 (1987).

[0437] 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.

[0438] For expression of DNA coding regions in rodent cells, the transcriptional promoter can be a viral LTR sequence, and the transcriptional promoter enhancer can be any one or both of the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer, as well as the polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are combined with the expression elements listed above to achieve protein expression in mammalian cells.

[0439] Each coding region or gene fusion is assembled into an expression vector or inserted into an expression vector. Subsequently, recipient cells capable of expressing the CD70 variable region or antigen-binding portion thereof are transfected with a gene encoding a CD70 antibody or antibody polypeptide or antigen-binding portion thereof, alone or co-transfected with polynucleotides encoding the VH and VL chain coding regions. The transfected recipient cells are cultured under conditions that permit expression of the incorporated coding regions and the expressed antibody chains or intact antibodies or antigen-binding portions are recovered from the culture.

[0440] Nucleic acids containing coding regions encoding binding units can be assembled into separate expression vectors, which are then used to co-transfect recipient host cells. Each vector may contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, the first selectable gene being designed for selection in bacterial systems and the second selectable gene being designed for selection in eukaryotic systems, wherein each vector has a set of coding regions. This strategy produces vectors that first direct the production of nucleotide sequences in bacterial systems and permit their amplification. The DNA vectors thus produced and amplified in the bacterial host are then used to co-transfect eukaryotic cells, allowing selection of co-transfected cells carrying the desired transfected nucleic acid (e.g., containing the CD70 antibody heavy and light chains). Non-limiting examples of selectable genes used in bacterial systems are genes that confer ampicillin resistance and genes that confer chloramphenicol resistance. Selectable genes used in eukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Alternatively, fusion nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0441] In order to transfect the expression vector and produce the binding unit, 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 infected immunoglobulin genes and have the glycosylation machinery of immunoglobulins. For example, in some embodiments, the recipient cell is a recombinant Ig-producing myeloma cell SP2 / 0. SP2 / 0 cells only produce immunoglobulins encoded by the transfected gene. Myeloma cells can be grown in culture or in the peritoneal cavity of mice, where the secreted immunoglobulins can be obtained from the ascites fluid.

[0442] The expression vector encoding the binding unit can be introduced into an appropriate host cell by any of a variety of suitable means, including biochemical means, such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran; and mechanical means, such as electroporation, direct microinjection, and microprojectile bombardment. Johnston et al., 240 Science 1538 (1988), as known to those skilled in the art.

[0443] Yeast has certain advantages over bacteria for producing immunoglobulin heavy and light chains. Yeast undergoes post-translational peptide modifications including glycosylation. There are a variety of recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used to produce the desired protein in yeast. Yeast recognizes the leader sequence of cloned mammalian gene products and secretes polypeptides (i.e., propolypeptides) carrying the leader sequence. See, for example, Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0444] Yeast gene expression systems can be routinely evaluated for the production, secretion, and stability of antibodies and assembled binding units. Different yeast gene expression systems can be used, including promoters and termination elements, which are derived from actively expressed genes encoding saccharolytic enzymes produced in large quantities when yeast is grown in a medium rich in glucose. Known saccharolytic genes can provide very effective transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be used. Another example is the translation elongation factor 1α promoter, such as a promoter from Chinese hamster cells. Various methods can be used to evaluate the optimal expression plasmid for expressing immunoglobulins in yeast. See IIDNA Clone 45 (Glover, ed., IRL Press, 1985) and, for example, U.S. Publication No. US2006 / 0270045A1.

[0445] Bacterial strains can also be used as hosts for producing antibody molecules or antigen-binding portions thereof as described herein. Escherichia coli K12 strains, such as Escherichia coli W3110, can be used; Bacillus; enteric bacteria, such as Salmonella typhimurium or Serratia marcescens; and various Pseudomonas species. Plasmid vectors containing replicons and control sequences derived from species compatible with the host cells are used in conjunction with these bacterial hosts. The vectors carry replication sites and specific genes that can provide phenotypic selection in transformed cells. A variety of methods can be used to evaluate expression plasmids for producing binding units in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).

[0446] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin molecules, including leader peptide removal, folding and assembly of VH and VL chains, glycosylation of antibody molecules, and secretion of functional antibodies and / or antigen-binding portions thereof.

[0447] In addition to the lymphoid cells described above, mammalian cells that can be used as hosts for producing antibody proteins include fibroblast-derived cells, 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 COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; PERC6 cells, including CHO-S and DG44 cells; and PERC6 cells. TM cells (Crucell); and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modification of the heavy and / or light chains. For example, in some embodiments, CHO cells produce polypeptides that have higher glycosylation levels than the same polypeptides produced in 293 cells.

[0448] One or more binding units can be produced in vivo according to any suitable method in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding a polypeptide.

[0449] Antibodies or antigen-binding portions thereof are produced in cell-free systems. Non-limiting exemplary cell-free systems are described in, e.g., 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).

[0450] Many vector systems are available for the expression of VH and VL chains in mammalian cells (see Glover, 1985). Various methods can be followed to obtain complete antibodies. As discussed above, VH and VL chains, and optionally the associated constant regions, can be co-expressed in the same cell to achieve intracellular binding and linkage of the VH and VL chains into a fully tetrameric H2 L2 antibody or its antigen-binding portion. Co-expression can be performed using 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 and then transfected into cells, thereby directly selecting cells expressing both chains. Alternatively, cells can first be transfected with a plasmid encoding one chain (e.g., the VL chain), and the resulting cell line is then transfected with a VH chain plasmid containing a second selectable marker. Cell lines producing antibodies, antigen-binding portions thereof, via either route can be transfected with plasmids encoding additional copies of the peptide, VH, VL, or VH plus VL chains, along with other selectable markers, to generate cell lines with enhanced properties, such as higher yields of assembled binding units, or enhanced stability of the transfected cell line.

[0451] In addition, plants have become a convenient, safe and economical alternative expression system for the production of recombinant antibodies based on large-scale culture of microorganisms or animal cells. Binding units can be expressed in plant cell cultures or conventionally grown plants. Expression in plants can be systemic, restricted to subcellular plasmids, or restricted to seeds (endosperm). See, for example, U.S. Patent 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).

[0452] 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 from various human cells (e.g., immortalized B cells) according to well-known procedures (WO 87 / 02671). The CD70 binding antibody may contain both light and heavy chain constant regions. The heavy chain constant region may include CH1, hinge, CH2, CH3, and optionally, a CH4 region. In some embodiments, the CH2 domain may be deleted or omitted.

[0453] Techniques described for the production of single-chain antibodies (see, e.g., U.S. Patent 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 by amino acid bridges, thereby producing a single-chain polypeptide. Techniques for assembling functional Fv portions in E. coli can also be used (see, e.g., Skerra et al., Science 242:1038-1041 (1988); which are incorporated herein by reference in their entireties).

[0454] In some embodiments, the binding unit includes one or more scFvs. ScFv can be a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) variable regions of an antibody are connected to a short linker peptide of ten to about 25 amino acids. The linker is usually 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. Although the constant region is removed and a linker is introduced, this protein retains the specificity of the original antibody. ScFv antibodies are described in, for example, Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Methods for making scFv molecules and designing suitable peptide linkers are described, for example, in U.S. Pat. No. 4,704,692; U.S. Pat. No. 4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9:132-137 (1991). ScFv-Fc has been described by Sokolowska-Wedzina et al., Mol. Cancer Res. 15(8):1040-1050, 2017.

[0455] In some embodiments, the binding unit is a single domain antibody, which is an antibody portion consisting of a single monomeric variable antibody domain. Single domain antibodies can be derived from the variable domain of the antibody heavy chain of camelids (e.g., nanobodies or VHH portions). In addition, single domain antibodies can be autonomous human heavy chain variable domains (aVH) or VNAR portions derived from sharks (see, e.g., Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0456] Techniques for producing single-domain antibodies (DABs or VHHs) are known in the art, as disclosed in, for example, 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, for example, 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 capacity and can interact with antigenic determinants that are inaccessible to conventional VH-VL pairs (see, for example, Muyldermans et al., 2001). Alpaca serum IgG contains only approximately 50% camelid heavy chain IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens, and VHHs that bind to and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries, which can be used to isolate antibody fragments by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).

[0457] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)); WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168; Carter (2001), J Immunol Methods 248, 7-15). Multispecific antibodies can also be prepared by engineering electrostatic steering effects to prepare antibody Fc-heterodimers (see, e.g., WO 93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991)). 2009 / 089004A1); cross-linking of 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)); use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); use of "diabody" technology to prepare bispecific antibodies. and the use of single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of trispecific antibodies, e.g., as described in Tutt et al., J. Immunol. 147:60 (1991).

[0458] Engineered antibodies, including Octopus antibodies, with three or more functional antigen binding sites can also be binding units (see, eg, US 2006 / 0025576 A1).

[0459] In some embodiments, binding units (e.g., antibodies or antigen-binding portions) also include "dual-acting Fabs" or "DAFs," which include antigen-binding sites that bind to two different antigens (see, e.g., US 2008 / 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).

[0460] 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 comprised in two different polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization yields several possible combinations of the two polypeptides. To improve the yield and purity of bispecific molecules during recombinant production, it would be advantageous to introduce modifications into the Fc domain of the binding unit that promote binding of the desired polypeptide.

[0461] In general, this approach 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 unfavorable but heterodimerization is electrostatically favored.

[0462] In some embodiments, the binding unit is a "bispecific T cell trap" or BiTE (see, for example, WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain can include two single-chain Fv (scFv) portions, each having a variable heavy chain (VH) domain and a variable light chain (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular binding between the two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFvs. Each scFv recognizes a different antigenic determinant, and these antigenic determinants can be specific for different proteins, such that both proteins bind to the BiTE.

[0463] Since it is a single polypeptide, the bispecific T cell capture agent can be expressed using any prokaryotic or eukaryotic cell expression system known in the art (e.g., CHO cell line). However, specific purification techniques (see, e.g., EP1691833) may be necessary to separate monomeric bispecific T cell capture agents from other multimeric species, which may have biological activity other than the expected activity of the monomer. In an exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted with a gradient of imidazole concentrations. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a gradient of sodium chloride concentrations. Finally, size exclusion chromatography is performed on this eluate to separate monomers from the polymer. In some embodiments, the binding unit of the bispecific antibody is composed of a single polypeptide chain comprising two single-chain FV portions (scFV) fused to each other via a peptide linker.

[0464] In some embodiments, the binding unit is 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 their preparation have been 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.

[0465] IgG-like dual variable domain antibodies (DVD-Ig) have been described by 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 have been described by Chelius et al., MAbs 2(3):309-319, 2010. Two-in-one IgGs have been described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. Tanden antibodies or TandAbs have been described by Kontermann et al. (supra). ScFv-HSA-scFv antibodies have also been described by Kontermann et al. (supra).

[0466] Intact (e.g., whole) antibodies, dimers thereof, individual light and heavy chains, or antigen-binding portions thereof (i.e., binding units) can be recovered and purified by known techniques, such as immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination of such methods. See generally Scopes, Protein Purification (Springer-Verlag, NY, 1982). For pharmaceutical applications, substantially pure binding units having a homogeneity of at least about 90% to 95% are advantageous, as are binding agents having a homogeneity of 98% to 99% or greater. Once partially purified or purified to the desired homogeneity, the intact binding unit can be used therapeutically or in the development and performance of assays, immunofluorescence staining, and the like. See generally Volumes I and II of Immunol. Meth. (Lefkovits and Pernis, eds., Acad. Press, NY, 1979 and 1981).

[0467] Antibody-drug conjugates

[0468] 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 attached to at least one linker, and at least one drug unit attached to each linker. As used herein, the term "drug unit" refers to cytotoxic agents (e.g., chemotherapeutic agents or drugs), immunomodulators, nucleic acids (including siRNA), growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), radioactive isotopes, PROTACs, and other compounds that are active against target cells when delivered to target cells.

[0469] Cytotoxic agents

[0470] In some embodiments, the CD70 ADC includes at least one drug unit that is a cytotoxic agent. A "cytotoxic agent" refers to an agent that has a cytotoxic effect on cells. A "cytotoxic effect" refers to the depletion, elimination, and / or killing of target cells. Cytotoxic agents include, for example, tubulin-disrupting agents, topoisomerase inhibitors, DNA minor groove binders, and DNA alkylating agents.

[0471] Tubulin disrupting agents include, for example, auristatins, dolastatin, tubulysin, colchicine, vinca alkaloids, taxanes, cryptophycin, maytansines, hemiasterlin, and other tubulin disrupting agents. Auristatins are derivatives of the natural product dolastatin 10. Exemplary auristatins include MMAE (N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), MMAF (N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), and AFP (see WO2004 / 010957 and WO2007 / 008603). Other auristatin compounds are disclosed in, for example, published U.S. application Nos. US2021 / 0008099, US2017 / 0121282, US2013 / 0309192, and US2013 / 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 published U.S. application US2001 / 0018422). Other dolastatin derivatives contemplated for use herein are disclosed in U.S. Pat. No. 9,345,785, which is incorporated herein by reference.

[0472] Tubulysin includes but is not limited to tubulysin D, tubulysin M, tubuphenylalanine and tubulysine. WO2017 / 096311 and WO / 2016-040684 describe tubulysin analogs including tubulysin M.

[0473] Colchicine includes, but is not limited to, colchicine and CA-4.

[0474] Vinca alkaloids include, but are not limited to, vinblastine (VBL), vinorelbine (VRL), vincristine (VCR), and vindesine (VOS).

[0475] Taxanes include, but are not limited to, paclitaxel and docetaxel.

[0476] Nostocs include, but are not limited to, nostoc-1 and nostoc-52.

[0477] Maytansines include, but are not limited to, maytansine, maytansinol, DM1, DM3 and DM4 forms of maytansine analogs and ansamitocin-2. Exemplary maytansine drug moieties include those with modified aromatic rings, such as: C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by reduction of ansamitocin P2 with lithium aluminum hydride); 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); and compounds with modifications at other positions.

[0478] The maytansine class of drugs also includes drugs with the following modifications, for example: C-9-SH (U.S. Patent No. 4,424,219) (prepared by the reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Patent No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Patent No. 4,450,254) (prepared by Nocardia); C-15-hydroxy / acyloxy (U.S. Patent No. 4,364,866) (prepared by transformation of maytansinol by Streptomyces); C-15-methoxy (U.S. Patent Nos. 4,313,946 and 4,315,929) (prepared by Trewia spp. nudlflora); C-18-N-desmethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol by Streptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by titanium trichloride / LAH reduction of maytansinol).

[0479] Hemiasterlins include, but are not limited to, hemiasterlin and HTI-286.

[0480] Other tubulin disrupting agents include taccalonolide A, taccalonolide B, taccalonolide AF, taccalonolide AJ, taccalonolide Al-epoxide, discodermolide, epothilone A, epothilone B, and laulimalide.

[0481] In some embodiments, the cytotoxic agent may be a topoisomerase inhibitor, such as camptothecin. Exemplary camptothecins include, for example, camptothecin, irinotecan (also known as CPT-11), belotecan, (7-(2-(N-isopropylamino)ethyl)camptothecin), topotecan, 10-hydroxy-CPT, SN-38, exatecan (SS form), diastereomers of exatecan, RS form and exatecan analogs DXd (see US20150297748) comprising exatecan and DXd analogs comprising RS exatecan. Other camptothecins are disclosed in WO1996 / 021666, WO00 / 08033, US2016 / 0229862 and WO2020 / 156189.

[0482] In some embodiments, the cytotoxic agent is duocarmcycin, including the synthetic analogs KW-2189 and CBI-TMI.

[0483] Immunomodulators

[0484] In some embodiments, the drug unit is an immunomodulator. The immunomodulator can be, for example, a TLR7 and / or TLR8 agonist, a STING agonist, a RIG-I agonist, or other immunomodulators.

[0485] 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 the group consisting of imidazoquinoline, imidazoquinolineamine, 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 thiadiazine-2,2-dioxide, benzonaphthyridine, guanosine analogs, adenosine analogs, thymidine homopolymers, ssRNA, CpG-A, PolyG10, and PolyG3. In some embodiments, the TLR7 agonist is selected from imidazoquinoline, imidazoquinolineamine, thiazoquinoline, aminoquinoline, aminoquinazoline, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine, tetrahydropyridopyrimidine, heteroarylthiadiazine-2,2-dioxide or benzonaphthyridine. 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.

[0486] In some embodiments, the TLR8 agonist is selected from benzazepines, imidazoquinolines, thiazoquinolines, aminoquinolines, aminoquinazolines, 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 TLR8 agonist is selected from benzazepines, imidazoquinolines, thiazoquinolines, aminoquinolines, aminoquinazolines, pyrido[3,2-d]pyrimidine-2,4-diamine, pyrimidine-2,4-diamine, 2-aminoimidazole, 1-alkyl-1H-benzimidazol-2-amine and tetrahydropyridopyrimidine. In some embodiments, the TLR8 agonist is a non-naturally occurring compound. Examples of TLR8 agonists include motomod, resiquimod, 3M-051, 3M-052, MCT-465, IMO-4200, VTX-763, VTX-1463.

[0487] In some embodiments, the TLR8 agonist may be any of the compounds described in WO2018 / 170179, WO2020 / 056198, and WO2020056194.

[0488] Other TLR7 and TLR8 agonists are disclosed in, for example, WO2016142250, WO2017046112, WO2007024612, WO2011022508, WO2011022509, WO2012045090, WO2012097173, WO2012097177, WO2017079283, US20160008374, US20160194350, US20160289229, U.S. Patent No. 6043238, US20180086755, WO2017216054, WO2017190669, WO2017202704, WO2017202703, WO20170071944, US 20140045849, US20140073642, WO2014056953, WO2014076221, WO2014128189, US2 0140350031, WO2014023813, US20080234251, US20080306050, US20100029585, US2 0110092485, US20110118235, US20120082658, US20120219615, US20140066432, US20140088085, US20140275167, and US20130251673, WO2018198091, and US20170131421.

[0489] In some embodiments, the immunomodulator is a STING agonist. Examples of STING agonists include, for example, those disclosed in WO2020059895, WO2015077354, WO2020227159, WO2020075790, WO2018200812, and WO2020074004.

[0490] 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.

[0491] toxin

[0492] In some embodiments, the Drug Unit is an enzymatically active toxin or a 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, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, scutellaria baicalensis toxin, scutellaria serrata ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes.

[0493] radioactive isotopes

[0494] In some embodiments, the drug unit is a radioactive atom. A variety of radioisotopes can be used to produce radioconjugates. Examples include I131, I125, Y90, Re186, Re188, Sm153, Bi213, P32, Pb212, and radioisotopes of lutetium (e.g., Lu177).

[0495] PROTAC

[0496] In some embodiments, the drug unit is a proteolytic targeting chimera (PROTAC). PROTACs are described in, for example, published US application numbers 20210015942, 20210015929, 20200392131, 20200216507, US20200199247, and US20190175612; the disclosures of which are incorporated herein by reference.

[0497] connector

[0498] The CD70 conjugate typically includes at least one linker, each linker having at least one drug unit attached thereto. Typically, the conjugate includes a linker between a CD70 antibody (or antigen-binding portion thereof (ie, binding unit)) and a drug unit. In various embodiments, the linker can include 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 a 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)), a non-cleavable linker (see, e.g., WO2007 / 008603), a photolabile linker, and / or a hydrophilic linker (see, e.g., WO2015 / 123679).

[0499] In some embodiments, the joint is a cleavable joint that can be cleaved under intracellular conditions so that the cracking of the joint releases the drug unit from the binding unit and / or the joint in the intracellular environment. For example, in some embodiments, the joint can be cleaved by a cleavage agent present in the intracellular environment (such as lysosomes or endoplasmic bodies or caveolae). The joint may include, for example, a peptidyl joint that is cleaved by an intracellular peptidase or protease (including but not limited to lysosomes or endosomal proteases) (see, for example, WO2004 / 010957, US20150297748, US2008 / 0166363, US20120328564, and US20200347075). Typically, the peptidyl joint is at least one amino acid long or at least two amino acids long. Intracellular cleavage agents may include cathepsin B and cathepsin D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). The most typical peptidyl linker is one that is cleaved by an enzyme present in cells expressing the target antigen. For example, a peptidyl linker (e.g., Phe-Leu or Gly-Phe-Leu-Gly linker) that is cleaved by the thiol-associated protease cathepsin B, which is highly expressed in cancer tissue, may be used. Other such linkers are described, for example, in U.S. Patent No. 6,214,345. In certain embodiments, the peptidyl linker that can be cleaved by intracellular proteases 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., US 2015 / 0297748). One advantage of using intracellular proteolysis to release the drug unit is that the drug is generally attenuated upon binding and the serum stability of the conjugate is generally higher. See U.S. Patent No. 9,345,785.

[0500] As used herein, the terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or reaction within a cell of an antibody drug conjugate whereby the covalent bond (e.g., linker) between the drug (e.g., cytotoxic agent) and the antibody is broken, resulting in free drug or other metabolites of the conjugate that dissociate from the antibody within the cell. Thus, the cleaved portion of the conjugate is an intracellular metabolite.

[0501] In some embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, pH sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, or its analogue) that can be hydrolyzed in lysosomes can be used. (See, e.g., U.S. Patent 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., the pH of blood), but are unstable below pH 5.5 or 5.0 (approximately the pH of lysosomes). In some embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to the drug via an acylhydrazone bond (see, e.g., U.S. Patent No. 5,622,929)).

[0502] In some embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). Various disulfide linkers are known, including, for example, linkers 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 (CW Vogel, ed., Oxford U. Press, 1987. See U.S. Pat. No. 4,880,935).

[0503] In some embodiments, the linker is a malonate 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. Publication No. 2005 / 0238649).

[0504] In some embodiments, the joint is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in the context of a joint refers to when an antibody drug conjugate (ADC) is present in an extracellular environment (e.g., plasma), no more than about 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the joint is cleaved in a sample of the ADC. Whether the joint is substantially insensitive in the extracellular environment can be determined by: cultivating (a) ADC ("ADC sample") and (b) an equimolar amount of unbound antibody or drug ("control sample") independently with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unbound antibody or drug present in the ADC sample to the amount present in the control sample, for example, by high performance liquid chromatography.

[0505] 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 a linker-drug moiety of an ADC as described herein). In other embodiments, the linker promotes cellular internalization when conjugated to both a drug and a CD70 antibody (i.e., in the context of an ADC as described herein).

[0506] In other embodiments, CD70 ADCs can be prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imino esters (e.g., dimethyl diimidoadipate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Chelating agents for conjugating radionucleotides to binding units have been described, for example, in WO 94 / 11026.

[0507] CD70 ADC conjugates include, but are not limited to, those prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0508] In some embodiments, the linker is connected to the end of the amino acid sequence of the antibody or its antigen-binding portion (i.e., binding unit), or can be connected to a side chain modification of the antibody or its antigen-binding portion, such as a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, a glutamine or glutamic acid residue. The connection between the antibody or its antigen-binding portion and the linker or drug unit can be via any of a number of bonds, such as, but not limited to, an amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single bond or triple bond, a disulfide bond or a thioether bond. Functional groups that can form such bonds include, for example, amino, carboxyl, aldehyde, azido, alkynyl and alkene groups, ketones, carbonates, carbonyl functional groups (e.g., cyano and succinimidyl) bonded to a leaving group, and hydroxyl groups.

[0509] In some embodiments, the linker is connected to the binding unit at an interchain disulfide. In some embodiments, the linker is connected to the binding unit at a hinge cysteine residue. In some embodiments, the linker is connected to the binding unit at an engineered cysteine residue. In some embodiments, the linker is connected to the binding unit at a lysine residue. In some embodiments, the linker is connected to the binding unit at an engineered glutamine residue. In some embodiments, the linker is connected to the binding unit at a heavy chain engineered non-natural amino acid.

[0510] In some embodiments, the linker is connected to the binding unit through a sulfhydryl group. In some embodiments, the linker is connected to the binding unit through a primary amine. In some embodiments, the linker is connected through a connection formed by reacting a non-natural amino acid on the binding unit with an oxime bond, which is formed by modifying a keto group with an alkoxyamine on the drug.

[0511] In some embodiments, the linker is attached to the binding unit via a sortase A linker. The sortase A linker can be generated by sortase A enzyme fusing an LPXTG recognition motif (SEQ ID NO: 33) to an N-terminal GGG motif to regenerate a native amide bond.

[0512] In some embodiments, the linker has the following formula (I):

[0513] ~L1–(AA) s –L2≈

[0514] (I)

[0515] or a salt thereof, wherein:

[0516] L1 is a stretching unit having a connection site to a binding unit;

[0517] AA is an amino acid unit having 1 to 12 subunits;

[0518] s is 0 or 1;

[0519] L2 is a linker subunit having 1 to 4 attachment sites for drug units;

[0520] The wavy (~) line indicates the attachment site to the Binding unit, and the double wavy (≈) line indicates the attachment site to the Drug unit;

[0521] wherein at least one polar unit is present in an amino acid unit, a stretcher unit, a linker subunit, or a combination thereof, and wherein the polar unit is selected from a sugar unit, a PEG unit, a carboxyl unit, and a combination thereof.

[0522] In some embodiments, the linker has the following formula (I):

[0523] ~L1–(AA)s –L2≈

[0524] (I)

[0525] or a salt thereof, wherein:

[0526] L1 is a stretching unit having a connection site to a binding unit;

[0527] AA is an amino acid unit having 1 to 12 subunits;

[0528] s is 0 or 1;

[0529] L2 is a linker subunit having 1 to 4 attachment sites for drug units;

[0530] The wavy (~) line indicates the attachment site to the Binding unit, and the double wavy (≈) line indicates the attachment site to the Drug unit;

[0531] wherein at least one polar unit is present in an amino acid unit, a linker subunit, a stretcher unit, or a combination thereof, and wherein the polar unit is selected from a sugar unit, a PEG unit, a carboxyl unit, and a combination thereof.

[0532] Sugar unit

[0533] In some embodiments, the linker comprises a saccharide unit having the formula:

[0534]

[0535] or a salt thereof, wherein:

[0536] Each X is independently selected from NH or O;

[0537] each R is independently selected from hydrogen, acetyl, monosaccharide, disaccharide, and polysaccharide;

[0538] Each X1 is independently selected from CH2 and C(O);

[0539] Each X2 is independently selected from H, OH and OR;

[0540] k is 1 to 10; and

[0541] L3a is selected from C1-C 10 Alkylene and polyethylene glycol having 1 to 24 ethylene glycol subunits;

[0542] p and o are independently 0 to 2; and

[0543] Each * and each # indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1).

[0544] In some embodiments, the linker comprises a saccharide unit having a formula selected from:

[0545]

[0546] or a stereoisomer or salt thereof, wherein:

[0547] each R is independently selected from hydrogen, a monosaccharide, a disaccharide, and a polysaccharide;

[0548] p and o are independently 0 to 2;

[0549] m is 1-8;

[0550] n is 0 to 4; and

[0551] Each * and each # indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1).

[0552] PEG unit

[0553] In some embodiments, the linker comprises a PEG unit having a formula selected from:

[0554] (a)

[0555] ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25

[0556] (XX)

[0557] or a salt thereof, wherein:

[0558] R 20 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0559] R 21 and R 22 Each independently is, optionally C1-C3 alkylene;

[0560] R 24 and R 25 Each independently selected from H; polyhydroxy group; substituted polyhydroxy group; -C(O)-polyhydroxy group; substituted -C(O)-polyhydroxy 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; -C(O)-R28 , where R 28 is a sugar unit of formula (XII) or (XIII); or -NR 24 R 25 Together they form a C3-C8 heterocycle;

[0561] The condition is R 24 and R 25 Not all are H;

[0562] The wavy line (~) indicates the same as R 20 Attachment site; and

[0563] n20 is 1 to 26;

[0564] or

[0565] (b)

[0566] ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25

[0567] (XX)

[0568] or a salt thereof, wherein:

[0569] R 20 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0570] R 21 and R 22 Each independently is, optionally C1-C3 alkylene;

[0571] R 24 and R 25 One selected from H; polyhydroxy group; substituted polyhydroxy group; -C (O) - polyhydroxy group; substituted -C (O) - polyhydroxy 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; -C(O)-R 28 , where R 28 is a sugar unit of formula (XII) or (XIII); and R 24 and R 25 The other is polyethylene glycol, optionally having 1 to 24 ethylene glycol subunits;

[0572] The wavy line (~) indicates the same as R 20 Attachment site; and

[0573] n20 is 1 to 26;

[0574] or

[0575] (c)

[0576] ~R 20 -[-R 26 -[R 29 -[O-CH2-CH2-] n20 R 29 ] n21 -R 27 -] n27 -NR 24 R 25

[0577] (XXI)

[0578] or a salt thereof, wherein:

[0579] R 20 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0580] R 26 and R 27 Each is optional and independently selected from 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)- and -C(O)-C1-C 12 Alkylene-NH-;

[0581] R 24 and R 25 One selected from H; polyhydroxy group; substituted polyhydroxy group; -C (O) - polyhydroxy group; substituted -C (O) - polyhydroxy 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; -C(O)-R 28 , where R 28 is a sugar unit of formula (XII) or (XIII); and R 24 and R25 The other is selected from H; polyhydroxy group; substituted polyhydroxy group; -C (O) -polyhydroxy group; substituted -C (O) -polyhydroxy 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; -C(O)-R 28 , where R 28 is a saccharide unit of formula (XII) or (XIII); and polyethylene glycol, optionally having 1 to 24 ethylene glycol subunits; or -NR 24 R 25 Together they form a C3-C8 heterocycle;

[0582] The condition is R 24 and R 25 Not all are H;

[0583] 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;

[0584] The wavy line (~) indicates the same as R 20 The attachment site;

[0585] n20 is 1 to 26;

[0586] n21 is 1 to 4; and

[0587] n27 is 1 to 4.

[0588] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 are not all H. In some embodiments, a linker is provided wherein the R 24 and R 25 are each independently selected from H and polyhydroxy groups, provided that R 24 and R 25 Not all are H.

[0589] In some embodiments, a conjugate comprising a linker is provided, wherein the polyhydroxy group is a linear monosaccharide, optionally selected from a C6 or C5 sugar, a sugar acid, or an amino sugar. In some embodiments, a conjugate comprising a linker is provided, wherein:

[0590] C6 or C5 sugars selected from glucose, ribose, galactose, mannose, arabinose, 2-deoxyglucose, glyceraldehyde, erythrose, threose, xylose, lyxose, allose, altrose, gulose, idose, talose, aldose, and ketose;

[0591] The sugar acid is selected from the group consisting of gluconic acid, aldonic acid, uronic acid and ketonic acid; or

[0592] The amino sugar is selected from the group consisting of glucosamine, N-acetylglucosamine, galactosamine and N-acetylgalactosamine.

[0593] In some embodiments, a conjugate is provided comprising a linker wherein the PEG unit is selected from the group consisting of:

[0594]

[0595]

[0596] where R 39 is selected from H, a linear monosaccharide, and polyethylene glycol, optionally having 1 to 24 ethylene glycol subunits; and the wavy line on the left indicates the site of attachment to a subunit of an Amino Acid unit, a Stretcher unit, and / or a portion of a Linker subunit.

[0597] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 One of the two is a linear monosaccharide and the other is a cyclic monosaccharide.

[0598] In some embodiments, a conjugate is provided comprising a linker wherein the PEG unit is selected from the group consisting of:

[0599]

[0600] where R 41 is a cyclic monosaccharide; and the wavy line on the left indicates the site of attachment to a portion of the subunit of the Amino Acid unit, the Stretcher unit, and / or the Linker subunit.

[0601] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit is selected from the following, or a stereoisomer or salt thereof:

[0602]

[0603] Each R 45 is selected from H and a monosaccharide, a disaccharide or a polysaccharide; and R 46is selected from a cyclic monosaccharide, disaccharide or polysaccharide; and the wavy line on the right indicates the site of attachment to a portion of the subunit of the Amino Acid unit, the Stretcher unit and / or the Linker subunit.

[0604] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 is independently selected from linear monosaccharides and substituted linear monosaccharides, wherein the substituted linear monosaccharides are substituted with monosaccharides, disaccharides or polysaccharides. In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit is selected from the following, or a stereoisomer or salt thereof:

[0605]

[0606] where R 47 is a straight-chain monosaccharide; and each R 49 is selected from the group consisting of a monosaccharide, a disaccharide, and a polysaccharide; and the wavy line on the left indicates the site of attachment to a portion of a subunit of the Amino Acid unit, a Stretcher unit, and / or a Linker subunit.

[0607] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 Independently selected from linear monosaccharides and substituted monosaccharides, wherein the substituted linear monosaccharides are substituted with one or more substituents selected from the group consisting of alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide, and are optionally further substituted with monosaccharides, disaccharides, or polysaccharides. In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit is selected from the group consisting of the following, or a stereoisomer or salt thereof:

[0608]

[0609] 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; and the wavy line on the left indicates the site of attachment to a portion of the Amino Acid unit's subunit, Stretcher unit and / or Linker subunit.

[0610] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 One of them is a -C(O)-polyhydroxy group or a substituted -C(O)-polyhydroxy group, and R 24 and R 25The other of the hydroxyl groups is H, -C(O)-polyhydroxyl, substituted -C(O)-polyhydroxyl, polyhydroxyl or substituted polyhydroxyl; wherein the substituted -C(O)-polyhydroxyl and polyhydroxyl groups are substituted with monosaccharides, disaccharides, polysaccharides, alkyl, -O-alkyl, aryl, carboxyl, ester, or amide. In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit is selected from the following, or a stereoisomer or salt thereof:

[0611]

[0612] The wavy line on the left indicates the site of attachment to a subunit of the Amino Acid unit, a Stretcher unit and / or a portion of the Linker subunit.

[0613] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 independently selected from H, substituted-C1-C8 alkyl, substituted-C1-C4 alkyl or substituted-C1-C3 alkyl; provided that R 24 and R 25 wherein the substituted -C1-C8 alkyl, -C1-C4 alkyl and -C1-C3 alkyl are substituted with hydroxyl and / or carboxyl groups. In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit is selected from the following, or a stereoisomer or salt thereof:

[0614]

[0615]

[0616] where R 48 is selected from H, OH, CH2OH, COOH or a -C1-C6 alkyl group substituted with a hydroxyl group or a carboxyl group; and the wavy line on the left indicates the site of attachment to a portion of the subunit of the Amino Acid unit, the Stretcher unit and / or the Linker subunit.

[0617] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 One of the following 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 of the alkyl group 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 groups. In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit is selected from the following, or a stereoisomer or salt thereof:

[0618]

[0619]

[0620] The wavy line on the left indicates the site of attachment to a subunit of the Amino Acid unit, a Stretcher unit and / or a portion of the Linker subunit.

[0621] In some embodiments, a conjugate comprising a linker is provided wherein R of the PEG unit is 24 and R 25 independently selected from H and a chelating agent, wherein the chelating agent is optionally linked to -NR via an alkylene, arylene, carbocyclyl, heteroarylene or heterocarbocyclyl group. 24 R 25 of nitrogen; provided that R 24 and R 25 Not all are H. In some embodiments, a conjugate comprising a linker is provided, wherein the chelator is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), triethylenetetraaminehexaacetic 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 piperazine. In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit is selected from the following, or a stereoisomer or salt thereof:

[0622]

[0623] The wavy line on the left indicates the site of attachment to a subunit of the Amino Acid unit, a Stretcher unit and / or a portion of the Linker subunit.

[0624] In some embodiments, a conjugate is provided comprising a linker wherein each monosaccharide of the saccharide unit or PEG unit is independently selected from:

[0625] C5 or C6 sugars selected from the group consisting of 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;

[0626] a sugar acid selected from the group consisting of gluconic acid, aldonic acid, uronic acid, and ketonic acid; or

[0627] An amino sugar selected from the group consisting of glucosamine, N-acetylglucosamine, galactosamine and N-acetylgalactosamine.

[0628] In some embodiments, a conjugate comprising a linker is provided wherein R 20 Selected from carboxyl, amino, alkynyl, azido, hydroxy, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate or protected forms thereof.

[0629] In some embodiments, a conjugate comprising a linker is provided wherein R 20 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

[0630] In some embodiments, a conjugate comprising a linker is provided wherein R 20 Selected from carboxyl, amino, alkynyl, azido, hydroxy, carbonyl, carbamate, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate or protected forms thereof.

[0631] In some embodiments, a conjugate comprising a linker is provided wherein R 20 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

[0632] In some embodiments, the linker comprises a PEG unit having a formula selected from:

[0633] ~R 40 -(R 43 -R 41 -[O-CH2-CH2]n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42

[0634] (XL)

[0635] or a salt thereof, wherein:

[0636] R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0637] R 41 and R 42 Not present or each independently represents a C1-C6 alkylene group;

[0638] Each R 43 Independently absent or selected from 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 them is H or C1-C 12 Alkylene and the other is C1-C 12 alkylene;

[0639] R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates;

[0640] The condition is R 44 and R45 Not all are H;

[0641] The wavy line (~) indicates the same as R 40 The attachment site;

[0642] n40 is 1 to 26;

[0643] n41 is 1 to 6; and

[0644] n42 is 1 to 6.

[0645] In some embodiments, the linker comprises a PEG unit having a formula selected from:

[0646] ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42

[0647] (XLI)

[0648] or a salt thereof, wherein:

[0649] R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0650] R 41 and R 42 Not present or each independently represents a C1-C6 alkylene group;

[0651] R 43 Not present or selected from 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)NR46 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 alkylene;

[0652] R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates;

[0653] The condition is R 44 and R 45 Not all are H;

[0654] The wavy line (~) indicates the same as R 40 The attachment site;

[0655] n40 is 1 to 26;

[0656] n41 is 1 to 6; and

[0657] n42 is 1 to 6.

[0658] In some embodiments, the linker comprises a PEG unit having a formula selected from:

[0659] ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42

[0660] (XLII)

[0661] or a salt thereof, wherein:

[0662] R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0663] R 41 and R 42 Not present or each independently represents a C1-C3 alkylene group;

[0664] R 43 Not present or selected from C1-C6 alkylene, -NH-C1-C 12Alkylene, -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 them is H or C1-C6 alkylene and the other is C1-C 12 alkylene;

[0665] R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates;

[0666] The condition is R 44 and R 45 Not all are H;

[0667] The wavy line (~) indicates the same as R 40 The attachment site;

[0668] n40 is 1 to 26;

[0669] n41 is 1 to 4; and

[0670] n42 is 1 to 4.

[0671] In some embodiments, a conjugate comprising a linker is provided wherein R 40 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

[0672] In some embodiments, a conjugate comprising a linker is provided wherein R 20 or R 40 Has one of the following structures:

[0673]

[0674]

[0675] Where R=H or C 1-6 alkyl; and

[0676] n = 0 to 12

[0677] or its stereoisomers, wherein (*) indicates R 20 or R 40 A site of attachment to a subunit of an Amino Acid unit, a Stretcher unit and / or a portion of a Linker subunit L2 and Indicator R 20 or R 40 The site of attachment to the remainder of the PEG unit.

[0678] In some embodiments, a conjugate comprising a linker is provided wherein R 20 or R 40 Has one of the following structures:

[0679]

[0680] Where n = 0 to 12

[0681] or its stereoisomers, wherein (*) indicates R 20 or R 40 A site of attachment to a subunit of an Amino Acid unit, a Stretcher unit and / or a portion of a Linker subunit L2 and Indicator R 20 or R 40 The site of attachment to the remainder of the PEG unit.

[0682] In some embodiments, a conjugate comprising a linker is provided wherein R 43 -(NR 44 R 45 ) n41 , when R 43 When present, has one of the following structures:

[0683]

[0684] Where R=H、C 1-6 Alkyl, polyhydroxy or substituted polyhydroxy

[0685] or a stereoisomer thereof, wherein Indicator R 43 The site of attachment to the remainder of the PEG unit.

[0686] In some embodiments, a conjugate comprising a linker is provided wherein R 43 -(NR 44 R 45 ) n41 , when R 43When present, has one of the following structures:

[0687]

[0688] or a stereoisomer thereof, wherein Indicator R 43 The site of attachment to the remainder of the PEG unit.

[0689] In some embodiments, a conjugate comprising a linker is provided wherein -NR 44 R 45 Has one of the following structures:

[0690]

[0691]

[0692] or a stereoisomer thereof, wherein Instructions-NR 44 R 45 The site of attachment to the remainder of the PEG unit.

[0693] In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit, prior to being linked to an Amino Acid unit, a Stretcher unit, and / or a portion of the Linker subunit L2, has one of the following structures, or a stereoisomer thereof:

[0694]

[0695]

[0696]

[0697]

[0698] wherein R is H or alkyl, and each n is independently 1 to 12, and wherein the functional group moiety of the attachment site of the PEG unit can be selected from carboxyl, hydroxyl, aminyl, azidyl, hydrazine, alkynyl, formyl, or triazolyl as described above.

[0699] In some embodiments, the linker comprises a PEG unit having a formula selected from:

[0700] ~R 40 -(R 43 -R 41 --[O-CH2-CH2] n40 -R 46 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR44 R 45 ) n41 ) n42

[0701] (XLIII)

[0702] or a salt thereof, wherein:

[0703] R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2;

[0704] R 41 and R 42 Not present or each independently represents a C1-C6 alkylene group;

[0705] Each R 43 Independently absent or selected from 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 them is H or C1-C 12 Alkylene and the other is C1-C 12 alkylene;

[0706] R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates;

[0707] R 46 Selected from amino, amino-alkyl-amino or -NH-C(O)-NH-S(O)2-NH-;

[0708] The wavy line (~) indicates the same as R 40 The attachment site;

[0709] n40 is 1 to 26;

[0710] n41 is 1 to 6; and

[0711] n42 is 1 to 6.

[0712] In some embodiments, a conjugate comprising a linker is provided, wherein the PEG unit, prior to being linked to an Amino Acid unit, a Stretcher unit, and / or a portion of the Linker subunit L2, has one of the following structures:

[0713]

[0714] wherein R is H or an alkyl group, and n is 1 to 12, and wherein the functional group moiety of the attachment site of the PEG unit can be selected from a carboxyl group, a hydroxyl group, an amine group, or an azide group.

[0715] In some embodiments, the linker comprises a PEG unit having a formula selected from:

[0716]

[0717]

[0718] or a stereoisomer or salt thereof, wherein:

[0719] Each Y is independently R 76 or Each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2) v -OS(=O)2(OH);

[0720] Each R a and R b are independently H or R a and R b Together with the carbon to which they are attached, they form an oxo group;

[0721] Each q is independently 1-26;

[0722] Each m is independently 1 to 4;

[0723] each n is independently 1 to 4;

[0724] each v is independently 1 to 6; and

[0725] Each * indicates a site of attachment to a subunit of the Amino Acid Unit (AA), a Linker Subunit L2, or a Stretcher Unit (L1).

[0726] In some embodiments, the linker comprises a PEG unit having a formula selected from:

[0727]

[0728] or a stereoisomer or salt thereof, wherein:

[0729] Each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2) v S(=O)2(OH);

[0730] Each q is independently 1-26;

[0731] Each m is independently 1 to 4;

[0732] each n is independently 1 to 4;

[0733] each v is independently 1 to 6; and

[0734] Each * indicates a site of attachment to a subunit of the Amino Acid Unit (AA), a Linker Subunit L2, or a Stretcher Unit (L1).

[0735] In some embodiments, the linker comprises a PEG unit having a formula selected from:

[0736]

[0737]

[0738] or a stereoisomer or salt thereof, wherein:

[0739] Each q is independently 1-26;

[0740] Each m is independently 1 to 4;

[0741] each n is independently 1 to 4; and

[0742] Each * indicates a site of attachment to a subunit of the Amino Acid Unit (AA), a Linker Subunit L2, or a Stretcher Unit (L1).

[0743] In some embodiments, a conjugate comprising a linker is provided wherein Y is R 76 .

[0744] In some embodiments, a conjugate comprising a linker is provided wherein Y is

[0745] In some embodiments, a conjugate comprising a linker is provided wherein each R a and R b are independently H.

[0746] In some embodiments, a conjugate comprising a linker is provided wherein R a and R b Together with the carbon to which they are attached they form an oxo group.

[0747] In some embodiments, a conjugate comprising a linker is provided, wherein q is 10-20.

[0748] In some embodiments, a conjugate comprising a linker is provided, wherein q is 12.

[0749] Carboxyl unit

[0750] In some embodiments, the linker comprises a carboxyl unit having the formula:

[0751]

[0752] or a salt thereof, wherein:

[0753] (a)

[0754] L 70 Selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, -C(O)-C1-C8 alkylene- and -C(O)-C1-C8 alkylene-C(O)-;

[0755] R 70 for NR 71 (R 72 -R 73 ), where R 71 Selected from 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, and R 73 is carboxyl or polycarboxyl, wherein the polycarboxyl group comprises 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are linked to each other through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino and / or amide groups;

[0756] Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and

[0757] p1 and o1 are each independently selected from 0 to 2;

[0758] or

[0759] (b)

[0760] L 70 Selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, -C(O)-C1-C8 alkylene- and -C(O)-C1-C8 alkylene-C(O)-;

[0761] R 70 for NR 71 (R 75 -(R 73 )2), where R 71 Selected from 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, a 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 independently carboxyl or polycarboxyl, wherein the polycarboxyl group comprises 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amide groups;

[0762] Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and

[0763] p1 and o1 are each independently selected from 0 to 2;

[0764] or

[0765] (c)

[0766] L 70 Selected from C1-C8 alkylene, C1-C8 alkylene-C(O)-, -C(O)-C1-C8 alkylene- and -C(O)-C1-C8 alkylene-C(O)-;

[0767] R 70 is ~N(R 74 -R 73 )(R 72 -R 73 ), where R 72 and R 74are each independently selected from optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl or optionally substituted heteroaryl, and each R 73 is independently carboxyl or polycarboxyl, comprising 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amide groups;

[0768] Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and

[0769] p1 and o1 are each independently selected from 0 to 2.

[0770] In some embodiments, a conjugate comprising a linker is provided, which comprises at least one saccharide unit. In some embodiments, a conjugate comprising a linker is provided, which comprises at least one PEG unit. In some embodiments, a conjugate comprising a linker is provided, which comprises at least one carboxyl unit. In some embodiments, a conjugate comprising a linker is provided, which comprises at least two polar units, each polar unit being selected from a saccharide unit, a PEG unit, and a carboxyl unit. In some embodiments, a conjugate comprising a linker is provided, which comprises at least one saccharide unit and a PEG unit or a carboxyl unit. In some embodiments, a conjugate comprising a linker is provided, which comprises at least one carboxyl unit and a PEG unit.

[0771] In some embodiments, a conjugate comprising a linker is provided, wherein an Amino Acid unit (AA) is present (s=1). In some embodiments, a conjugate comprising a linker is provided, wherein the Amino Acid unit comprises at least one polar unit.

[0772] In some embodiments, a conjugate comprising a linker is provided, wherein L2 or AA-L2 has one of the following structures, or a stereoisomer thereof:

[0773]

[0774] The wavy line above the amino group indicates the site of attachment to the Stretcher or Amino Acid unit, and the Drug unit is attached to the benzyl alcohol.

[0775] In some embodiments, a conjugate comprising a linker is provided, wherein the linker comprises AA-L2-, which has a formula selected from the group consisting of:

[0776] ~[SU–aa]-L2≈,

[0777] ~[aa1(PEG)–aa]-L2≈, or

[0778] ~[CU–aa]-L2≈

[0779] wherein square brackets indicate an amino acid unit, each aa is an optional subunit of AA, L2 is a linker subunit, each wavy line (~) indicates a site of attachment to 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 indicates a site of attachment to a drug unit, wherein aa and aa1 are independently selected from α, β, and γ amino acids and derivatives thereof.

[0780] In some embodiments, a conjugate comprising a linker is provided, wherein the linker comprises AA-L2-, which has a formula selected from the group consisting of:

[0781]

[0782] wherein square brackets indicate amino acid units, each aa is an amino acid subunit of AA, L2 is a linker subunit connected to the side chain of aa, a wavy line (~) indicates the site of attachment to the stretcher unit; aa1(PEG) is a PEG unit connected to aa, SU is a sugar unit connected to aa, CU is a carboxyl unit connected to aa, and a double wavy (≈) line indicates the site of attachment to the drug unit; wherein aa and aa1 are independently selected from α, β and γ amino acids and derivatives thereof.

[0783] In some embodiments, a conjugate comprising a linker is provided, wherein the Amino Acid unit comprises at least two Polar units.

[0784] In some embodiments, a conjugate comprising a linker is provided, wherein the linker comprises AA-L2-, which has a formula selected from the group consisting of:

[0785] ~[SU–aa–SU]–L2≈,

[0786] ~[aa1(PEG)–aa–aa2(PEG)]–L2≈, or

[0787] ~[CU–aa–CU]–L2≈

[0788] wherein square brackets indicate an amino acid unit, aa is an optional subunit of AA, L2 is a linker subunit, and a wavy line (-) indicates the site of attachment to the stretcher unit; aa1(PEG) and aa2(PEG) are each a PEG unit connected to aa or another PEG unit; each SU is a sugar unit connected to aa or another sugar unit, each CU is a carboxyl unit connected to aa or another carboxyl unit, and a double wavy (≈) line indicates the site of attachment to the drug unit; wherein aa, aa1, and aa2 are independently selected from α, β, and γ amino acids and derivatives thereof.

[0789] In some embodiments, a conjugate comprising a linker is provided, wherein the linker comprises AA-L2-, which has a formula selected from the group consisting of:

[0790]

[0791] wherein square brackets indicate amino acid units, aa is an amino acid subunit of AA, L2 is a linker subunit connected to the side chain of aa, each wavy line (~) indicates a site of attachment to a stretcher unit; aa1(PEG) and aa2(PEG) are each a PEG unit connected to aa, each SU is a sugar unit connected to aa; each CU is a carboxyl unit connected to aa; and a double wavy (≈) line indicates a site of attachment to a drug unit; wherein aa, aa1, and aa2 are each independently selected from α, β, and γ amino acids and derivatives thereof.

[0792] In some embodiments, a conjugate comprising a linker is provided, wherein the linker subunit L2 is a cleavable linker unit. In some embodiments, a conjugate comprising a linker is provided, wherein the linker subunit L2 comprises a peptide cleavable by an intracellular protease. In some embodiments, a conjugate comprising a linker is 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.

[0793] In some embodiments, a conjugate comprising a linker is provided, wherein the linker subunit L2 comprises at least one polar unit. In some embodiments, a conjugate comprising a linker is provided, wherein the polar unit is a sugar unit (SU). In some embodiments, a conjugate comprising a linker is provided, wherein the cleavable peptide comprises a SU-valine-citrulline peptide, a SU-valine-lysine peptide, a SU-valine-alanine peptide, a SU-phenylalanine-lysine peptide, or a SU-glycine-glycine-phenylalanine-glycine peptide.

[0794] In some embodiments, a conjugate comprising a linker is provided, wherein the polar unit is a carboxyl unit (CU). In some embodiments, a conjugate comprising a linker is 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 the CU-lysine is a carboxyl unit comprising a lysine residue.

[0795] In some embodiments, a conjugate comprising a linker is provided, wherein the polar unit is a PEG unit (PEG). In some embodiments, a conjugate comprising a linker is 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 Phe-Lys(PEG)) peptide, or a Lys(PEG)-glycine-glycine-phenylalanine-glycine peptide, wherein Lys(PEG) and Cit(PEG) comprise a PEG unit attached to a lysine residue or a citrulline residue, respectively.

[0796] In some embodiments, a conjugate is provided comprising a linker wherein the cleavable peptide is linked to a p-aminobenzyl alcohol self-immolative group (PABA).

[0797] In some embodiments, a conjugate comprising a linker is provided, wherein the amino acid unit is bound to the linker subunit L2 via a non-peptide linker. In some embodiments, a conjugate comprising a linker is provided, wherein the non-peptide linker is selected from the group consisting of C1-C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylidene or polyethylene glycol.

[0798] In some embodiments, a conjugate comprising a linker is provided, further comprising a stretcher unit. In some embodiments, a conjugate comprising a linker is provided, wherein the stretcher unit is selected from the following:

[0799]

[0800] where R 17 -C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-, -C3-C8 carbocyclyl-, -O-(C1-C8 alkylene)-, -(CH2-O-CH2) b-C1-C8 alkylene-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-(wherein b is 1 to 26), -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-, -C3-C8 heterocycle-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C1-C8alkylene-(CH2-O-CH2) b -C(=O)-(wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C3-C8 carbocyclyl-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-C(=O)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocycle-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-C(=O)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C1-C8alkylene-(CH2-O-CH2) b -NH- (wherein b is 1 to 26), -(CH2-O-CH2) b-C1-C8 alkylene-NH-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-NH-(wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -NH-(wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C3-C8 carbocyclyl-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 carbocyclyl)-NH-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-NH-, -C3-C8 heterocycle-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-NH-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclyl-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 carbocyclyl)-S-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-S-, -C3-C8 heterocycle-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-S- or -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-S-; or

[0801] The extension unit includes a maleimide group (C1-C 10 Alkylene-C(O)-, maleimide (CH2OCH2)p2 (C1-C 10 Alkylene) C(O)-, maleimide (C1-C 10 Alkylene)(CH2OCH2) p2 C(O)-, or a ring-opened form thereof, wherein p2 is 1 to 26.

[0802] In some embodiments, a conjugate comprising a linker is provided, wherein the Stretcher unit is selected from the group consisting of:

[0803]

[0804] The wavy line The attachment site of the Stretcher unit to the Amino Acid unit or Linker subunit L2 is indicated, and the attachment site to the Binding unit is at a maleimide, primary amine, or alkyne functional group.

[0805] In some embodiments, a conjugate is provided that comprises a linker having one of the following structures, or a stereoisomer thereof:

[0806]

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825] wherein each Z is attached at * and is independently selected from:

[0826]

[0827] Wherein the Drug unit is linked to the Linker subunit L2, the terminal acid group or benzyl alcohol, or where the wavy (≈) line indicates the site of attachment to the Drug unit.

[0828] In some embodiments, a conjugate having the structure

[0829] T-linker-drug unit,

[0830] or a pharmaceutically acceptable salt thereof;

[0831] Where T is the binding unit;

[0832] wherein the linker has the following formula (I):

[0833] L1–(AA) s –L2

[0834] (I)

[0835] in:

[0836] (i) L1 is a stretching unit connected to a binding unit,

[0837] (ii) AA is an amino acid unit having 1 to 12 subunits;

[0838] (iii) s is 0 or 1;

[0839] (iv) L2 is a Linker subunit connected to the Drug unit, wherein the Linker subunit is a Cleavable Linker unit comprising a Cleavable Peptide;

[0840] (v) the Drug unit is selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, a radioisotope, and a chelating ligand; and

[0841] (vi) at least one PEG unit,

[0842] wherein at least one PEG unit is present in the Amino Acid unit, the Linker subunit, or a combination thereof, and wherein at least one PEG unit has the formula:

[0843]

[0844] Each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2) v S(=O)2(OH);

[0845] Each q is independently 1-26;

[0846] Each m is independently 1 to 4;

[0847] each n is independently 1 to 4;

[0848] each v is independently 1 to 6; and

[0849] Each * indicates the site of attachment of the subunits, amino acid units, or both of the linker subunits

[0850] In some embodiments, a drug-linker having the following formula (III) is provided

[0851] [L1–(AA) s –L2]–D t

[0852] (III)

[0853] or a salt thereof, wherein:

[0854] (i) L1 is an extension unit;

[0855] (ii) AA is an amino acid unit having 1 to 12 subunits;

[0856] (iii) s is 0 or 1;

[0857] (iv) L2 is a Linker subunit linked to the Drug unit (D), wherein the Linker subunit is a Cleavable Linker unit comprising a Cleavable Peptide, and wherein t is 1 to 4;

[0858] (v) the Drug unit is selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, a radioisotope, and a chelating ligand; and

[0859] (vi) one or more PEG units,

[0860] wherein the one or more PEG units are present in the Amino Acid unit, the Linker subunit, or a combination thereof, and wherein at least one of the one or more PEG units has the formula:

[0861]

[0862] Each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2)v S(=O)2(OH);

[0863] Each q is independently 1-26;

[0864] Each m is independently 1 to 4;

[0865] each n is independently 1 to 4;

[0866] each v is independently 1 to 6; and

[0867] Each * is a site of attachment to a linker subunit, an amino acid unit, or both.

[0868] In some embodiments, for the conjugates, the binding unit is an antibody or an antigen-binding portion thereof.

[0869] In some embodiments, for the conjugate, (1) the binding unit comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region comprising complementarity determining regions HCDR1, HCDR2, and HCDR3 positioned in a heavy chain variable region framework region and the VL region comprising LCDR1, LCDR2, and LCDR3 positioned in a light chain variable region framework region, the VH and VL CDRs having an amino acid sequence selected from the group of amino acid sequences set forth as 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; ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:18; 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.

[0870] In some embodiments, the binding unit is as described elsewhere herein for the conjugate.

[0871] In some embodiments, for the conjugate or drug-linker, each of the one or more PEG units has the formula:

[0872]

[0873] In some embodiments, for the conjugate or drug-linker, each of the one or more PEG units has the formula:

[0874]

[0875] In some embodiments, for a conjugate or drug-linker, (vi) has one PEG unit. In some cases, (vi) has two PEG units.

[0876] In some embodiments, for the 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.

[0877] In some embodiments, for drug-linkers, the Stretcher is capable of forming a bond with a sulfur atom. In some cases, the Stretcher comprises a maleimido group (C1-C 10 Alkylene)-C(O)-, maleimide (CH2OCH2) p2 (C1-C 10 Alkylene) C(O)-, maleimide (C1-C 10 Alkylene)(CH2OCH2) p2 C(O)-, or its open ring form, wherein p2 is 1 to 26. In some cases, the stretcher unit includes a maleimide group (C1-C 10 In some cases, the stretching unit is a maleimido (C1-C 10 In some cases, the stretcher unit is

[0878] In some embodiments, for a conjugate or drug-linker, s is 0.

[0879] In some embodiments, for conjugates or drug-linkers, 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. 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 attached to a lysine residue or a citrulline residue, respectively, wherein the PEG unit is represented by Formula (XVIb).

[0880] In some embodiments, for a conjugate or drug-linker, the cleavable peptide comprises a self-immolative group. In some cases, the cleavable peptide comprises a p-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.

[0881] 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.

[0882] In some embodiments, for a conjugate or drug-linker, s is 1.

[0883] In some embodiments, for a 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, aminoalkane diacid, aminobenzoic acid, amino-heterocyclic-alkanoic acid, heterocyclic-carboxylic acid, citrulline, and diaminoalkanoic acid; wherein one or more PEG units are attached 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 has a formula selected from the following:

[0884] In some embodiments, for conjugates or drug-linkers, L2 is a cleavable peptide. In some cases, L2 is a cleavable peptide substituted with one or more PEG units.

[0885] In some embodiments, for a Drug-Linker, the Stretcher unit is selected from

[0886]

[0887] The wavy line Indicates the site of attachment of the Stretcher unit to the Amino Acid unit (if s is 1) or to L2 (if s is 0).

[0888] In some embodiments, for a 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.

[0889] In some embodiments, a conjugate having the following formula (III*C) is provided

[0890] T–[L1–AA–L2–D] S

[0891] (III*C)

[0892] or a salt thereof, wherein:

[0893] (vii) T is a binding unit;

[0894] (viii) s is p load , where p load Selected from about 1 to about 16;

[0895] (i) L1 is where R 17 is C1-C8 alkylene-C(O)-;

[0896] (ii) AA is an amino acid unit having 1 to 5 subunits;

[0897] (iii) wherein 1 to 5 subunits of the Amino Acid unit are selected from the group consisting of 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, aminoalkanediacid, aminobenzoic acid, amino-heterocyclic-alkanoic acid, heterocyclic-carboxylic acid, citrulline, and diaminoalkanoic acid;

[0898] (iv) L2 is a cleavable peptide covalently linked to a self-immolative group;

[0899] (v) D is selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, a radioisotope, and a chelating ligand, wherein D is covalently linked to a self-immolative group of L2; and

[0900] (vi) wherein one of the 1 to 5 subunits of the Amino Acid unit is covalently linked to a PEG unit, wherein the PEG unit has the formula:

[0901]

[0902] wherein each q is independently 1-26;

[0903] wherein each m is independently 1 to 4;

[0904] wherein each n is independently 1 to 4; and

[0905] where * indicates the site of attachment to one of the 1 to 5 subunits of the amino acid unit.

[0906] In some embodiments, for the conjugates, the binding unit is an antibody or an antigen-binding portion thereof.

[0907] In some embodiments, for the conjugate, (1) the binding unit comprises a heavy chain variable (VH) region and a light chain variable (VL) region, the VH region comprising complementarity determining regions HCDR1, HCDR2, and HCDR3 positioned in a heavy chain variable region framework region and the VL region comprising LCDR1, LCDR2, and LCDR3 positioned in a light chain variable region framework region, the VH and VL CDRs having an amino acid sequence selected from the group of amino acid sequences set forth as 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; ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:18; 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.

[0908] In some embodiments, the binding unit can be as described anywhere else herein.

[0909] In some embodiments, for the conjugate, 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 About 16.

[0910] In some embodiments, a drug-linker having the following formula (III*) is provided

[0911] L1–AA–L2–D

[0912] (III*)

[0913] or a salt thereof, wherein:

[0914] (i) L1 is a maleimide group (C1-C 10 alkylene)-C(O)-;

[0915] (ii) AA is an amino acid unit having 1 to 5 subunits;

[0916] (iii) wherein 1 to 5 subunits of the Amino Acid unit are selected from the group consisting of 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, aminoalkanediacid, aminobenzoic acid, amino-heterocyclic-alkanoic acid, heterocyclic-carboxylic acid, citrulline, and diaminoalkanoic acid;

[0917] (iv) L2 is a cleavable peptide covalently linked to a self-immolative group;

[0918] (v) D is selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, a radioisotope, and a chelating ligand, wherein D is covalently linked to a self-immolative group of L2; and

[0919] (vi) wherein one of the 1 to 5 subunits of the Amino Acid unit is covalently linked to a PEG unit, wherein the PEG unit has the formula:

[0920]

[0921] wherein each q is independently 1-26;

[0922] wherein each m is independently 1 to 4;

[0923] wherein each n is independently 1 to 4; and

[0924] where * indicates the site of attachment to one of the 1 to 5 subunits of the amino acid unit.

[0925] In some embodiments, for a 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 In some cases, D is

[0926] 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. In some cases, the cleavable peptide is selected from In some cases, the cleavable peptide is In some cases, the cleavable peptide is In some cases, the cleavable peptide is

[0927] In some embodiments, for the conjugate or drug-linker, the self-immolative group is selected from the group consisting of p-aminobenzyl alcohol self-immolative group (PABA) and p-amino-benzyloxycarbonyl self-immolative group. In some cases, the self-immolative group is selected from the group consisting of

[0928] In some embodiments, L2 is selected from In some cases, L2 is In some cases, L2 is In some cases, L2 is

[0929] In some embodiments, for a drug-linker, L1 is

[0930] In some embodiments, for a conjugate or drug-linker, 1 to 5 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, 1 to 5 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 1 subunit. In some cases, the Amino Acid unit has 2 subunits. In some cases, the Amino Acid unit has 3 subunits.

[0931] In some embodiments, for a conjugate or drug-linker, AA is selected from In some cases, AA is selected from In some cases, q is independently 1-16. 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.

[0932] In some embodiments, a conjugate is provided, wherein the conjugate comprises a drug-linker of formula (III*) and a binding unit. In some cases, the binding unit comprises a reactive substituent that reacts with the maleimide group of the drug-linker of formula (III*) to form a new covalent bond, thereby forming a conjugate.

[0933] In some embodiments, a conjugate is provided, wherein the conjugate comprises a drug-linker of formula (III) and a binding unit, wherein the drug-linker of formula (III) In some cases, the binding unit comprises a reactive substituent that reacts with the maleimide group of the drug-linker of formula (III) to form a new covalent bond, thereby forming a conjugate.

[0934] In some embodiments, a conjugate comprising a linker is provided, further comprising at least one drug unit connected to the linker subunit L2 to form a drug-linker. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the drug unit is selected from a cytotoxic agent, an immunomodulator, a nucleic acid, a growth inhibitory agent, a PROTAC, a toxin, a radioisotope, and a chelating ligand. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the drug unit is a cytotoxic agent. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is selected from auristatin, a maytansine, a camptothecin, a duocarmycin, and a calicheamicin. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is auristatin. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is MMAE or MMAF. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is camptothecin. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is exatecan or SN-38. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is exatecan (SS). In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is a diastereomer of exatecan, the RS form. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is calicheamicin. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is a maytansine. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the cytotoxic agent is a maytansine. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the maytansine is maytansine, maytansinol or ansamitocin-2.

[0935] In some embodiments, a conjugate comprising a drug-linker is provided, wherein the drug unit is an immunomodulator. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the immunomodulator is selected from a TRL7 agonist, a TLR8 agonist, a STING agonist, or a RIG-I agonist. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the immunomodulator is a TLR7 agonist. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the TLR7 agonist is an imidazoquinoline, an imidazoquinolineamine, a thiazoquinoline, an aminoquinoline, an aminoquinazoline, a pyrido[3,2-d]pyrimidine-2,4-diamine, a pyrimidine-2,4-diamine, a 2-aminoimidazole, a 1-alkyl-1H-benzimidazol-2-amine, a tetrahydropyridopyrimidine, a heteroaromatic thiadiazine-2,2-dioxide, a benzonaphthyridine, a guanosine analog, an adenosine analog, a thymidine homopolymer, ssRNA, CpG-A, PolyG10 or PolyG3. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the immunomodulator is a TLR8 agonist. In some embodiments, a conjugate comprising a drug-linker is provided, wherein the TLR8 agonist is selected from an imidazoquinoline, a thiazoquinoline, an aminoquinoline, an aminoquinazoline, a pyrido[3,2-d]pyrimidine-2,4-diamine, a pyrimidine-2,4-diamine, a 2-aminoimidazole, a 1-alkyl-1H-benzimidazol-2-amine, a tetrahydropyridopyrimidine, or an 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 conjugate comprising a drug-linker is provided, wherein the RIG-I agonist is selected from KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, and KIN2000.

[0936] In some embodiments, a drug-linker is provided wherein the drug unit is a chelating ligand. In some embodiments, a drug-linker is provided 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); radioactive isotopes 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.

[0937] In some embodiments, a conjugate is provided, comprising a binding unit connected to any of the drug-linkers described herein. In some embodiments, a conjugate is provided, wherein the binding unit is selected from an antibody or an antigen-binding portion thereof. In some embodiments, a conjugate is provided, 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. In some embodiments, a conjugate is provided, wherein the binding unit is monospecific. In some embodiments, a conjugate is provided, wherein the binding unit is divalent. In some embodiments, a conjugate is provided, wherein the binding unit is bispecific.

[0938] In some embodiments, a conjugate is provided wherein the average drug loading (p load ) is about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16.

[0939] In some embodiments, a conjugate is provided that is selected from the group consisting of:

[0940]

[0941]

[0942]

[0943]

[0944]

[0945]

[0946]

[0947]

[0948]

[0949]

[0950]

[0951]

[0952]

[0953]

[0954]

[0955]

[0956]

[0957]

[0958]

[0959]

[0960]

[0961]

[0962]

[0963]

[0964]

[0965]

[0966]

[0967]

[0968]

[0969] wherein each Z is attached at * and is independently selected from:

[0970]

[0971]

[0972] wherein each Z is attached at * and is independently selected from:

[0973]

[0974] or a stereoisomer thereof, wherein Ab represents a binding unit and n can be selected from p load , for example, where p load 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.

[0975] In some embodiments, a conjugate as described above is provided, wherein the binding unit is antibody 2E7 and the drug-linker is LD038. In a specific embodiment, the binding unit is antibody 2E7 (VH SEQ ID NO: 7 and VL SEQ ID NO: 8).

[0976] In some embodiments, the conjugate has the following structure:

[0977]

[0978] and wherein Ab is 2E7 and n can be selected from p load , for example, where p load 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.

[0979] In some embodiments, the conjugate has the following structure:

[0980]

[0981] and wherein Ab is 2E7 and n can be selected from p load , for example, where p load 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.

[0982] Exemplary Linker-Drug Combinations

[0983] In some embodiments, conjugates are provided wherein the drug unit (e.g., a tubulin disrupting agent, e.g., auristatin) is linked to a linker via a C-terminal carboxyl group that forms an amide bond with the linker subunit L2. In some embodiments, conjugates are provided wherein the linker comprises at least one amino acid.

[0984] In some embodiments, conjugates are provided wherein the linker comprises a Stretcher unit and / or an Amino Acid unit in addition to the Linker subunit L2.

[0985] In some embodiments, the extension unit can be connected to the binding unit, amino acid unit or linker subunit L2 through the sulfhydryl group of the binding unit. The sulfhydryl group can be generated by, for example, reducing the interchain disulfide bond of the binding unit. For example, the extension unit can be connected to the binding unit by the sulfur atom generated by the reduction of the interchain disulfide bond of the binding unit. In some embodiments, the extension unit is connected to the binding unit only by the sulfur atom generated by the reduction of the interchain disulfide bond of the binding unit. In some embodiments, the sulfhydryl group can be generated by reacting the amino group of the binding unit lysine portion with 2-iminothiolane (Traut reagent) or other sulfhydryl group generating reagents. In some embodiments, the binding unit is a recombinant antibody and is engineered to include one or more additional lysines. In some embodiments, the recombinant binding unit is engineered to include additional sulfhydryl groups, such as additional cysteine, such as engineered cysteine.

[0986] 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 of preparing antibody drug conjugates are described, for example, in U.S. Publication Nos. 2006 / 0074008 and 2009 / 0010945, each of which is incorporated herein by reference in its entirety.

[0987] Representative Stretcher units are described within the square brackets of Formulas IIIa and IIIb in US Patent No. 9,211,319, incorporated herein by reference.

[0988] In some embodiments, the CD70 conjugate comprises monomethyl auristatin E (MMAE) and a protease-cleavable linker. In some embodiments, the CD70 cleavage agent comprises exatecan and a protease-cleavable linker. It is contemplated that the protease-cleavable linker comprises a sulfhydryl-reactive spacer and a dipeptide. In various embodiments, the protease-cleavable linker comprises a sulfhydryl-reactive maleimidocaproyl spacer, a valine-citrulline (val-cit) dipeptide, and a p-aminobenzyloxycarbonyl or PAB spacer.

[0989] The abbreviation "PAB" refers to a self-immolative spacer:

[0990]

[0991] The abbreviation "MC" refers to the extending group maleimidocaproyl:

[0992]

[0993] In some embodiments, conjugates are provided wherein 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, DXdor, or a diastereomer of SN-38.

[0994] Representative structures are shown below.

[0995]

[0996] CPT:R1=R2=R3=H

[0997] 10-Hydroxy-CPT: R1=OH;R2=R3=H

[0998] CPT-11: R2=ethyl;R3=H

[0999] SN-38: R1=OH; R2=ethyl; R3=H

[1000] Topotecan: R1=OH; R2=H; R3=CH2—N(CH3)2

[1001]

[1002] Connection of drug-linker to binding unit

[1003] The techniques for connecting 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 connected to a drug unit (e.g., a cytotoxic agent) and the drug-linker is then connected to the binding unit. In some embodiments, conjugates are provided in which a linker is first connected to a binding unit and the drug unit is then connected to the linker. In the following discussion, the term "drug-linker" is used to illustrate the connection of a linker or drug-linker to a binding unit; one skilled in the art will understand that the selected connection method can be determined based on the linker and the drug unit (e.g., a cytotoxic agent or other drug unit). In some embodiments, conjugates are provided in which a drug unit is connected to a binding unit via a linker in a manner that reduces the activity of the drug unit until it is released from the conjugate (e.g., by hydrolysis, proteolytic degradation, or by a cleavage agent).

[1004] In general, conjugates can be prepared by several routes, using organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group of a Binding unit with a divalent linker reagent to form a Binding unit-linker intermediate via a covalent bond, which is then reacted 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 divalent linker reagent to form a drug-linker via a covalent bond, which is then reacted with a nucleophilic group of a Binding unit. Exemplary methods for preparing conjugates by the latter route are described in U.S. Patent No. 7,498,298, which is incorporated herein by reference.

[1005] Nucleophilic groups on binding units include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups, such as lysine, (iii) side chain sulfhydryl groups, such as cysteine, and (iv) sugar hydroxyl or amino groups when the antibody is glycosylated. Amine, sulfhydryl, and hydroxyl groups are all nucleophilic groups that can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, including: (i) active esters, such as NHS esters, HOBt esters, haloformates, and acyl halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyls, and maleimide groups. Certain binding units have reducible interchain disulfide bonds, i.e., cysteine bridges. Binding units can be rendered reactive for conjugation with linker reagents by treatment with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), resulting in complete or partial reduction of the binding unit. Thus, in theory, each cysteine bridge will form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into binding units by modifying lysine residues, for example, by reacting lysine residues with 2-iminothiolane (Traut's reagent) to convert amines into thiols. Reactive sulfhydryl groups can also be introduced into binding units by introducing one, two, three, four, or more cysteine residues (for example, by preparing binding units comprising one or more non-natural cysteine amino acid residues).

[1006] Conjugates can also be prepared by reaction between an electrophilic group (e.g., an aldehyde or ketone carbonyl) on a binding unit and a nucleophilic group on a linking reagent or a drug unit. Available nucleophilic groups on linker reagents include, but are not limited to, hydrazides, oximes, amino groups, hydrazines, thiosemicarbazones, carboxylic acid hydrazines, and arylhydrazides. In one embodiment, the binding unit is modified to introduce an electrophilic moiety capable of reacting with a nucleophilic substituent on a linker reagent or a drug unit. In another embodiment, the sugar of a glycosylated binding unit can be oxidized, for example, with a periodate oxidant, to form an aldehyde or ketone group, which can react with an amine group on a linker reagent or a drug unit moiety. The resulting imine Schiff base group can form a stable linkage or can be reduced, for example, by a borohydride reagent, to form a stable amine bond. In one embodiment, the carbohydrate portion of a glycosylated binding unit is reacted with galactose oxidase or sodium metaperiodate to generate carbonyl groups (aldehydes and ketones) in the binding unit, which can react with corresponding groups on the drug unit (see, for example, Hermanson, Bioconjugate Techniques). In another embodiment, a binding unit containing an N-terminal serine or threonine residue can be reacted with sodium metaperiodate to generate an aldehyde to replace the first amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3: 138-146; US 5362852). Such an aldehyde can react with a cytotoxic agent or a linker.

[1007] Exemplary nucleophilic groups on the Drug unit (e.g., cytotoxic agent) include, but are not limited to, amine, sulfhydryl, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arohydrazide groups capable of reacting 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, carboxyls, and maleimido groups.

[1008] In some embodiments, conjugates are provided wherein the drug-linker is attached to an interchain cysteine residue of a binding unit. See, for example, WO2004 / 010957 and WO2005 / 081711. In these embodiments, the linker typically comprises a maleimido group for attachment to the cysteine residue of the interchain disulfide bond. In some embodiments, conjugates are provided wherein the linker or drug-linker is attached to a cysteine residue of a binding unit as described in U.S. Patent Nos. 7,585,491 or 8,080,250.

[1009] In some embodiments, conjugates are provided wherein the linker or drug-linker is attached to a lysine or cysteine residue of the binding unit as described in WO2005 / 037992 or WO2010 / 141566.

[1010] In some embodiments, conjugates are provided wherein an engineered cysteine residue, a polyhistidine sequence, a glycoengineered tag, or a transglutaminase recognition sequence can be used to site-specifically attach a linker or drug linker to a binding unit.

[1011] In some embodiments, conjugates are provided wherein the drug-linker is attached to an engineered cysteine residue on an Fc residue other than an interchain disulfide bond. In some embodiments, conjugates are provided wherein the drug-linker is at position 118, 221, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 275, 276, 278, 280, 281, 283, 285, 286, 291, 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 are linked to an engineered cysteine introduced into an IgG (typically IgG1), and / or at positions 106, 108, 142 (light chain), 149 (light chain) and / or position V205 to the light chain, according to EU numbering as in Kabat. An exemplary substitution for site-specific conjugation using an engineered cysteine is S239C (see, e.g., US20100158909; the numbering of the Fc region is according to the EU index).

[1012] In some embodiments, conjugates are provided wherein a linker or drug linker is attached to one or more introduced cysteine residues of a binding unit as described in WO2006 / 034488, WO2011 / 156328 and / or WO2016040856.

[1013] In some embodiments, conjugates are provided wherein exemplary substitutions for site-specific conjugation using bacterial transglutaminase are N297S or N297Q of the Fc region. In some embodiments, conjugates are provided wherein a linker or drug-linker is attached to a glycan or modified glycan of a binding unit. See, e.g., WO2017 / 147542, WO2020 / 123425, WO2020 / 245229, WO2014 / 072482; WO2014 / 065661, WO2015 / 057066, and WO2016 / 022027; the disclosures of which are incorporated herein by reference.

[1014] Drug loading

[1015] Each binding unit of a CD70 ADC can contain one or more drug units. The number of drug units per binding unit is called the drug load. The drug load of a CD70 ADC is expressed as p load represents the average number of drug units (drug molecules (e.g., cytotoxic agents)) per binding unit (e.g., antibody or antigen-binding portion) in the CD70 ADC. For example, if p load is about 4, then taking into account all binding units (e.g., antibodies or antigen binding moieties or non-antibody scaffolds or non-antibody proteins) present in the composition, the average drug loading is about 4. In some embodiments, p load In some embodiments, p load Can be about 3, about 4, or about 5. In some embodiments, p load In some embodiments, p load Can be about 6, about 7, or about 8. In some embodiments, p load The range is from about 8 to about 16.

[1016] The average number of drug units per binding unit (e.g., antibody or antigen binding portion) in the formulation can be characterized by conventional methods, such as UV, mass spectrometry, capillary electrophoresis (CE), and HPLC. The concentration of the conjugate at p load In some cases, the p can be analyzed by methods such as reverse phase HPLC or hydrophobic interaction chromatography (HIC) HPLC. load Homogeneous conjugates of a specific value and conjugates with other drug loads were isolated, purified, and characterized.

[1017] pharmaceutical preparations

[1018] Other aspects relate to compositions comprising the CD70 conjugates described herein. In some embodiments, the compositions are pharmaceutical compositions. As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and a pharmaceutically acceptable carrier generally recognized in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[1019] The preparation of pharmacological compositions containing an active ingredient dissolved or dispersed therein is well understood in the art and is not necessarily limited based on any particular formulation. Typically, such compositions are prepared in the form of injectable liquid solutions or suspensions; however, solid forms suitable for rehydration or suspension in a liquid prior to use can also be prepared. The formulation can also be emulsified or presented as a liposomal composition. The CD70 conjugate can be mixed with a pharmaceutically acceptable excipient compatible with the active ingredient in an amount suitable for the treatment methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if necessary, the pharmaceutical composition may contain small amounts of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, and the like, which enhance or maintain the effectiveness of the active ingredient (e.g., CD70 conjugate). The pharmaceutical compositions described herein may include pharmaceutically acceptable salts of the components thereof. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the polypeptide) formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, tartaric acid, mandelic acid, and the like). Salts formed with free carboxyl groups may 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 to those skilled in the art. An exemplary liquid carrier is a sterile aqueous solution containing the active ingredient (e.g., a CD70 conjugate) and water, and may contain a buffer such as sodium phosphate, saline, or both at physiological pH, such as phosphate-buffered saline. Furthermore, aqueous carriers may 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 may also contain a liquid phase other than water or excluding water. Other exemplary liquid phases of this type are glycerin, vegetable oils (eg, cottonseed oil), and water-oil emulsions.The amount of active agent effective to treat a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques.

[1020] In some embodiments, a pharmaceutical composition comprising a CD70 conjugate described herein can be a lyophilisate.

[1021] In some embodiments, a syringe is provided that contains a therapeutically effective amount of a CD70 conjugate described herein or a pharmaceutical composition thereof.

[1022] Cancer treatment

[1023] In some embodiments, the CD70 conjugates described herein can be used in a method comprising administering a CD70 conjugate described herein to a subject in need thereof, eg, a subject having cancer.

[1024] In some embodiments, a method of treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have amino acid sequences selected from 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, a method of treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions have amino acid sequences selected from 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, a method for treating cancer is provided, comprising administering a CD70 conjugate 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 shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate 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 shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate 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 shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. In some embodiments, a method of treating cancer is provided, comprising administering a CD70 conjugate 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 shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[1025] In some embodiments, a method of treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions respectively have an amino acid sequence set forth in an amino acid sequence pair selected from 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; wherein the heavy chain 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 in the framework regions, and wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified.In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate 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 shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein the heavy chain 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 in the framework regions, and wherein the CDRs of the heavy chain or light chain variable regions are not modified.

[1026] In some embodiments, a method of treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions respectively have an amino acid sequence set forth in an amino acid sequence pair selected from 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; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified. In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having the amino acid sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable region are not modified.In some embodiments, a method for treating cancer is provided, comprising administering a CD70 conjugate 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 shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; wherein the heavy chain 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 in the framework regions, wherein the CDRs of the heavy chain or light chain variable regions are not modified. In some embodiments, a method of treating cancer is provided, comprising administering a CD70 conjugate comprising a heavy chain variable 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 any one of SEQ ID NOs: 3, 5, 7, 9, or 11. In some embodiments, a method of treating cancer is provided comprising administering a CD70 conjugate comprising 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%,...

Claims

1. A conjugate comprising: a Binding unit bound to one or more Drug units via 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 bound to one or more Drug units via one or more Linkers, wherein: (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 disposed in the heavy chain variable region framework region, and the VL region comprises LCDR1, LCDR2 and LCDR3 disposed in the light chain variable region framework region, and the VH and VL CDRs each have an amino acid sequence selected from the group consisting of: 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; 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; 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; 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; 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; (2) Each joint has the following formula (I): ~L1–(AA) s –L2≈ (I) or a salt thereof, wherein: L1 is a stretching unit covalently bound to the binding unit, wherein the wavy (~) line indicates the attachment site to 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 a Drug unit, wherein a double wavy (≈) line indicates an attachment site for the Drug unit; and wherein at least one polar unit is present in the Amino Acid unit, the Linker subunit, the Stretcher unit, or a combination thereof, and wherein the polar unit is selected from a sugar unit, a PEG unit, a carboxyl unit, and a combination thereof; and (3) Each Drug unit is covalently linked to each Linker subunit at (≈).

4. The conjugate of claim 3, wherein the VH and VL regions of the binding unit each have an amino acid sequence selected from the amino acid sequence pair shown in the group consisting of: 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.

5. The conjugate of claim 3, wherein the VH and VL regions of the binding unit each have an amino acid sequence selected from the amino acid sequence pair shown in the group consisting of: 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, wherein the heavy and light chain framework regions are optionally modified with 1 to 8 amino acid substitutions, deletions or insertions in the framework regions.

6. The conjugate of any one of claims 3 to 5, wherein the HCDR1, HCDR2, and HCDR3 and the LCDR1, LCDR2, and LCDR3 of the binding unit 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 region of the binding unit is a human framework region.

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. 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 has a heavy chain variable region further comprising a heavy chain constant region.

11. The conjugate of claim 10, wherein the heavy chain constant region of the binding unit is of the IgG isotype. The conjugate of claim 11 , wherein the heavy chain constant region of the binding unit is an IgG1 constant region. The conjugate of claim 10 , wherein the heavy chain constant region of the binding unit is an IgG4 constant region. The conjugate of claim 12 , wherein the IgG1 constant region of the binding unit has the amino acid sequence of SEQ ID NO:

28. The conjugate of any one of claims 3 to 14 , wherein the binding unit has a light chain variable region further comprising a light chain constant region.

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

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

29.

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 divalent.

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

22. The conjugate of any one of claims 3 to 21, wherein the sugar unit of the linker has the formula: or a salt thereof, wherein: 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; and L3a is selected from C1-C 10 Alkylene and polyethylene glycol having 1 to 24 ethylene glycol subunits; p and o are independently 0 to 2; and Each * and each # indicates a site of attachment to another subunit of the Amino Acid Unit (AA), a Linker Subunit (L2), or a Stretcher Unit (L1).

23. The conjugate of any one of claims 3 to 22, wherein the PEG unit of the linker has a formula selected from: (a) ~R 20 -R 21 -[O-CH2-CH2] n20 -R 22 -NR 24 R 25 (XX) or a salt thereof, wherein: R 20 A functional group that is a part of the subunit, the stretcher unit and / or the linker subunit L2 for connection to the Amino Acid unit; R 21 and R 22 Each independently is, optionally C1-C3 alkylene; R 24 and R 25 Each independently selected from H; polyhydroxy group; substituted polyhydroxy group; -C(O)-polyhydroxy group; substituted -C(O)-polyhydroxy 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; -C(O)-R 28 , where R 28 is a sugar unit of formula (XII) or (XIII); or -NR 24 R 25 Together they form a C3-C8 heterocycle; The condition is R 24 and R 25 Not all are H; The wavy line (~) indicates the same as R 20 Attachment site; and n20 is 1 to 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 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2; R 21 and R 22 Each independently is, optionally C1-C3 alkylene; R 24 and R 25 One selected from polyhydroxy groups; substituted polyhydroxy groups; -C(O)-polyhydroxy groups; substituted -C(O)-polyhydroxy groups; 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; -C(O)-R 28 , where R 28 is a sugar unit of formula (XII) or (XIII); and R 24 and R 25 The other is polyethylene glycol, optionally having 1 to 24 ethylene glycol subunits; The wavy line (~) indicates the same as R 20 Attachment site; and n20 is 1 to 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 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2; R 26 and R 27 Each is optional and independently selected from 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)- and -C(O)-C1-C 12 Alkylene-NH-; R 24 and R 25 One selected from H; polyhydroxy group; substituted polyhydroxy group; -C (O) - polyhydroxy group; substituted -C (O) - polyhydroxy 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; -C(O)-R 28 , where R 28 is a sugar unit of formula (XII) or (XIII); and R 24 and R 25 The other is selected from H; polyhydroxy group; substituted polyhydroxy group; -C (O) -polyhydroxy group; substituted -C (O) -polyhydroxy 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; -C(O)-R 28 , where R 28 is a saccharide unit of formula (XII) or (XIII); and polyethylene glycol, optionally having 1 to 24 ethylene glycol subunits; or -NR 24 R 25 Together they form a C3-C8 heterocycle; The condition is R 24 and R 25 Not all are 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 (~) indicates the same as R 20 The attachment site; n20 is 1 to 26; n21 is 1 to 4; and n27 is 1 to 4.

24. The conjugate of claim 23, wherein R 24 and R 25 are each independently selected from H and polyhydroxy groups, provided that R 24 and R 25 Not all are H.

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

26. The conjugate of claim 25, wherein: 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 ketonic acid; or The amino sugar is selected from the group consisting of glucosamine, N-acetyl glucosamine, galactosamine and N-acetyl galactosamine.

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

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

29. The conjugate of claim 23, wherein R 24 and R 25 Independently selected from linear monosaccharides and substituted linear monosaccharides, wherein the substituted linear monosaccharides are substituted with monosaccharides, disaccharides or polysaccharides.

30. The conjugate of claim 23, wherein R 24 and R 25 Independently selected from linear monosaccharides and substituted monosaccharides, wherein the substituted linear monosaccharides are substituted with one or more substituents selected from the group consisting of alkyl, O-alkyl, aryl, O-aryl, carboxyl, ester, or amide, and are optionally further substituted with a monosaccharide, disaccharide, or polysaccharide.

31. The conjugate of claim 23, wherein R 24 and R 25 One of them is a -C(O)-polyhydroxy group or a substituted -C(O)-polyhydroxy group, and R 24 and R 25 The other is H, -C(O)-polyhydroxy group, substituted -C(O)-polyhydroxy group, polyhydroxy group or substituted polyhydroxy group; wherein the substituted -C(O)-polyhydroxy group and polyhydroxy group are substituted with monosaccharide, disaccharide, polysaccharide, alkyl, -O-alkyl, aryl, carboxyl, ester, or amide.

32. The conjugate of claim 23, wherein R 24 and R 25 independently selected from H, substituted-C1-C8 alkyl, substituted-C1-C4 alkyl or substituted-C1-C3 alkyl; provided that R 24 and R 25 Not all are H; wherein the substituted -C1-C8 alkyl, -C1-C4 alkyl and -C1-C3 alkyl are substituted with hydroxyl and / or carboxyl; provided that R 24 and R 25 Not all are H.

33. The conjugate of claim 23, wherein R 24 and R 25 One of the following 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 The other of the group 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 groups; provided that R 24 and R 25 Not all are H.

34. The conjugate of any one of claims 24 to 31, wherein each monosaccharide is independently selected from: C5 or C6 sugars selected from the group consisting of 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 the group consisting of gluconic acid, aldonic acid, uronic acid, and ketonic acid; or An amino sugar selected from the group consisting of glucosamine, N-acetylglucosamine, galactosamine, and N-acetylgalactosamine.

35. The conjugate of any one of claims 23 to 31, wherein R 20 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

36. The conjugate of claim 23, wherein R 20 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

37. The conjugate of any one of claims 3 to 22, comprising a PEG unit having a formula selected from the group consisting of: ~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, wherein: R 40 A functional group that is part of a subunit for linking to an Amino Acid unit, a Stretcher unit and / or a Linker subunit L2; R 41 and R 42 Not present or each independently represents a C1-C6 alkylene group; Each R 43 Independently absent or selected from 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 them is H or C1-C 12 Alkylene and the other is C1-C 12 alkylene; R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates; provided that R 44 and R 45 Not all are H; The wavy line (~) indicates the same as R 40 The attachment site; n40 is 1 to 26; n41 is 1 to 6; and n42 is 1 to 6.

38. The conjugate of any one of claims 3 to 22, comprising a PEG unit having a formula selected from the group consisting of: ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLI) or a salt thereof, wherein: R 40 A functional group that is a part of the subunit, the stretcher unit and / or the linker subunit L2 for connection to the Amino Acid unit; R 41 and R 42 Not present or each independently represents a C1-C6 alkylene group; R 43 Not present or selected from 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 them is H or C1-C 12 Alkylene and the other is C1-C 12 alkylene; R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates; provided that R 44 and R 45 Not all are H; The wavy line (~) indicates the same as R 40 The attachment site; n40 is 1 to 26; n41 is 1 to 6; and n42 is 1 to 6.

39. The conjugate of any one of claims 3 to 22, comprising a PEG unit having a formula selected from the group consisting of: ~R 40 -(R 41 -[O-CH2-CH2] n40 -R 42 -R 43 -(NR 44 R 45 ) n41 ) n42 (XLII) or a salt thereof, wherein: R 40 A functional group that is a part of the subunit, the stretcher unit and / or the linker subunit L2 for connection to the Amino Acid unit; R 41 and R 42 Not present or each independently represents a C1-C3 alkylene group; R 43 Not present or selected from 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 them is H or C1-C6 alkylene and the other is C1-C 12 alkylene; R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates; provided that R 44 and R 45 Not all are H; The wavy line (~) indicates the same as R 40 The attachment site; n40 is 1 to 26; n41 is 1 to 4; and n42 is 1 to 4.

40. The conjugate of any one of claims 37 to 39, wherein R 40 is selected from halogen, aldehyde, carboxyl, amino, alkynyl, azido, hydroxyl, carbonyl, carbamate, thiol, urea, thiocarbamate, thiourea, sulfonamide, acylsulfonamide, alkylsulfonate, triazole, azadibenzocyclooctyne, hydrazine, carbonylalkylheteroaryl, or a protected form thereof.

41. The conjugate of any one of claims 37 to 40, wherein R 20 or R 40 Has one of the following structures: Where R=H or C 1-6 alkyl; and n = 0 to 12 or a stereoisomer thereof, wherein the (*) indicates R 20 or R 40 The attachment site to the subunit of the amino acid unit, the stretcher unit and / or a portion of the linker subunit L2 and the Indicator R 20 or R 40 The site of attachment to the remainder of the PEG unit.

42. The conjugate of claim 41, wherein R 20 or R 40 Has one of the following structures: Where n = 0 to 12 or a stereoisomer thereof, wherein the (*) indicates R 20 or R 40 The attachment site to the subunit of the amino acid unit, the stretcher unit and / or a portion of the linker subunit L2 and the Indicator R 20 or R 40 The site of attachment to the remainder of the PEG unit.

43. The conjugate of any one of claims 37 to 42, wherein R 43 -(NR 44 R 45 ) n41 , when NR 43 When present, has one of the following structures: Where R=H、C 1-6 Alkyl, polyhydroxy or substituted polyhydroxy or a stereoisomer thereof, wherein said Indicator R 43 The site of attachment to the remainder of the PEG unit.

44. The conjugate of claim 43, wherein R 43 -(NR 44 R 45 ) n41 , when NR 43 When present, has one of the following structures: or a stereoisomer thereof, wherein said Indicator R 43 The site of attachment to the remainder of the PEG unit.

45. The conjugate of any one of claims 37 to 44, wherein -NR 44 R 45 Has one of the following structures: or a stereoisomer thereof, wherein said Instructions-NR 44 R 45 The site of attachment to the remainder of the PEG unit.

46. The conjugate of any one of claims 3 to 22, comprising a PEG unit having a formula selected from the group consisting of: ~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, wherein: R 40 is a functional group for connecting 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 Not present or each independently represents a C1-C6 alkylene group; Each R 43 Independently absent or selected from 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 them is H or C1-C 12 Alkylene and the other is C1-C 12 alkylene; R 44 and R 45 are each independently H, a polyhydroxy group, a substituted polyhydroxy group, a -C(O)-polyhydroxy group, or a substituted -C(O)-polyhydroxy group, wherein the optional substituents are selected from sulfates, phosphates, alkyl sulfates, and alkyl phosphates; provided that R 44 and R 45 Not all are H; R 46 Selected from amino, amino-alkyl-amino or -NH-C(O)-NH-S(O)2-NH-; The wavy line (~) indicates the same as R 40 The attachment site; n40 is 1 to 26; n41 is 1 to 6; and n42 is 1 to 6.

47. The conjugate of any one of claims 3 to 22, comprising a PEG unit having a formula selected from the group consisting of: or a stereoisomer or salt thereof, wherein: Each Y is independently R 76 or 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, they form an oxo group; Each q is independently 1-26; Each m is independently 1 to 4; each n is independently 1 to 4; each v is independently 1 to 6; and Each * indicates a site of attachment to the subunit of the Amino Acid unit (AA), the Linker subunit L2 or the Stretcher unit (L1).

48. The conjugate of claim 47, wherein the PEG unit has a formula selected from: or a stereoisomer or salt thereof, wherein: Each R 76 are independently H, acetyl, -P(=O)(OH)2 or -(CH2) v S(=O)2(OH); Each q is independently 1-26; Each m is independently 1 to 4; each n is independently 1 to 4; each v is independently 1 to 6; and Each * indicates a site of attachment to the subunit of the Amino Acid unit (AA), the Linker subunit L2 or the Stretcher unit (L1).

49. The conjugate of claim 47 or 48, wherein the PEG unit has a formula selected from: or a stereoisomer or salt thereof, wherein: Each q is independently 1-26; Each m is independently 1 to 4; each n is independently 1 to 4; and Each * indicates a site of attachment to a subunit of the Amino Acid Unit (AA), a Linker Subunit L2, or a Stretcher Unit (L1).

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

51. The conjugate of claim 47, wherein Y is 52. The conjugate of claim 47, wherein each R a and R b are independently H.

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

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

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

56. The conjugate of any one of claims 3 to 22, comprising a carboxyl unit having the formula: or a salt thereof, wherein: (a) L 70 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 for NR 71 (R 72 -R 73 ), where R 71 Selected from 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, and R 73 is carboxyl or polycarboxyl, wherein the polycarboxyl group comprises 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino and / or amide groups; Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and p1 and o1 are each independently selected from 0 to 2; or (b) L 70 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 for NR 71 (R 75 -(R 73 )2), where R 71 Selected from 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, a 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 group comprises 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amide groups; Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and p1 and o1 are each independently selected from 0 to 2; or (c) L 70 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 is ~N(R 74 -R 73 )(R 72 - R 73 ), where R 72 and R 74 are each independently selected from optionally substituted C1-C3 alkylene, optionally substituted ether, optionally substituted thioether, optionally substituted ketone, optionally substituted amide, polyethylene glycol (optionally having 1 to 12 ethylene glycol subunits), optionally substituted carbocycle, optionally substituted aryl or optionally substituted heteroaryl, and each R 73 is independently carboxyl or polycarboxyl, comprising 1 to 10, or 1 to 6, or 1 to 4 carboxyl groups, wherein the carboxyl groups are interconnected through alkyl, alkylene, substituted alkyl, substituted alkylene, heteroalkyl, heteroalkylene, amino, and / or amide groups; Each wavy line (~) indicates a site of attachment to another subunit of the Amino Acid Unit (AA), the Linker Subunit L2, or the Stretcher Unit (L1); and p1 and o1 are each independently selected from 0 to 2.

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

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

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

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

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

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

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

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

65. The conjugate of any one of claims 3 to 64, wherein L2 or AA-L2 has one of the following structures, or a stereoisomer thereof: Wherein the wavy line above the amino group indicates the site of attachment to the Stretcher unit or Amino Acid unit and the Drug unit is attached to benzyl alcohol.

66. The conjugate of any one of claims 3 to 63, wherein the linker comprises ~AA-L2~ having a formula selected from the group consisting of: ~[SU–aa]-L2≈, ~[aa1(PEG)–aa]-L2≈, or ~[CU–aa]-L2≈ Wherein square brackets indicate the Amino Acid unit, each aa is an optional subunit of AA, L2 is the Linker subunit, each wavy line (~) indicates the attachment site to the Extender unit; aa1(PEG) is a PEG unit attached to the Amino Acid subunit of AA, SU is a sugar unit attached to a subunit of AA or L2, and CU is a Carboxyl unit attached to a subunit of AA or L2; and the double wavy (≈) line indicates the attachment site to the Drug unit, wherein aa and aa1 are independently selected from α, β and γ amino acids and derivatives thereof.

67. The conjugate of any one of claims 3 to 63, wherein the linker comprises ~AA-L2~ having a formula selected from the group consisting of: wherein square brackets indicate the amino acid unit, each aa is an amino acid subunit of AA, L2 is a linker subunit connected to the side chain of aa, a wavy line (~) indicates the attachment site to the stretcher unit; aa1(PEG) is a PEG unit connected to aa, SU is a sugar unit connected to aa, CU is a carboxyl unit connected to aa, and a double wavy (≈) line indicates the attachment site to the drug unit; wherein aa and aa1 are independently selected from α, β and γ amino acids and derivatives thereof.

68. The conjugate of any one of claims 3 to 63, wherein the Amino Acid unit comprises at least two Polar units.

69. The conjugate of claim 68, wherein the linker comprises ~AA-L2~ having a formula selected from the group consisting of: ~[SU–aa–SU]–L2≈, ~[aa1(PEG)–aa–aa2(PEG)]–L2≈, or ~[CU–aa–CU]–L2≈ wherein square brackets indicate an amino acid unit, aa is an optional subunit of AA, L2 is a linker subunit, and a wavy line (-) indicates the site of attachment to the stretcher unit; aa1(PEG) and aa2(PEG) are each a PEG unit connected to aa or another PEG unit; each SU is a sugar unit connected to aa or another sugar unit, each CU is a carboxyl unit connected to aa or another carboxyl unit, and a double wavy (≈) line indicates the site of attachment to the drug unit; wherein aa, aa1, and aa2 are independently selected from α, β, and γ amino acids and derivatives thereof.

70. The conjugate of claim 68, wherein the linker comprises ~AA-L2~ having a formula selected from the group consisting of: wherein square brackets indicate the amino acid unit, aa is the amino acid subunit of AA, L2 is the linker subunit connected to the side chain of aa, each wavy line (~) indicates the attachment site to the stretcher unit; aa1(PEG) and aa2(PEG) are each a PEG unit connected to aa, each SU is a sugar unit connected to aa; each CU is a carboxyl unit connected to aa; and the double wavy (≈) line indicates the attachment site to the drug unit; wherein aa, aa1 and aa2 are each independently selected from α, β and γ amino acids and derivatives thereof.

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

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

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

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

76. The conjugate of claim 75, wherein the cleavable peptide comprises a SU-valine-citrulline peptide, a SU-valine-lysine peptide, a SU-valine-alanine peptide, a SU-phenylalanine-lysine peptide, or a SU-glycine-glycine-phenylalanine-glycine peptide.

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

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

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)-Phe-Lysine peptide, a Phe-Lys(PEG) peptide, or a Lys(PEG)-Gly-Gly-Phe-Gly peptide, wherein Lys(PEG) and Cit(PEG) comprise a PEG unit attached to a lysine residue or a citrulline residue, respectively.

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

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

83. The conjugate of any one of claims 3 to 82, wherein the Amino Acid unit is bonded to the Linker subunit L2 via a non-peptide linker.

84. The conjugate of claim 83, wherein the non-peptide linker is selected from the group consisting of C1-C 10 Alkylene, C2-C 10 Alkenylene, C2-C 10 Alkynylidene or polyethylene glycol.

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

86. The conjugate of claim 85, wherein the Stretcher unit is selected from the group consisting of: where R 17 -C1-C 10 Alkylene-, -C1-C 10 Heteroalkylene-, -C3-C8 carbocyclyl-, -O-(C1-C8 alkylene)-, -(CH2-O-CH2) b -C1-C8 alkylene-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-(wherein b is 1 to 26), -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-, -C3-C8 heterocycle-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, C1-C 10 Heteroalkylene-C(=O)-, -C1-C8alkylene-(CH2-O-CH2) b -C(=O)-(wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C3-C8 carbocyclyl-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclyl)-C(=O)-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocycle-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-C(=O)-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10 Heteroalkylene-NH-, -C1-C8alkylene-(CH2-O-CH2) b -NH- (wherein b is 1 to 26), -(CH2-O-CH2) b -C1-C8 alkylene-NH-(wherein b is 1 to 26), -C1-C8 alkylene-(CH2-O-CH2) b -C1-C8 alkylene-NH-(wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-(C(=O))-NH-(CH2-O-CH2) b -C1-C8 alkylene-C(=O)-(wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -NH-(wherein b is 1 to 26), -C1-C8 alkylene-NH-(C(=O))-(CH2-O-CH2) b -C1-C8 alkylene-NH- (wherein b is 1 to 26), -C3-C8 carbocyclyl-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 carbocyclyl)-NH-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-NH-, -C3-C8 heterocycle-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-NH-, -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, C1-C 10 Heteroalkylene-S-, -C3-C8 carbocyclyl-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 carbocyclyl)-S-, -(C3-C8 carbocyclyl)-C1-C 10 Alkylene-S-, -C3-C8 heterocycle-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclyl)-S- or -(C3-C8 heterocyclyl)-C1-C 10 Alkylene-S-; or The extending unit includes a maleimide group (C1-C 10 Alkylene-C(O)-, maleimide (CH2OCH2) p2 (C1-C 10 Alkylene) C(O)-, maleimide (C1-C 10 Alkylene)(CH2OCH2) p2 C(O)-, or a ring-opened form thereof, wherein p2 is 1 to 26.

87. The conjugate of claim 85, wherein the Stretcher unit is selected from the group consisting of: The wavy line The attachment site of the Stretcher unit to the Amino Acid unit or Linker subunit L2 is indicated, and the attachment site to the Binding unit is on a maleimide, primary amine, or alkyne functional group.

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

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

90. The conjugate of any one of claims 3 to 89, wherein the average drug loading (p load ) is about 1 to about 8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 3 to about 5, about 6 to about 8, or about 8 to about 16.

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

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

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

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

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

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

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

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

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

100. The conjugate of claim 99, wherein the maytansinoid is maytansine, maytansinol, or a maytansine analog in the form of DM1, DM3, and DM4, and ansamitocin-2.

101. The conjugate of claim 88, wherein the Drug unit is an immunomodulatory agent.

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

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

104. The conjugate of claim 103, wherein the TLR7 agonist is an imidazoquinoline, an imidazoquinolineamine, a thiazoquinoline, an aminoquinoline, an aminoquinazoline, a pyrido[3,2-d]pyrimidine-2,4-diamine, a pyrimidine-2,4-diamine, a 2-aminoimidazole, a 1-alkyl-1H-benzimidazol-2-amine, a tetrahydropyridopyrimidine, a heteroaromatic thiadiazine-2,2-dioxide, a benzonaphthyridine, a guanosine analog, an adenosine analog, a thymidine homopolymer, ssRNA, CpG-A, PolyG10, or PolyG3.

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

106. The conjugate of claim 105, wherein the TLR8 agonist is selected from an imidazoquinoline, a thiazoquinoline, an aminoquinoline, an aminoquinazoline, a pyrido[3,2-d]pyrimidine-2,4-diamine, a pyrimidine-2,4-diamine, a 2-aminoimidazole, a 1-alkyl-1H-benzimidazol-2-amine, a tetrahydropyridopyrimidine, or an ssRNA.

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

108. 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 the group consisting of KIN1148, SB-9200, KIN700, KIN600, KIN500, KIN100, KIN101, KIN400, and KIN2000.

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

111. The conjugate 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); radioactive isotopes 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 conjugate of claim 71, wherein the linker comprises mc-VC-PAB, CL2, CL2A, or (succinimidyl-3-yl-N)-(CH2) n -C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-, wherein n=1 to 5.

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

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

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

116. The conjugate of claim 112, wherein the linker comprises (succinimidyl-3-yl-N)-(CH2) n -C(=O)-Gly-Gly-Phe-Gly-NH-CH2-O-CH2-(C=O)-.

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

118. The conjugate of any one of claims 3 to 21, selected from the group consisting of: wherein each Z is attached at * and is independently selected from: wherein each Z is attached at * and is independently selected from: or a stereoisomer thereof, wherein 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.

119. The conjugate of claim 3, wherein the conjugate has the structure: and wherein Ab is 2E7 and n is p load , where p load 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.

120. The conjugate of claim 3, wherein the conjugate has the structure: and wherein Ab is 2E7 and n is p load , where p load 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.

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

122. A method of treating a CD70+ cancer, 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

140. The method of claim 139, wherein the 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 to 140, wherein the subject's treatment outcome is improved.

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

143. The method of claim 141, wherein the improved therapeutic outcome is a reduction in tumor burden.

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

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

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

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

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

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

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

152. The method of claim 151, wherein the improved therapeutic outcome is slowing disease progression or lessening disease severity.

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

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