Nuclear localization polypeptides and conjugates and uses thereof

Abstract

Disclosed herein are nuclear localization polypeptides and conjugates comprising the same. Exemplary conjugates include antibody-drug conjugates, comprising an antibody or an antibody fragment conjugated to a therapeutic payload via a linker comprising a nuclear localization polypeptide. The conjugates provided herein may be useful for treating a disease or disorder, such as cancer.

Description

NUCLEAR LOCALIZATION POLYPEPTIDES AND CONJUGATES AND USES THEREOFCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 492,125 filed March 24, 2023, which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 21, 2024, is named 65657-703_601_SL.xml and is 56,962 bytes in size.FIELD OF THE INVENTION

[0003] Provided herein are nuclear localization sequence (NLS) polypeptides, and conjugates comprising the same, as well as compositions comprising the same for use in delivering a payload to the interior (e.g., nucleus) of a cell. Also provided herein are methods of using the NLS polypeptide precursor molecules and conjugates provided herein, for the manufacture of a medicament for treating diseases or disorders such as cancer. In certain embodiments, an NLS-linked payload is retained intracellularly, thereby enhancing the therapeutic window of the NLS-linked payload relative to unconjugated payload.BACKGROUND OF THE INVENTION

[0004] Antibody-drug conjugates (ADCs) are targeting technologies that can direct a therapeutic payload to a cell type of interest by virtue of linking it to a targeting antibody which binds to a cellspecific target antigen (e.g., a tumor-associated antigen). Many antibodies are currently not suitable for delivering payloads to cancer cells, because they are not efficiently internalized upon binding and / or are not trafficked to lysosomal compartments, where the payload can be released. Antibody-drug conjugates (ADCs) are often trapped in endosomes or subsequently pumped extracellularly or degraded in the endosomal lysosomal pathway, leading to a reduction in intracellular accumulation.SUMMARY OF THE INVENTION

[0005] In one aspect, provided herein is a composition comprising a nuclear localization sequence (NLS), for example, an antibody -drug conjugate (ADC) comprising an NLS. A NLS is an amino acid sequence that labels a protein tor import into the nucleus of a cell, e.g., by nuclear transport. A NLS can be recognized by a corresponding nuclear transporter, which can interact with nucleoporins to help NLS-containing proteins reach the nucleus through the nuclear pore complex (NPC). This NLS- dependent protein recognition, a process necessary for cargo proteins to pass the nuclear envelope through the nuclear pore complex (NPC), is facilitated by members of the importin superfamily. Due to the complex roles of nuclear proteins, NLS-mediated nuclear transport is a highly regulated process.

[0006] In one aspect, provided herein is a nuclear localization sequence (e.g., a polypeptide) which, after being internalized within a cell, is transported into the cell’s nucleus. In some embodiments, the NLS polypeptide is conjugated (e.g., covalently bonded to) a therapeutic payload which, by virtue of being conjugated to the NLS polypeptide, is transported into the nucleus of the cell. In some embodiments, provided herein is a conjugate comprising an NLS polypeptide, and a therapeutic payload bound covalently thereto, which may be used for delivering the therapeutic payload to a nuclear target. In some embodiments, the therapeutic payload is released from the polypeptide (e.g., inside the target cell, or inside the target cell nucleus). In some embodiments, the therapeutic payload is stably bound to the polypeptide such that the therapeutic payload produces a desired (e.g., therapeutic) effect without release from the polypeptide. In some embodiments, the therapeutic payload is stably bound to the polypeptide, thereby reducing unwanted release, which can lead to off-target toxicity and side-effects that limit some antibody-drug conjugates’ effectiveness in the clinic.

[0007] In some embodiments, a therapeutic payload is transported intracellularly (e.g., into a cell’s nucleus). In some embodiments, a therapeutic payload is retained intracellularly (e.g., within a cell’s nucleus). In some embodiments, a therapeutic payload stably conjugated to a NLS polypeptide (e.g., a polypeptide (PP) disclosed herein) accumulates within a cell’s nucleus, thereby enriching the local concentration of the payload within the nucleus. In certain embodiments, a therapeutic payload conjugated to an NLS (e.g., a peptide disclosed herein) has a wider therapeutic window than the therapeutic payload alone, by virtue of the conjugated payload being enriched within the nucleus of a cell (e.g., a target cell such as a tumor cell).

[0008] In some embodiments, an NLS polypeptide (PP) is conjugated to an antibody, or an antigen - binding fragment thereof. In some embodiments, the antibody is a non-binding antibody. In some embodiments, the antibody modulates (e.g., enhances) one or more pharmacokinetic properties of a conjugate relative to an antibody -free conjugate (e.g., half-life, metabolism, volume of distribution, etc.). In some embodiments, the antibody is an antigen -binding antibody. For example, an antigen may be a tumor-associated antigen (i.e., a protein that is over-expressed (2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, or more) in a tumor ceil compared to a non-tumor cell). In some embodiments, the antibody is internalized within a target cell, following binding of the antibody to the target antigen.

[0009] In some embodiments, provided herein is an antibody-drug conjugate comprising a NLS polypeptide conjugated to a monoclonal antibody and a therapeutic payload. Preferably, the antibodydrug conjugate is internalized within a cell, following binding of the antibody to a target antigen (e.g., a tumor antigen). In some embodiments, the antibody is processed intracellularly. In some embodiments, the antibody-drug conjugate releases a polypeptide-payload conjugate upon being processed intracellularly, wherein the polypeptide-payload conjugate has therapeutic activity (i.e., on a nuclear target). In some embodiments, the therapeutic payload is toxic to the cell, e.g., resulting in cell death, or inhibition of, or reversal of growth. In some embodiments, the therapeutic payload is a cytotoxic payload. In some embodiments, the antibody-drug conjugate (comprising a NLS polypeptide linker) increases the therapeutic window of a cytotoxic payload, due to the antibody-mediated tumor-specific internalizationof the conjugate, in combination with the nuclear transport and retention of the cytotoxic payload mediated by the NLS polypeptide conjugated thereto.

[0010] In some embodiments, provided herein is a conjugate comprising an antibody or an antigenbinding fragment and a therapeutic payload or a detectable group, conjugated via a linker, wherein the linker comprises a polypeptide, and wherein the polypeptide comprises a nuclear localization sequence.

[0011] In some embodiments, the conjugate is of the formula:AB-[(LI)m-PP-(L2)„-DG]Pwherein:AB is the antibody or antigen-binding fragment;LI is a linker;PP is the polypeptide;L2 is a linker;DG is the detectable group; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0012] In some embodiments, the conjugate is of Formula I:AB-[(Ll)m-PP-(L2)n-TP]p Formula I wherein:AB is the antibody or antigen-binding fragment;LI is a linker;PP is the polypeptide;L2 is a linker;TP is the therapeutic payload; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0013] In some embodiments, the polypeptide comprises the sequence:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0014] In some embodiments, x1is G or A; x2is G or A; x3is A or V; x4is L or I; x5is G or A; and a is 0 or 1. In some embodiments, the polypeptide comprises a sequence selected from SEQ ID NOs. 1-8: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG. In some embodiments, the polypeptide comprises SEQ ID NO 3: PAAKRVKLD. In some embodiments, the polypeptide comprises SEQ ID NO4: PAAKRVKLDG. In some embodiments, the detectable group is a dye. In some embodiments, the therapeutic payload is a cytotoxic or cytostatic agent. In some embodiments, the antibody or antigenbinding fragment thereof binds to a target antigen. In some embodiments, the target antigen is CD 19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, or tissue factor. In some embodiments, the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, belantamab, loncastuximab, tisotumab, mirvetuximab, or a biosimilar thereof. Also provided herein is a pharmaceutical composition comprising a conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0015] Also provided herein is a method of delivering a therapeutic payload to an intracellular target within a tumor cell, wherein the therapeutic payload is conjugated to a polypeptide comprising a nuclear localization sequence. In some embodiments, the polypeptide comprises the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0016] In some embodiments, the polypeptide comprises a sequence selected from SEQ ID Nos. 1-8: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG. In some embodiments, the therapeutic payload is cytotoxic.

[0017] Also provided herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a conjugate disclosed herein. In some embodiments, the disease or disorder is a cancer. In some embodiments, the cancer is a hematological cancer or a solid tumor.

[0018] Also provided herein is a compound or a pharmaceutically acceptable salt thereof having the structure of Formula V, Formula VI, or Formula VIEAC-(Ll)m-PP-(L2)„-TP Formula VAC-(Ll)m-PP Formula VIAC-(Ll)m-PP-(L2)n-DG Formula VII wherein:AC is an antibody-coupling group configured to form a covalent bond with a sulfur or nitrogen atom of an amino acid side-chain of the antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide comprising the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein:x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1 L2 is a linker;TP is a therapeutic payload;DG is a detectable group; m is 0 or 1 ; and n is 0 or 1.

[0019] In some embodiments, x1is G or A; x2is G or A; x3is A or V; and x4is L or I. In some embodiments, x5is G or A. In some embodiments, x5is absent. In some embodiments, the polypeptide comprises any one of SEQ ID Nos. 1-8: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG. In some embodiments, the detectable group is a dye. In some embodiments, the detectable group is an Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, HiLyte™ Fluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte™ Fluor 555, Alexa Fluor® 561, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, HiLyte™1Fluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, or Alexa Fluor® 750, HiLyte™ Fluor 750, or a functional equivalent thereof. In some embodiments, the therapeutic payload is an antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA-alkylating drug, a DNA-binding drug, a DNA-cleaving drug, or an RNA polymerase inhibitor.

[0020] Also considered within the scope of the invention is the use of the compound according to Formula V, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament. Also provided herein is the use of the compound according to Formula V, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating cancer.

[0021] In one aspect, provided herein is a composition (e.g., medicament or pharmaceutical composition, or a kit for preparing the same) comprising a NLS polypeptide, wherein the NLS polypeptide comprises the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0022] In some embodiments, the composition comprises a polypeptide having the sequence SEQ ID NO 11:P-x1-x2-K-R-x3-K-x4-D SEQ ID NO 11 wherein: x1is G or A; x2is G or A; x3is A or V; and x4is L or I.

[0023] In some embodiments, the composition comprises a polypeptide having the sequence SEQ ID NO 12:P-x'-x2-K-R-x3-K-x4-D-x5SEQ ID NO 12 wherein: x1is G or A; x2is G or A; x3is A or V; x4is L or I; and x5is G or A.

[0024] In some embodiments, the polypeptide comprises the sequence:P-x1-x2-K-R-V-K-L-D-(G)aSEQ ID NO 13 wherein: x1is G or A; x2is G or A; and a is 0 or 1.

[0025] In some embodiments, the polypeptide comprises a sequence selected from SEQ ID NOs: 1-8: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG. In some embodiments, the polypeptide comprises SEQ ID NO 1: PGGKRVKLD. In some embodiments, the polypeptide comprises SEQ ID NO 2: PGGKRVKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 3: PAAKRVKLD. In some embodiments, the polypeptide comprises SEQ ID NO 4: PAAKRVKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 5: PAGKRVKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 6: PGAKRVKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 7: PAAKRAKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 8: PAAKRVKIDG.

[0026] Also provided herein, is aNLS polypeptide comprising the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0027] In some embodiments, the NLS polypeptide is further conjugated to a therapeutic payload and / or an antibody or antigen-binding fragment thereof, as provided herein.

[0028] In another aspect, provided herein is an antibody-drug conjugate of Formula I:AB-[(Ll)m-PP-(L2)n-TP]p Formula I wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a NLS polypeptide;L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0029] In some embodiments, the antibody-drug conjugate is of Formula I- 10:AB-[(Ll)m-PP-(L2)n-TP]p Formula I- 10 wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide comprising SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein x1, x2,x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1;L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0030] In some embodiments, x1is G or A; x2is G or A; x3is A or V; and x4is L or I. In some embodiments, x5is G or A. In some embodiments, x5is absent. In some embodiments, the polypeptide (PP) comprises a sequence selected from SEQ ID NOs: 1-8.

[0031] In some embodiments, TP comprises a duocarmycin, auristatin, maytansinoid, uncialamycin, dynemicin, thailanstatin, camptothecin, exatecan, or tubulysin compound. In some embodiments, TP comprises an antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA-alkylating drug, a DNA-binding drug, a DNA-cleaving drug, or an RNA polymerase inhibitor. In some embodiments, the therapeutic payload (TP) comprises lurbinectedin, trabectedin, safracin, lenalidomide, eribulin, a vinca alkaloid (e.g., vincristine, vinblastine, vindesine, or vinorelbine); an epothilone, a taxane (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), a cryptophycin, a hemiasterlin, an anthracyclin, a bisnaphthylamide (e.g., elinafide), or a cytotoxic molecular glue / PROTAC compound.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof, is a non-binding antibody (e.g., a non-binding IgGl antibody). In some embodiments, the antibody or antigen-binding fragment thereof binds to a target antigen. In some embodiments, the target antigen is expressed on a tumor cell. In some embodiments, the target antigen is CD19, CD22, CD30, CD33, CD79b, HER2,TR0P2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, tissue factor. In some embodiments, the target antigen is AXL, BCMA, CA9, CCR7, CD123, CD166, CD19, CD20, CD205, CD22, CD25, CD276, CD30, CD33, CD37, CD46, CD70, CD74, CD79b, CEACAM5, CLDN18.2, CLDN6, CXCR4, DLL3, EFNA4, EGFR, EGFRvIII, ENPP3, EPCAM, EPHA2, F3, FOLR1, GPC3, GPNMB, GUCY2C, HER2, HER3, IGF1R, LI CAM, LIV1, LRRC15, MET, MSLN, MUC1, MUC16, NCAM1, NECTIN4, PSMA, PTK7, R0R1, R0R2, SDC1, SLC1A5, SLC34A2, TFRC, TIM1, TPBG, or TROP2. In some embodiments, the target antigen corresponds to a gene that is TNFRSF17, ALCAM, MS4A1, LY75, B7H3, TNFRSF8, CD142, SLC39A6, CD138, CD71, or HAVCR1.

[0033] In some embodiments, provided herein is a conjugate according to Formula I, wherein the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, belantamab, loncastuximab, tisotumab, mirvetuximab, or a biosimilar thereof.

[0034] In another aspect, provided herein is a pharmaceutical composition comprising an antibody-drug conjugate of Formula I, and a pharmaceutically acceptable excipient. In some embodiments, the antibody-drug conjugate is of Formula I- 10. In some embodiments, PP of Formula I is selected from SEQ ID NO 1-8.

[0035] In still another aspect, provided herein is a method of delivering a therapeutic payload to an intracellular target within a tumor cell, the method comprising contacting the tumor cell with a conjugate disclosed herein. In some embodiments, the conjugate is an antibody -drug conjugate of Formula I (e.g., Formula I- 10). In some embodiments, the polypeptide (PP) comprises a sequence selected from SEQ ID NOs: 1-8. In some embodiments, the therapeutic payload is cytotoxic. In some embodiments, the therapeutic payload is internalized into a nucleus of the tumor cell. In some embodiments, the intracellular target is within the nucleus of the tumor cell.

[0036] In another aspect, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibodydrug conjugate according to Formula I.AB-[(Ll)m-PP-(L2)n-TP]p Formula I wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a NLS polypeptide;L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0037] In some embodiments, the polypeptide (PP) comprises SEQ ID NO 10. In some embodiments, PP comprises SEQ ID NO 11 and / or SEQ ID NO 12. In some embodiments, the method comprisesadministering a therapeutically effective amount of an antibody -drug conjugate of Formula I, wherein PP comprises one or more of SEQ ID NO 1-8. In some embodiments, the disease or disorder is a cancer. In some embodiments, the disease or disorder is a hematological cancer. In some embodiments, the disease or disorder comprises a solid tumor. In some embodiments, the disease or disorder is a cancer associated with overexpression of CD 19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, tissue factor.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0039] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0040] FIG. 1 depicts relative luminescence units in SKBR3 (breast cancer adenocarcinoma) cells following administration of Example ADCs 12 and 13, wherein each is conjugated to either an IgGl antibody or anti-HER2 antibody (trastuzumab).

[0041] FIG. 2 depicts relative luminescence units in HCC1954 (epithelial breast cancer / ductal carcinoma) cells following administration of Example ADCs 12 and 13, wherein each is conjugated to either an IgGl antibody or anti-HER2 antibody (trastuzumab).

[0042] FIG. 3 depicts relative luminescence units in human neutrophils following administration of Example ADCs 12 and 13, wherein each is conjugated to either an IgGl antibody or anti-HER2 antibody (trastuzumab).

[0043] FIG. 4 depicts relative luminescence units in human megakaryocytes following administration of Example ADCs 12 and 13, wherein each is conjugated to either an IgGl antibody or anti-HER2 antibody (trastuzumab).

[0044] FIG. 5 depicts images from 2h, 8h, 24h, and 48h increments following administration of an antibody labeled with green dye (A, top four images), or an antibody with the green label and a red dye (B, bottom four images), wherein the red dye is conjugated via an NLS linker.DETAILED DESCRIPTION OF THE INVENTION

[0045] Described herein are nuclear localization peptides and compositions comprising nuclear localization peptides. Exemplary compositions comprise antibody-drug conjugates comprising a nuclear localization peptide connected to an antibody or antibody fragment, e.g., optionally via one or more linker moieties. In some cases, the antibody or antibody fragment has been modified to reduce or eliminate effector function. In some cases, the antibody drug conjugate comprises a therapeutic payload, for instance, that is effective in killing a cancer cell. Non-limiting example antibodies and therapeutic payloads are provided herein.

[0046] Various antibody drug conjugates described herein selectively target and kill cancer cells as compared to non-cancer cells. Various drug conjugates described herein comprise therapeutic payloads (e.g., cytotoxic moieties) that are delivered to cancer cells expressing an antigen to which the antibody drug conjugate specifically binds. In some cases, the drug conjugates herein have an improved therapeutic window as compared to drug conjugates lacking a nuclear localization signal.

[0047] Antibody drug conjugates comprising a nuclear localization peptide may be used to treat a disease or condition in a subject in need thereof. Generally, a method of treatment comprises administering to the subject a composition (e.g., a pharmaceutical composition) comprising a conjugate (e.g., an antibody-drug conjugate) disclosed herein. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate according to Formula I (e.g., I- 10), or an antibody- polypeptide conjugate of Formula (II), or a polypeptide-drug conjugate of Formula (III), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0048] In various instances, connection of an antibody or antibody fragment to a cytotoxic moiety via a nuclear localization signal (NLS) as described herein has an effect of increasing the therapeutic window or otherwise improving specific delivery of the cytotoxic moiety to a cancer cells.

[0049] Before the present methods and compositions are described, it is to be understood that this disclosure is not limited to a particular method or composition described. The terminology used is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. Examples are put forth so as to provide those of ordinary skill in the art with a disclosure and description of how to make and use the present compositions and methods, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.Definitions

[0050] Unless otherwise stated, the following terms used in this application have the definitions given below.

[0051] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof. As used herein, the term “about” a number may refer to that number plus or minus 15% of that number. The term “about” a range may refer to that range minus 15% of its lowest value and plus 15% of its greatest value. The use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0052] The term “and / or” as used in a phrase with a list of members is intended to include all members individually and all combination of full or partial list of members. For example, a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0053] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0054] An “alkyl” group generally refers to an aliphatic hydrocarbon group. The alkyl group may be branched or a straight chain. In some embodiments, the “alkyl” group has 1 to 80 carbon atoms, i.e., a Ci-Csoalkyl. In some embodiments, the “alkyl” group has 1 to 12 carbon atoms, i.e., a Ci-Cnalkyl. Whenever it appears herein, a numerical range such as “1 to 12” refers to each integer in the given range; e.g., “1 to 12 carbon atoms” means that the alkyl group consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 12 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated. In some embodiments, an alkyl is a Ci-Ce alkyl. In one aspect the alkyl is methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, or t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyl, neopentyl, or hexyl.

[0055] The term “heteroalkyl” generally refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from an atom other than carbon, e.g. , oxygen, nitrogen (e.g., NH, N-alkyl, N- oxide), sulfur (e.g., S, SO, SO2), or combinations thereof. A heteroalkyl is generally attached to the rest of the molecule at a carbon atom of the heteroalkyl. In one aspect, a heteroalkyl is a Ci-Ce heteroalkyl. Heteroalkyl may include nitriles, amides, esters, ethers, amines, thioethers, thioesters, carbamates, carbonates, polyethers, polyamines, and the like. In some embodiments, a heteroalkyl group comprises one or more units or groups such as -CH2CH2O-, -CH2CH2NH-, -CH2CH2NHC(O)-, -CH2CH2C(O)NH-, - NHCH2CH2NH-, and the like.

[0056] A "conjugate" is herein defined as a compound wherein one binding molecule (e.g., an antigen binding molecule) is covalently connected to a payload via a linker. In some embodiments, a conjugate comprises a binding molecule and one or more payloads. In some embodiments, the binding molecule is a small molecule (<1000 Da), a polypeptide (comprising 2 to about 20 amino acids), a protein (e.g., an antibody), or a protein fragment (e.g., an antigen -binding portion of a protein). In some embodiments, the binding molecule is an antibody or an antigen -binding fragment thereof.

[0057] As used herein, the term “target protein” generally refers to a protein that is expressed on the surface of a cell (e.g., a cancer cell), which can efficiently bind a small molecule binder. As used herein, the term “target antigen” generally refers to an antigen that is expressed on the surface of a cell (e.g., a cancer cell), which can efficiently bind an antibody or antigen-binding fragment thereof. Efficiently isused herein to generally refer to a compound with micromolar potency or better (e.g., sub -micromolar, nanomolar, sub-nanomolar, etc. as used in the art).

[0058] In accordance with the present invention, the term “antibody” is to be understood in its broadest meaning and comprises immunoglobulin molecules, for example intact or modified monoclonal antibodies, polyclonal antibodies or multispecific antibodies (e.g., bispecific antibodies). An antibody preferably comprises a molecule having four peptide chains, two heavy chains (H chains) and two light chains (L chains) which are typically linked by disulfide bridges. Each heavy chain comprises a variable domain of the heavy chain (abbreviated VH) and a constant domain of the heavy chain. The constant domain of the heavy chain may, for example, comprise three domains CHI, CH2 and CH3. Each light chain comprises a variable domain (abbreviated VL) and a constant domain. The constant domain of the light chain comprises a domain (abbreviated CL). The VH and VL domains may be subdivided further into regions having hypervariability, also referred to as complementarity determining regions (abbreviated CDR) and regions having low sequence variability (framework region, abbreviated FR). Typically, each VH and VL region is composed of three CDRs and up to four FRs. For example, from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibody may be obtained from any suitable species, e.g., rabbit, llama, camel, mouse or rat. In one embodiment, the antibody is of human or murine origin. An antibody may, for example, be human, humanized or chimeric.

[0059] The terms “specifically bind to,” “specific binding,” and analogous terms when used in the context of one molecule binding to the other, means that one molecule binds to the other molecule with significantly higher affinity than to any cross-reactive antigen or off-target antigen (together as non-target antigen) as determined using experimental techniques, such as Surface Plasmon Resonance (SPR), fluorescence activated cell sorting (FACS) analysis, Kinetic Exclusion Assay (KinExA), isothermal titration calorimetry (ITC), radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs). Typically, a specific or selective reaction will be at least twice non-target signal or noise of non-target binding and may be more than 10 times non-target binding. An antibody or antigen binding fragment which binds a target of interest is one that binds the target with sufficient affinity such that the antibody or antigen binding fragment is useful as a therapeutic agent in targeting a cell or tissue expressing the target, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody or antigen binding fragment to a “non-target” protein will be less than about 10% of the binding of the antibody or antigen binding fragment to its particular target protein. Specific binding of an antibody typically describes an antibody having an affinity of at least 10-7M (as Kd value; i.e. preferably those with Kd values smaller than 10-7M), with the antibody having an at least two times higher affinity for the predetermined antigen / target molecule than for a non-specific antigen / target molecule (e.g. bovine serum albumin, or casein) which is not the predetermined antigen / target molecule or a closely related antigen / target molecule. Specific binding of an antibody or binder does not exclude the antibody binding to a plurality of antigens / target molecules (e.g., orthologs of different species). Specific binding can be measured, for example, by determining binding of a moleculecompared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. As such, the term “specific binding,” “specifically binds to,” “specifically inhibits,” “specifically blocks” or “is specific for” a particular target as used herein refers to binding, blocking, or inhibition where a molecule binds to, blocks, or inhibits a particular target without substantially binding to or inhibiting a non-target.

[0060] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0061] “Pharmaceutically acceptable,” as used herein, refers a material, such as a salt, a solvate, a carrier, or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material is administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0062] The term “pharmaceutically acceptable salt” refers to a form of a therapeutically active agent that consists of a cationic form of the therapeutically active agent in combination with a suitable anion, or in alternative embodiments, an anionic form of the therapeutically active agent in combination with a suitable cation. In some embodiments, pharmaceutically acceptable salts are obtained by reacting a compound described herein with a base. In such situations, an acidic proton of the compound described herein is replaced by a metal ion, e.g., a lithium, sodium, potassium, magnesium ion. In some embodiments, pharmaceutically acceptable salts are obtained by reacting a compound described herein with an acid. In some embodiments, a compound described herein is prepared as a chloride salt, sulfate salt, bromide salt, mesylate salt, maleate salt, citrate salt or phosphate salt.

[0063] The term “active agent” generally refers to any small molecule payload (<2000 Da) that can be conjugated to a linker or polypeptide (e.g., via an amide bond, a disulfide bond, a thiophosphate bond) and produce an effect from the interior of a cell. In some embodiments, the interior of the cell refers to the nucleus of the cell. An active agent may be a therapeutic payload (TP), indicating it produces a physiological effect on the cell it enters. In some embodiments, the therapeutic payload is cytotoxic or otherwise damaging to a cell, or binds to a target in the interior (nucleus) of the cell. In some embodiments, a therapeutic payload is an agent that stops the proliferation, differentiation, growth,angiogenesis, or other aberrant activity such as is found in cancer cells, or the cells of other hyperproliferative disorders. Examples of therapeutic agents are provided throughout the present disclosure. An active agent may also be a non-therapeutic payload. Example of non-therapeutic payloads include, for example, detectable groups (DG).

[0064] In some embodiments, the active agent (e.g., detectable group) is a dye. In some embodiments, the active agent emits a detectable signature (e.g., radioactivity). In some embodiments, the active agent is a gamma emitter, beta emitter, or alpha emitter. In some embodiments, the active agent responds to a stimulus (e.g., a light, a laser, or other energy source) with an emitted response (e.g., fluorescence, phosphorescence, or other detectable light). In some embodiments, the active agent is a donor or acceptor of a FRET pair, or a BRET pair. In some embodiments, the active agent is a chromophore, fluorescent labels, biolumine scent label, or chemiluminescent label. In some embodiments, the active agent is an Alexa Fluor dye, or other functional equivalent (e.g., a coumarin, rhodamine, or cyanine dye). In some embodiments, the active agent is an Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, HiLyte™ Fluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte™ Fluor 555, Alexa Fluor® 561, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, HiLyte™ Fluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, or HiLyte™ Fluor 750.

[0065] In another embodiment, the compounds described herein are labeled isotopically (e.g., with a radioisotope) or by another other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0066] As used herein, a polypeptide (e.g., PP) generally refers to a peptide comprising at least two, and preferably no more than twenty, amino acids (also known as an oligopeptide). Unless otherwise stated, the term “polypeptide” as used herein is differentiated from a protein (e.g., an antibody), which may comprise hundreds or thousands of amino acids. In some embodiments, a polypeptide (PP) provided herein comprises six, seven, eight, nine, ten, eleven, or twelve amino acids. In some embodiments, a polypeptide (PP) comprises nine or ten amino acids. Unless otherwise specified, a polypeptide defined as having “x” amino acids is not adjacent to another peptide or amino acid.

[0067] As used herein, the term “non-cleavable linker” refers to a linking unit of atoms (e.g., 1 to 200 atoms selected from C, H, N, O, S, and halogen) that is not known to be chemically or biologically unstable. The term “non-cleavable linker” is intended to differentiate from cleavable linkers (e.g., protease -cleavable linkers, self-immolative linkers, pH-sensitive linkers, etc.). A non-cleavable linker may be an alkyl or heteroalkyl linker, optionally interrupted by one or more cyclyl or heterocyclyl groups (e.g., click partners or artifacts therefrom). A non-cleavable linker may comprise a polymeric section (e.g., PEG), an alkyl section (e.g., C1-12 alkyl), and / or a heteroalkyl section (e.g., a C1-12 alkyl group wherein one or more carbon atoms are replaced or substituted by one or more heteroatoms selected from N, O, S, and P).

[0068] A "self-immolative group" is herein defined as a part of a linker in an antibody-drug conjugate with a function to conditionally release free drug at the site targeted by the ligand unit. The activatable self-immolative moiety comprises an activatable group (AG) and a self-immolative spacer unit. Self-immolative elimination occurs when a molecular system undergoes a spontaneous and irreversible disassembly into its constituent fragments through either an electronic cascade, via an elimination pathway or, alternatively, where self-immolation is achieved through a cyclisation-elimination event. Upon activation of the activatable group, for example by enzymatic conversion of an amide group to an amino group or by reduction of a disulfide to a free thiol group, a self-immolative reaction sequence is initiated that leads to release of free drug by one or more of various mechanisms, which may involve (temporary) 1,6-elimination of a p-aminobenzyl group to a p- quinone methide, optionally with release of carbon dioxide and / or followed by a second cyclization release mechanism. The self-immolative assembly unit can part of the chemical spacer connecting the antibody and the payload (via the functional group). Alternatively, the self-immolative group is not an inherent part of the chemical spacer, but branches off from the chemical spacer connecting the antibody and the payload.

[0069] As used herein, the term “therapeutic payload” generally refers to a chemical group, generally a small molecule group, having utility in treating or diagnosing a disease or disorder. For example, a therapeutic payload may comprise a drug, a detectable group (e.g., a dye, a radiolabel, a contrast agent), a radioisotope (e.g., technetium-99, iodine-131), a radiopharmaceutical, a chromophore, etc. A therapeutic payload may have multiple functions. For example, a radioisotope attached to a conjugate can undergo decay and produce specific amounts of radiation that can be used to diagnose or treat a disease or disorder. In some embodiments, a therapeutic payload is or comprises a drug. In some embodiments, the therapeutic payload is a cytotoxic or cytostatic compound. In some embodiments, the therapeutic payload is effective in killing or slowing the growth of cancer cells. In some embodiments, the therapeutic payload is a chemotherapeutic agent. In some embodiments, the therapeutic payload is a cytotoxic or cytostatic agent. In some embodiments, the therapeutic payload is a camptothecin compound or a derivative thereof. Specific examples of therapeutic payloads used in accordance with the invention are provided throughout the present disclosure.

[0070] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA™); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan)(HYCAMTIN®, CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall and anthracy clines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher such as folinic acid (leucovorin); aceglatone; anti-folate anti-neoplastic agents such as ALIMTA®, LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate, anti-metabolites such as 5 -fluorouracil (5-FU) and its prodrugs such as UFT, S-l and capecitabine, and thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX', TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2 ''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone -Poulenc Rorer, Antony, France); chloranbucil; gemcitabine)(GEMZAR®) ; 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine)(VELBAN®; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine)(ONCOVIN®; vinca alkaloid; vinorelbine)(NAVELBINE®; novantrone; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

[0071] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0072] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0073] The terms “therapeutically effective amount” or “effective amount,” as used herein, refer to a sufficient amount of an agent or a compound being administered, which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result includes reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses is the amount of the composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate “effective” amount in any individual case is optionally determined using techniques, such as a dose escalation study. The terms “therapeutically effective amount” or “effective amount” also refer to the amount of a compound that is sufficient to elicit the biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.

[0074] The term “therapeutic window” as used herein generally refers to the range of concentrations of an agent that provides safe, effective therapy with minimal adverse effects. The therapeutic window is generally between the minimum effective dose, and the dose at which toxicity or adverse side-effectsdevelop. Drugs with a narrow therapeutic window have a therapeutically effective amount that is similar to the toxic amount (i.e., wherein toxicity is observed). In some embodiments, a drug has a wide therapeutic window, which enables a broader range of effective doses to be evaluated without unacceptable toxicity. In some embodiments, a targeted drug (e.g., a conjugate) has a therapeutic window that is wider than a non-targeted drug, as the targeting agent increases the local concentration of the drug at a desired location or tissue type without producing unwanted exposure to healthy tissues, cells, or cellular compartments. One measurement of therapeutic window is the “therapeutic index”, which is the ratio between the LD50 (lethal dose in 50% of treated subjects) and the ED50 (effective dose in 50% of treated subjects). In some embodiments, a conjugate provided herein has a therapeutic window or a therapeutic index that is greater than that of a non -conjugate.

[0075] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Nuclear Localization Polypeptides

[0076] Intracellular protein delivery is a powerful tool for development of protein-based therapeutics and is a challenging task due to cell membrane impermeability to large biomolecules and endosomal entrapment. Most delivery strategies rely on endosomal uptake of the carrier. “Endosomal entrapment” occurs when a protein delivery vehicles enter into the cells via endocytosis but then the cargo becomes entrapped in the endosomes. Endosomally entrapped cargo generally exhibit low escape efficiency leading to eventual degradation. Endosomally entrapped proteins may eventually be degraded by proteases in the acidic endolysosomal compartments without being able to access the cytosol. This sequestration and degradation can limit the efficacy of protein delivery systems that are internalized into the cells by endocytic pathways. Access of delivered proteins to the cytosol is critical for intracellular activity, either directly for activity or as a gateway to the nucleus or subcellular organelles. Provided herein are solutions to the problem of delivering a protein (e.g., an antibody, or a conjugate thereof) to the nucleus of a cell via its conjugation to a NLS polypeptide.

[0077] In one aspect, provided herein are polypeptides having a sequence disclosed herein, which can function as a nuclear localization signal, i.e., a recognition motif that promotes internalization of the motif, and conjugates thereof, into a cell’s nucleus. Polypeptides disclosed herein may be useful in promoting the intracellular (i.e., nuclear) delivery of a second agent, which can be conjugated to the polypeptide. A second agent can include, by way of non -limiting example, a therapeutic payload, an antibody or antigen-binding antibody fragment, a detectable agent, a diagnostic, and the like.

[0078] In some embodiments, nuclear localization signals (NLS) are short peptides that act as a signal fragment that mediates the transport of proteins from the cytoplasm into the nucleus. In someembodiments, the nuclear localization signal or sequence (NLS) comprises one or more short sequences of positively charged lysines or arginines exposed on the protein surface. In some embodiments, the nuclear localization signals (NLS) are classical nuclear localization signals (cNLS). In some embodiments, the nuclear localization signals (NLS) are non-classical nuclear localization signals (ncNLS). In some embodiments, the nuclear localization signals (NLS) are other types of nuclear localization signals. In some embodiments, the NLS is a novel nuclear localization signal.

[0079] In some embodiments, provided herein is a nuclear localization signal (NLS) polypeptide conjugated to an antibody. In some embodiments, the NLS polypeptide is conjugated to a therapeutic payload (e.g., a cytotoxic agent). In some embodiments, the NLS polypeptide is conjugated to both an antibody, or an antigen-binding antibody fragment, and a therapeutic payload. In some embodiments, the NLS polypeptide connects the therapeutic payload and the antibody or antigen-binding fragment. In some embodiments, provided herein is an antibody-drug conjugate comprising a NLS polypeptide connected to an antibody or antigen binding fragment at one end, and connected to a therapeutic payload at the opposite end (of the polypeptide). In some embodiments, the NLS polypeptide connects the therapeutic payload and the antibody or antigen-binding fragment (i.e., in an antibody drug conjugate), such that the therapeutic payload or conjugate thereof can escape the endosomal-lysosomal pathway and reroute to the nucleus, thereby increasing its intracellular accumulation with high target cell selectivity. In some embodiments, the antibody drug conjugates accumulate intracellularly while retaining target cell selectivity and evading off-target effects due to unwanted release or localization of the payload. In some embodiments, the polypeptide is a linear polypeptide comprising an N- and C-terminus. In some embodiments, provided herein is an antibody-drug conjugate comprising an antibody and a therapeutic payload, wherein the antibody and therapeutic payload are connected via a linker, and wherein the linker comprises an NLS polypeptide (i.e., a polypeptide disclosed herein).

[0080] In some embodiments, the NLS polypeptide comprises a sequence according to SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0081] In some embodiments, x1is a non-polar amino acid. In some embodiments, x1is a flexible amino acid. In some embodiments, x1is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x1is an uncharged amino acid. In some embodiments, x1is G or A. In some embodiments, x1is G. In some embodiments, x1is A. In some embodiments, x2is a non-polar amino acid. In some embodiments, x2is a flexible amino acid. In some embodiments, x2is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x2is an uncharged amino acid. In certain embodiments, x2is G or A. In certain embodiments, x2is G. In certain embodiments, x2is A. In some embodiments, x3is a non-polar amino acid. In some embodiments, x3is a flexible amino acid. In some embodiments, x3is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x3is an uncharged amino acid. In some embodiments, x3is A, L, I, or V. In someembodiments, x3is A or V. In some embodiments, x3is A. In some embodiments, x3is V. In some embodiments, x4is a non-polar amino acid. In some embodiments, x4is a flexible amino acid. In some embodiments, x4is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x4is an uncharged amino acid. In some embodiments, x4is A, L, I, or V. In certain embodiments, x4is L or I. In certain embodiments, x4is L. In certain embodiments, x4is I. In some embodiments, x5is G or A. In some embodiments, x5is G. In some embodiments, x5is A. In some embodiments, a is 0 or 1. In some embodiments, a is 0. In some embodiments, a is 1.

[0082] In some embodiments, the polypeptide comprises SEQ ID NO 11:P-x1-x2-K-R-x3-K-x4-D SEQ ID NO 11

[0083] In some embodiments, x1is G or A. In some embodiments, x1is G. In some embodiments, x1is A. In certain embodiments, x2is G or A. In certain embodiments, x2is G. In certain embodiments, x2is A. In some embodiments, x3is A or V. In some embodiments, x3is A. In some embodiments, x3is V. In certain embodiments, x4is L or I. In certain embodiments, x4is L. In certain embodiments, x4is I.

[0084] In some embodiments, the polypeptide comprises SEQ ID NO 12:P-x'-x2-I<-R-x -I<-x4-D-x SEQ ID NO 12

[0085] In some embodiments, x1is G or A. In some embodiments, x1is G. In some embodiments, x1is A. In certain embodiments, x2is G or A. In certain embodiments, x2is G. In certain embodiments, x2is A. In some embodiments, x3is A or V. In some embodiments, x3is A. In some embodiments, x3is V. In certain embodiments, x4is L or I. In certain embodiments, x4is L. In certain embodiments, x4is I. In some embodiments, x5is G or A. In some embodiments, x5is G. In some embodiments, x5is A.

[0086] In some embodiments, the polypeptide comprises SEQ ID NO 13:P-x1-x2-K-R-V-K-L-D-(G)aSEQ ID NO 13

[0087] In some embodiments, x1and x2are each independently A or G. In some embodiments, x1and x2are each A. In some embodiments, x1and x2are each G. In some embodiments, x1is A and x2is G. In some embodiments, x1is G and x2is A. In some embodiments, a is 0 or 1. In some embodiments, a is 0. In some embodiments, a is 1.

[0088] In some embodiments, the polypeptide comprises a sequence selected from SEQ ID NOs: 1-8. In some embodiments, the polypeptide comprises SEQ ID NO 1: PGGKRVKLD. In some embodiments, the polypeptide comprises SEQ ID NO 2: PGGKRVKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 3: PAAKRVKLD. In some embodiments, the polypeptide SEQ ID NO 4: PAAKRVKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 5: PAGKRVKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 6: PGAKRVKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 7: PAAKRAKLDG. In some embodiments, the polypeptide comprises SEQ ID NO 8: PAAKRVKIDG.Conjugates

[0089] Also provided herein is a conjugate of aNLS polypeptide disclosed herein. Conjugates of the present disclosure include, but are not limited to, an antibody-polypeptide conjugate, a polypeptide -drug conjugate, and antibody -drug conjugate (wherein the antibody and drug are linked via the NLS polypeptide), as well as any metabolites or precursor molecules thereof. In some embodiments, the conjugate is an antibody-polypeptide conjugate. In some embodiments, the conjugate is an antibody- polypeptide conjugate, wherein the antibody is conjugated to the polypeptide via a proline residue of the polypeptide. In some embodiments, the polypeptide is linked to the antibody via a linker, wherein the linker forms a bond between the proline of the polypeptide, and a lysine or cysteine residue of the antibody. Also considered within the scope of the invention are conjugates comprising an NLS polypeptide herein (e.g., of Formula I) and a non-antibody binder (e.g., a peptide binder, a small molecule binder, and the like).

[0090] In some embodiments, the antibody or antigen-binding fragment is conjugated to the polypeptide via a bond. In some embodiments, the antibody or antigen-binding fragment is conjugated to the polypeptide via a linker. In some embodiments, the antibody or antigen-binding fragment is conjugated to P (Proline) of the polypeptide. In some embodiments, the linker comprises one or more polyethylene glycol or polyethyleneimine groups. In some embodiments, the linker comprises 1 to 12 polyethylene glycol groups. In some embodiments, the linker comprises 2 to 8 polyethylene glycol groups. In some embodiments, the linker comprises 3 to 6 polyethylene glycol groups.

[0091] In some embodiments, the linker comprises a cleavable group. In some embodiments, the linker is a chemically-labile group. In some embodiments, the linker is a pH-sensitive group. In some embodiments, the linker is a protease-cleavable group. In some embodiments, the linker is a self- immolative group. In some embodiments, the linker is a hydrolysable group. In some embodiments, the cleavable linker is a protease-cleavable group (e.g., a peptide). In some embodiments, the cleavable group is the peptide Val-Cit or Gly-Gly-Phe-Gly.

[0092] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker comprises a non-cleavable heteroalkyl and / or heterocyclic group. In some embodiments, the linker comprises a non-cleavable heteroalkyl and heterocyclic group. In some embodiments, the linker comprises a non-cleavable heteroalkyl group. In some embodiments, the linker comprises a non- cleavable heterocyclic group. In some embodiments, the linker comprises 1 to 12 amino acids. In certain embodiments, the antibody or antigen-binding fragment comprises a natural or unnatural amino acid, and wherein the linker is conjugated to the antibody or antigen-binding fragment via the natural or unnatural amino acid. In some embodiments, the antibody or antigen-binding fragment comprises a thiol or an amine. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid comprising a thiol side-chain or an amine side-chain. In some embodiments, the antibody or antigenbinding fragment comprises a cysteine, a lysine, or a glutamine. In some embodiments, the linker is conjugated to the antibody or antigen-binding fragment via the cysteine, lysine, or glutamine. In someembodiments, the antibody or antigen-binding fragment comprises a cysteine. In some embodiments, the linker is conjugated to the antibody or antigen -binding fragment via the cysteine.

[0093] In some embodiments, provided herein is a conjugate having a structure of Formula I:AB-[(Ll)m-PP-(L2)n-TP]p Formula I or a pharmaceutically acceptable salt thereof, wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a NLS polypeptide;L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1; and p is 1-20.

[0094] In another aspect, provided herein is a conjugate of the formula:AB-[(Ll)m-PP-(L2)„-DG]Por a pharmaceutically acceptable salt thereof, wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a NLS polypeptide;L2 is a linker;DG is a detectable group (e.g., a coumarin dye, a rhodamine dye, or a cyanine dye); m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0095] In some embodiments, PP is a polypeptide comprising (e.g., consisting of) SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 or a pharmaceutically acceptable salt thereof, wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0096] In some embodiments, a conjugate comprises a structure of Formula 1-10:AB-[(Ll)m-PP-(L2)n-TP]p Formula I- 10 or a pharmaceutically acceptable salt thereof, wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide comprising SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5} SEQ ID NO 10wherein x1, x2,x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1;L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1; and p is 1-20.

[0097] In some embodiments, x1of Formula 1-10 is a non-polar amino acid. In some embodiments, x1of Formula I is a flexible amino acid. In some embodiments, x1of Formula I is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x1is an uncharged amino acid. In some embodiments, x1is G or A.

[0098] In some embodiments, x2of Formula 1-10 is a non-polar amino acid. In some embodiments, x2of Formula I is a flexible amino acid. In some embodiments, x2of Formula I is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x2is an uncharged amino acid. In some embodiments, x2is G or A.

[0099] In some embodiments, x3of Formula 1-10 is a non-polar amino acid. In some embodiments, x3is a flexible amino acid. In some embodiments, x3is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x3is an uncharged amino acid. In some embodiments, x3is A, L, I, or V. In some embodiments, x3is A or V.

[0100] In some embodiments, x4of Formula 1-10 is a non-polar amino acid. In some embodiments, x4is a flexible amino acid. In some embodiments, x4is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x4is an uncharged amino acid. In some embodiments, x4is A, L, I, or V. In some embodiments, x4is L or I.

[0101] In some embodiments, of Formula I a is 0. In some embodiments of Formula I, a is 1-10. In some embodiments, x5is G or A.

[0102] In some embodiments, x1of Formula 1-10 is G or A. In some embodiments, x2is G or A. In some embodiments, x3is A or V. In some embodiments, x4is L or I. In some embodiments, a is 0. In some embodiments of Formula 1-10, a is 1. In some embodiments, x5is G or A.

[0103] As used herein, a non-polar amino acid generally refers to an amino acid having a non-polar side-chain. Examples of non-polar amino acids include, but are not limited to glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, tryptophan, and tyrosine. Examples of flexible amino acids include, but are not limited to, glycine, alanine, and beta-alanine. In some embodiments, a flexible amino acid is a glycine or an alanine. As used herein, an uncharged amino acid refers to an amino acid having a side-chain that does not form a cation or anion, or a salt thereof, in physiological pH. An uncharged amino acid may be polar but uncharged (e.g., serine, threonine, asparagine, glutamine and cysteine), or may be non-polar and uncharged (e.g., glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, tryptophan, and tyrosine).

[0104] In some embodiments, the polypeptide (PP) of Formula I comprises SEQ ID NO 11:P-x -x -I<-R-x -I<-x4-D SEQ ID NO 11 or a pharmaceutically acceptable salt thereof, wherein: x1is G or A; x2is G or A; x3is A or V; and x4is L or I.

[0105] In some embodiments, the polypeptide (PP) of Formula I comprises SEQ ID NO 12:P-x -x2-I<-R-x -K-x4-D-x SEQ ID NO 12 or a pharmaceutically acceptable salt thereof, wherein: x1is G or A; x2is G or A; x3is A or V; x4is L or I; and x5is G or A.

[0106] In some embodiments, the polypeptide (PP) of Formula I comprises SEQ ID NO 13:P-x1-x2-K-R-V-K-L-D-(G)aSEQ ID NO 13 or a pharmaceutically acceptable salt thereof, wherein: x1and x2are each independently A or G; and a is 0 or 1.

[0107] In some embodiments, x1and x2are each A. In some embodiments, x1and x2are each G. In some embodiments, x1is G and x2is A. In some embodiments, x1is A and x2is G. In some embodiments, a is 0. In some embodiments, a is 1.

[0108] In some embodiments, the provided herein is a conjugate of Formula I, or a pharmaceutically acceptable salt thereof, wherein the NLS polypeptide (PP) of Formula I comprises a sequence selected from SEQ ID NOS: 1-8. In some embodiment, PP comprises SEQ ID NO 1: PGGKRVKLD. In another embodiment, PP comprises SEQ ID NO 2: PGGKRVKLDG. In certain embodiments, PP comprises SEQ ID NO 3: PAAKRVKLD. In some embodiments, PP comprises SEQ ID NO 4: PAAKRVKLDG. In some embodiments, PP comprises SEQ ID NO 5: PAGKRVKLDG. In some embodiments, PP comprises SEQ ID NO 6: PGAKRVKLDG. In some embodiments, PP comprises SEQ ID NO 7: PAAKRAKLDG. In some embodiments, PP comprises SEQ ID NO 8: PAAKRVKIDG.

[0109] In some embodiments, LI comprises a cleavable linker. In some embodiments, LI comprises a non-cleavable linker. In some embodiments, LI comprises 0 to 12 (e.g., 0, 1, 2, 3, ... 12) amino acids. In certain embodiments, LI comprises 0 to 12 (e.g., 0, 1, 2, 3, ... 12) polyethylene glycol monomers. In some embodiments, LI comprises 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) polyethylene glycol monomers. In some embodiments, LI comprises a protein -coupling or protein-coupled group. In certain embodiments, LI comprises a lysine -reach ve / ly sine -reacted group, a cysteine-reactive / cysteine-reacted group, or aglutamine-reactive / glutamine-reacted group. In some embodiments, LI comprises a homoglycine or a Ci-12 alkylene group. In some embodiments, LI comprises the group:

[0110] In some embodiments, LI comprises the group:

[0111] In some embodiments, LI is:

[0112] In some embodiments, AB is an antibody or an antigen-binding fragment thereof. In some embodiments, AB is AC, wherein AC is an antibody-coupling group (alternatively, a protein-coupling group, protein-reactive group, antibody-reactive group). In some embodiments, AB represents a bond to an antibody. In some embodiments, AC represents a group configured to form a bond with an antibody. Specifically, an AC group may form a bond with a sulfur of a cysteine side -chain, or a nitrogen of a lysine side-chain. Alternatively, AC may form a bond with an unnatural amino acid, i.e., using bio- orthogonal conjugation chemistry. In some embodiments, AC comprises an alkyne (e.g., a cyclooctyne), a triazine, a succinimide, a maleimide, a carbamate, an ester (e.g., an NHS ester), an isocyanate, anisothiocyanate, or the like. In some embodiments, AB represents an antibody. In some embodiments, AB represents a monoclonal antibody.

[0113] In certain embodiments, the antibody comprises a constant region. In some embodiments, the linker is conjugated to the antibody via the constant region. In some embodiments, the antibody comprises an Fc region. In certain embodiments, the linker is conjugated to the antibody via the Fc region. In some embodiments, the Fc region has reduced effector function.

[0114] In some embodiments, the antibody or antigen-binding fragment binds to a target antigen. In certain embodiments, the target antigen is expressed on a tumor cell. In some embodiments, the target antigen is CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, or tissue factor. In some embodiments, the target antigen is AXL, BCMA, CA9, CCR7, CD123, CD166, CD19, CD20, CD205, CD22, CD25, CD276, CD30, CD33, CD37, CD46, CD70, CD74, CD79b, CEACAM5, CLDN18.2, CLDN6, CXCR4, DLL3, EFNA4, EGFR, EGFRvIII, ENPP3, EPCAM, EPHA2, F3, FOLR1, GPC3, GPNMB, GUCY2C, HER2, HER3, IGF1R, L1CAM, LIV1, LRRC15, MET, MSLN, MUC1, MUC16, NCAM1, NECTIN4, PSMA, PTK7, ROR1, ROR2, SDC1, SLC1A5, SLC34A2, TFRC, TIM1, TPBG, or TROP2.

[0115] In some embodiments, the target antigen is CD 19. In some embodiments, the target antigen is CD22. In some embodiments, the target antigen is CD30. In some embodiments, the target antigen is CD33. In some embodiments, the target antigen is CD79b. In some embodiments, the target antigen is HER2. In some embodiments, the target antigen is TROP2. In some embodiments, the target antigen is EGFR. In some embodiments, the target antigen is Nectin-4. In some embodiments, the target antigen is mesothelin. In some embodiments, the target antigen is BCMA. In some embodiments, the target antigen is folate receptor alpha. In some embodiments, the target antigen is tissue factor.

[0116] In some embodiments, the target antigen corresponds to a gene that is TNFRSF17, ALCAM, MS4A1, LY75, B7H3, TNFRSF8, CD142, SLC39A6, CD138, CD71, or HAVCR1. In some embodiments, the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, belantamab, loncastuximab, tisotumab, mirvetuximab, or a biosimilar thereof.

[0117] In some embodiments, the antibody is trastuzumab, or a biosimilar thereof. In some embodiments, the antibody is brentuximab, or a biosimilar thereof. In some embodiments, the antibody is gemtuzumab, or a biosimilar thereof. In some embodiments, the antibody is inotuzumab, or a biosimilar thereof. In some embodiments, the antibody is moxetumomab, or a biosimilar thereof. In some embodiments, the antibody is polatuzumab, or a biosimilar thereof. In some embodiments, the antibody is enfortumab, or a biosimilar thereof. In some embodiments, the antibody is sacituzumab, or a biosimilar thereof. In some embodiments, the antibody is belantamab, or a biosimilar thereof. In some embodiments, the antibody is loncastuximab, or a biosimilar thereof. In some embodiments, the antibody is tisotumab, or a biosimilar thereof. In some embodiments, the antibody is mirvetuximab, or a biosimilar thereof.

[0118] In some embodiments, the composition comprises a therapeutic payload, wherein the therapeutic payload and the polypeptide (PP) are conjugated via a bond, or via a second linker (e.g., where the first linker connects the polypeptide PP to the antibody or antigen-binding fragment thereof, if present, or to the protein-coupling group (AC). In certain embodiments, the therapeutic payload is conjugated to D (Aspartic acid) or x5of the polypeptide (PP). In some embodiments, the therapeutic payload is conjugated to the polypeptide (PP) via a bond. In some embodiments, the therapeutic payload is conjugated to the polypeptide (PP) via a second linker. In some embodiments, the second linker is a Ci- C12 alkyl or heteroalkyl linker. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is a polypeptide linker. In some embodiments, the second linker is L2.

[0119] In some embodiments, L2 comprises a cleavable linker. In certain embodiments, L2 comprises a non-cleavable linker. In some embodiments, L2 comprises a self-immolative group. In some embodiments, L2 comprises 0 to 12 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acids. In some embodiments, L2 comprises 0 to 12 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) polyethylene glycol monomers. In some embodiments, L2 comprises a glycine or C1-12 alkylene group. In some embodiments, n is 0 and L2 is absent.

[0120] In some embodiments, the therapeutic payload is a cytotoxic payload. In some embodiments, the therapeutic payload is cytotoxic to a tumor cell upon internalization into the tumor cell. In certain embodiments, the therapeutic payload comprises a duocarmycin, auristatin, maytansinoid, uncialamycin, dynemicin, thailanstatin, camptothecin, exatecan, or tubulysin compound. In some embodiments, the therapeutic payload comprises an antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA-alkylating drug, a DNA-binding drug, a DNA- cleaving drug, or an RNA polymerase inhibitor. In some embodiments, the therapeutic payload (TP) comprises ecubectedin, lurbinectedin, trabectedin, safracin, lenalidomide, eribulin, a vinca alkaloid (e.g., vincristine, vinblastine, vindesine, or vinorelbine); an epothilone, a taxane (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), a cryptophycin, a hemiasterlin, an anthracyclin, a bisnaphthylamide (e.g., elinafide), or a cytotoxic molecular glue / PROTAC compound.

[0121] In some embodiments, the payload of the conjugate is an active agent, e.g., a therapeutic payload such as a drug. In some embodiments, the therapeutic payload is cytotoxic to a tumor cell. In some embodiments, the antibody of the conjugate is linked to the polypeptide (PP) N-terminally. In certain embodiments, the antibody is linked to the polypeptide (PP) N-terminally via a bond or a linker. In some embodiments, the active agent of the conjugate is linked to the polypeptide (PP) C-terminally. In some embodiments, the active agent of the conjugate is linked to the polypeptide (PP) C-terminally via a bond or a linker.

[0122] In some embodiments, the antibody-drug conjugate has a structure that is:or a pharmaceutically acceptable salt thereof, wherein:AB is an antibody or antigen-binding fragment thereof; and p is 1-20.

[0123] In some embodiments, p is 1-10. In some embodiments, p is 2-8. In some embodiments, p is 4-8.In some embodiments, p is 6-8. In some embodiments, p is 1. In each of the preceding structures, the succinimide ring may exist in one or more of the ring -opened isomeric forms indicated below:

[0124] In some embodiments, provided herein is a conjugate having a structure of Formula II orFormula III:AB-[(Ll)m-PP]qFormula IIPP-(L2)n-TP Formula III or a pharmaceutically acceptable salt thereof, wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide (e.g., an NLS polypeptide disclosed herein);L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1; and q is 1-20.

[0125] In some embodiments, each of AB, LI, PP, L2, TP, m, and n, is as defined in Formula I; and q is 1-20. In some embodiments, q is 1-10. In some embodiments, q is 2-8. In some embodiments, q is 3-6.

[0126] In some embodiments, provided herein is an antibody-polypeptide conjugate of Formula (II).AB-[(Ll)m-PP]qFormula II or a pharmaceutically acceptable salt thereof, wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide (e.g., an NLS polypeptide disclosed herein); and m is 0 or 1; and q is 1-20.

[0127] In some embodiments, AB is an antibody. In some embodiments, LI is a polymeric linker. In some embodiments, LI comprises a PEG group and an antibody-coupled group. In some embodiments, LI is a linker disclosed herein. In some embodiments, LI is a PEG3-6 linker. In some embodiments, LI is a PEG3 or PEG4 linker. In some embodiments, PP comprises SEQ ID NO 10. In some embodiments, PP comprises SEQ ID NO 11. In some embodiments, PP comprises SEQ ID NO 12. In some embodiments, PP comprises SEQ ID NO 13. In some embodiments, PP comprises any one of SEQ ID NO 1-8. In some embodiments, PP comprises SEQ ID NO 1. In some embodiments, PP comprises SEQ ID NO 2. In someembodiments, PP comprises SEQ ID NO 3. In some embodiments, PP comprises SEQ ID NO 4. In some embodiments, PP comprises SEQ ID NO 5. In some embodiments, PP comprises SEQ ID NO 6. In some embodiments, PP comprises SEQ ID NO 7. In some embodiments, PP comprises SEQ ID NO 8. In some embodiments, m is 1. In some embodiments, q is an integer between 1 and 20. In some embodiments, q is1-10. In some embodiments, q is 1-8. In some embodiments, q is 2-6. In some embodiments, q is about 2, about 3, about 4, about 5, or about 6. In some embodiments, q is about 3. In some embodiments, q is about 4. In some embodiments, q is about 5. In some embodiments, q is about 6.

[0128] In some embodiments, a conjugate of Formula (II) is an antibody-polypeptide PP conjugate. In some embodiments, the conjugate of Formula (II) is useful in the delivery of an antibody to an intracellular (i.e., nuclear) target. In some embodiments, the antibody-polypeptide conjugate binds to target antigens which are specifically expressed on the cancer cells, the antibody-polypeptide conjugate is then endocytosed or internalized by cells to form an early endosome, followed a maturation into late endosomes and finally fused with lysosomes. In some embodiment, in the fused lysosomes, enzymatic cleavage of the linker or antibody degradation under the specific conditions (pH, presence of degrading enzymes) can lead to the formation of a metabolite. In some embodiments, entrapment and degradation in the endosomal lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. In some embodiments, the NLS polypeptide (PP) can facilitate the antibody-polypeptide conjugate to escape endosome entrapment and route the complex to the nucleus, resulting in the increased intracellular accumulation with high target cell selectivity.

[0129] In some embodiments, provided herein is a polypeptide -drug conjugate of Formula (III).PP-(L2)n-TP Formula III or a pharmaceutically acceptable salt thereof, wherein:PP is a polypeptide;L2 is a linker;TP is a therapeutic payload; and n is 0 or 1.

[0130] In some embodiments, PP is an NLS polypeptide. In some embodiments, PP is a polypeptide according to SEQ ID NO 10. In some embodiments, PP comprises a sequence according to SEQ ID NO 11 and / or SEQ ID NO 12. In specific embodiments, PP comprises a sequence according to SEQ ID NO 13. In more specific embodiments, PP is any one of SEQ ID NO 1-8. In some embodiments, L2 is a stable (non-cleavable) linker. In some embodiments, L2 is a Ci-6 alkyl or heteroalkyl linker. In some embodiments, L2 is a bond (alternatively phrased, L2 is absent, or n is 0). In some embodiments, the therapeutic payload is a cytotoxic agent. In some embodiments, the cytotoxic agent inhibits the growth of a cell (e.g., a tumor cell). In some embodiments, the therapeutic payload is an inhibitor of DNA transcription or translation. In some embodiments, the therapeutic payload is an inhibitor of RNA transcription or translation. In some embodiments, the therapeutic payload is an inhibitor of a nuclearenzyme (e.g., a nuclear enzyme involved in processing DNA or RNA). In some embodiments, the therapeutic payload inhibits DNA or RNA synthesis or replication. In some embodiments, the therapeutic payload is a topoisomerase inhibitor.

[0131] In some embodiments, a conjugate of Formula (III) is a polypeptide -drug conjugate (e.g., an NLS polypeptide-drug conjugate). In some embodiments, a conjugate of Formula (III) is useful in the delivery of a therapeutic payload to an intracellular (i.e., nuclear) target. In some embodiments, the therapeutic payload inhibits or interrupts DNA transcription, translation, or replication. In some embodiments, the therapeutic payload is cytotoxic to a cancer cell. In some embodiments, the conjugate of Formula (III) is retained intracellularly (e.g., in the nucleus). In some embodiments, the conjugate of Formula (III) is cytotoxic intracellularly to cancer cells (e.g., cells expressing a cancer-associated antigen). In some embodiments, the conjugate of Formula (III) produces less toxicity (e.g., off-target cytotoxicity) than a therapeutic payload alone. In some embodiments, the conjugate of Formula (I) has a wider therapeutic window (i.e., less off-target toxicity and / or higher on-target toxicity) than a reference conjugate lacking a polypeptide (e.g., NLS polypeptide) disclosed herein. In some embodiments, the conjugate of Formula (III) has a wider therapeutic window (i.e., less off-target toxicity and / or higher on-target toxicity) than a reference payload lacking a polypeptide (e.g., NLS polypeptide) disclosed herein (e.g., any one of SEQ ID NOS: 1-13).Metabolites

[0132] In some embodiments, a polypeptide (PP) comprises a linker adduct. As used herein, a linker adduct refers to a linker fragment produced by the cleavage (i.e., metabolic cleavage) of a conjugate or linker provided herein. In some embodiments, the linker adduct comprises a PEG group (i.e., comprising one, two, three, four, five, or six polyethylene glycol monomers). In some embodiments, the linker adduct further comprises a heteroalkyl or heterocycloalkyl group. In some embodiments, the linker adduct further comprises an amino acid. In some embodiments, provided herein is a compound having the structure of Formula IV:(LA)r-PP-(L2)n-TP Formula IV wherein:LA is a linker adduct;PP is a polypeptide;L2 is a linker;TP is a therapeutic payload; r is 0 or 1; and n is 0 or 1.

[0133] In some embodiments, PP of Formula IV is an NLS polypeptide. In some embodiments, PP is a polypeptide according to SEQ ID NO 10. In some embodiments, PP comprises a sequence according to SEQ ID NO 11 and / or SEQ ID NO 12. In specific embodiments, PP comprises a sequence according toSEQ ID NO 13. In more specific embodiments, PP is any one of SEQ ID NO 1-8. In some embodiments,LA is:or a fragment thereof.

[0134] In some embodiments, the therapeutic payload (TP) is a warhead (preferably a cytotoxic agent) as described herein.

[0135] In some embodiments, the compound of Formula IV represents an active metabolite of an antibody-drug conjugate according to Formula I. In some embodiments, the compound of Formula IV is generated within a cell (e.g., a tumor cell) following intracellular processing of the antibody (AB) of Formula I. Provided herein, in one aspect, is a method of inhibiting the transcription and / or translation of genetic information (e.g., DNA, RNA, etc.) in a cell comprising contacting a target protein with a compound of Formula IV. In some embodiments, the target protein is expressed intracellularly. In some embodiments, the target protein is involved in the transcription and / or translation of DNA and / or RNA. In some embodiments, the target protein is a topoisomerase (e.g., topoisomerase I). In some embodiments, provided herein is a method of killing or inhibiting the growth of a tumor cell in a subject, the method comprising administering to the subject an antibody-drug conjugate according to Formula I. In some embodiments, provided herein is a compound of Formula IV for use in a method of treating a disease or disorder in a subject. In some embodiments, the disease or disorder is a cancer. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an antibody-drug conjugate according to Formula I. In some embodiments, the antibody-drug conjugate is of Formula I- 10. In some embodiments, provided herein is a compound of Formula IV-10:(LA)r-PP-(L2)„-TP Formula IV-10 wherein:LA is a linker adduct;PP is a polypeptide comprising SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10wherein x1, x2,x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1;L2 is a linker;TP is a therapeutic payload; r is 0 or 1 ; and n is 0 or 1.

[0136] In some embodiments, x1is G or A; x2is G or A; x3is A or V; and x4is L or I. In some embodiments, x5is G or A. In some embodiments, x5is absent. In some embodiments, the polypeptide (PP) of Formula IV-10 is SEQ ID NO 11. In some embodiments, the polypeptide (PP) of Formula IV-10 is SEQ ID NO 12. In some embodiments, the polypeptide (PP) comprises a sequence selected from SEQ ID NOs: 1-8. In some embodiments, the polypeptide (PP) of Formula IV-10 is any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, provided herein is a compound of Formula IV-10 for use in a method of treating a disease or disorder (e.g., a cancer) in a subject in need thereof.Therapeutic Payloads

[0137] The term therapeutic payload generally refers to any payload having utility in the treatment, diagnosis, or detection of a disease, which may include drugs, diagnostics, detectable agents, radioligands, etc., and is not intended to be limiting to therapeutically active compounds. In some embodiments, the therapeutic payload (TP) is the warhead that exerts cytotoxicity after internalization of the antibody polypeptide -drug conjugate into cancer cells. In some embodiments, the therapeutic payload has at least acceptable potency to destroy the tumor cells. In some embodiments, the therapeutic payload is stable in physiological conditions and linked to antibodies via linker(s). A therapeutic payload can be, for example, a cytotoxic agent or an immunostimulatory agent. Preferably, the therapeutic payload is permeable to cell membranes (e.g., to tumor cell membranes). More preferably, the therapeutic payload is penetrant to tumor cells and produces cytotoxic or antiproliferative effects in a cell.

[0138] In some embodiments, the therapeutic payload (TP) is an anti-cancer drug. In some embodiments, the therapeutic payload (TP) is an antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA-alkylating drug, a DNA-binding drug, a DNA-cleaving drug, or an RNA polymerase inhibitor. In some embodiments, the therapeutic payload inhibits DNA or RNA synthesis or replication. In some embodiments, the therapeutic payload is a topoisomerase inhibitor. In some embodiments, the therapeutic payload is a microtubule inhibitor, a pyrrolobenzodiazepine, or a topoisomerase inhibitor.

[0139] In some embodiments, the therapeutic payload (TP) comprises an antitumor antibiotic. In some embodiments, the therapeutic payload (TP) comprises a microtubule inhibitor. In some embodiments, the therapeutic payload (TP) comprises a cytotoxic or cytostatic agent. In some embodiments, the therapeutic payload (TP) comprises a topoisomerase inhibitor. In some embodiments, the therapeutic payload (TP) comprises a pyrrolobenzodiazepine. In some embodiments, the therapeutic payload (TP) comprises a DNA-alkylating drug. In some embodiments, the therapeutic payload (TP) a DNA-binding drug. In someembodiments, the therapeutic payload (TP) a DNA-cleaving drug. In some embodiments, the therapeutic payload (TP) comprises an RNA polymerase inhibitor.

[0140] In some embodiments, the therapeutic payload (TP) is a kinase inhibitor. In some embodiments, the therapeutic payload (TP) is a MEK inhibitor. In some embodiments, the therapeutic payload (TP) is a KSP inhibitor. In some embodiments, the therapeutic payload (TP) is a maytansinoid compound. In some embodiments, the therapeutic payload (TP) is a uncialamycin compound. In some embodiments, the therapeutic payload (TP) is a dynemicin compound. In some embodiments, the therapeutic payload (TP) is a thailanstatin compound. In some embodiments, the therapeutic payload (TP) is a tubulysin compound. In some embodiments, the therapeutic payload (TP) is a vinca alkaloid compound. In some embodiments, the therapeutic payload is a camptothecin, an exatecan, a tubulysin, MMAE, a maytansinoid, a pyrrolobenzodiazepine, an uncialamycin, or a thailanstatin. In some embodiments, the therapeutic payload (TP) is a duocarmycin, auristatin, maytansinoid, uncialamycin, dynemicin, thailanstatin, camptothecin, exatecan, or tubulysin compound. In some embodiments, the therapeutic payload (TP) comprises a camptothecin, an exatecan, a tubulysin, MMAE, a maytansinoid, a pyrrolobenzodiazepine, an uncialamycin, thailanstatin, lurbinectedin, trabectedin, safracin, lenalidomide, eribulin, a vinca alkaloid (e.g., vincristine, vinblastine, vindesine, or vinorelbine); an epothilone, a taxane (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), a cryptophycin, a hemiasterlin, an anthracyclin, a bisnaphthylamide (e.g., elinafide), or a cytotoxic molecular glue / PROTAC compound.

[0141] In some embodiments, the therapeutic payload (TP) comprises an antiproliferative agent. In some embodiments, the therapeutic payload (TP) comprises a cytotoxic and / or cytostatic agent. In some embodiments, the therapeutic payload (TP) comprises an angiogenesis inhibitor, cell cycle progression inhibitor, RNA polymerase inhibitor, MAPK inhibitor, PI3K inhibitor, mTOR inhibitor, EGFR inhibitor, VEGFR inhibitor, HDAC inhibitor, PARP inhibitor, Wnt Hedgehog inhibitor, tubulin polymerization inhibitor, topoisomerase inhibitor, an antitumor antibiotic, an oxidative phosphorylation inhibitor, or a vinca alkaloid. In some embodiments, the therapeutic payload (TP) comprises a DNA-binder, DNA- intercalator, a DNA-alkylator (e.g., a calicheamicin, a dactinomycin, a mitomycin), a microtubule destabilizer, a topoisomerase inhibitor, a platinum -containing drug, an anthracycline (e.g., doxorubicin, epirubicin, idarubicin, daunorubicin, nemorubicin), or a protein synthesis inhibitor.

[0142] In some embodiments, the therapeutic payload (TP) is a pyrrolobenzodiazepine. In some embodiments, the therapeutic payload (TP) is a pyrrolobenzodiazepine monomer. In some embodiments, the therapeutic payload (TP) is a symmetric pyrrolobenzodiazepine dimer. In some embodiments, the therapeutic payload (TP) is an asymmetric pyrrolobenzodiazepine dimer.

[0143] In some embodiments, the therapeutic payload (TP) is a duocarmycin compound. In some embodiments, the therapeutic payload (TP) is duocarmycin A. In some embodiments, the therapeutic payload (TP) is duocarmycin B. In some embodiments, the therapeutic payload (TP) is duocarmycin B2. In some embodiments, the therapeutic payload (TP) is duocarmycin CI. In some embodiments, the therapeutic payload (TP) is duocarmycin C2. In some embodiments, the therapeutic payload (TP) is duocarmycin D. In some embodiments, the therapeutic payload (TP) is duocarmycin SA. In someembodiments, the therapeutic payload (TP) is CC-1065. In some embodiments, the therapeutic payload (TP) is adozelesin. In some embodiments, the therapeutic payload (TP) is bizelesin. In some embodiments, the therapeutic payload (TP) is carzelesin. In some embodiments, the therapeutic payload (TP) is duocarmycin A, duocarmycin B duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin, bizelesin, or carzelesin.

[0144] In some embodiments, the therapeutic payload (TP) is an auristatin compound. In some embodiments, the therapeutic payload (TP) is monomethyl auristatin E (MMAE). In some embodiments, the therapeutic payload (TP) is monomethyl auristatin F (MMAF). In some embodiments, the therapeutic payload (TP) is monomethyl auristatin F hydroxypropyl amide (MMAFHPA). In some embodiments, the therapeutic payload (TP) is auristatin F hydroxypropyl amide (AFHPA). In some embodiments, the therapeutic payload (TP) is auristatin F phenylenediamine (AFP). In some embodiments, the therapeutic payload (TP) is MMAE, MMAF, MMAFHPA, AFHPA, or AFP. In some embodiments, the therapeutic payload (TP) is dolastatin compound. In some embodiments, the therapeutic payload (TP) is dolastatin 10. In some embodiments, the therapeutic payload (TP) is dolastatin 15. In some embodiments, the therapeutic payload (TP) is dolastatin 10 or dolastatin 15.

[0145] In some embodiments, the therapeutic payload (TP) is a antitumor chemotherapeutic. In some embodiments, the therapeutic payload (TP) is a cytotoxic or cytostatic agent. In some embodiments, the therapeutic payload (TP) is a splicing inhibitor. In some embodiments, the therapeutic payload (TP) is a topoisomerase inhibitor. In some embodiments, the therapeutic payload (TP) is a topoisomerase I inhibitor. In some embodiments, the therapeutic payload (TP) is camptothecin, 10-hydroxycamptothecin, topotecan, irinotecan, belotecan, exatecan, diflomotecan, grimatecan, lurtotecan, silatecan, rubitecan, or SN-38. In some embodiments, the therapeutic payload (TP) is a camptothecin compound. In some embodiments, the therapeutic payload (TP) is camptothecin. In some embodiments, the therapeutic payload (TP) is CPT-1 1 (irinotecan). In some embodiments, the therapeutic payload (TP) is SN-38. In some embodiments, the therapeutic payload (TP) is topotecan. In some embodiments, the therapeutic payload (TP) is exatecan. In some embodiments, the therapeutic payload (TP) comprises the group:whereindenotes the point of attachment to a linker (e.g., L2 or PP). In some embodiments, the therapeutic payload (TP) is conjugated to L2 or PP via an amide bond.

[0146] In some embodiments, the therapeutic payload (TP) is an antitumor antibiotic. In some embodiments, the therapeutic payload (TP) is a calicheamicin. In some embodiments, TP is a calicheamicin residue, conjugated to a sulfur atom to L2. In some embodiments, the therapeutic payload (TP) is a calicheamicin, having the structure:whereindenotes the point of attachment to a linker (e.g., L2 or PP). In some embodiments, the therapeutic payload (TP) is conjugated to L2 or PP via a disulfide bond.

[0147] In some embodiments, the therapeutic payload (TP) is a DNA alkylating agent. In some embodiments, the therapeutic payload (TP) is a DNA groove-binding drug. In some embodiments, the therapeutic payload (TP) is an RNA polymerase II inhibitor. In some embodiments, the therapeutic payload (TP) is an ecteinascidin. In some embodiments, the therapeutic payload (TP) is trabectedin, ecubectedin, or lurbinectedin. In some embodiments, the therapeutic payload (TP) is:wherein denotes the point of attachment to a linker (e.g., L2 or PP). In some embodiments, the therapeutic payload (TP) is conjugated to L2 or PP via an amide, phosphate, thiophosphate, or phosphoramide.Linkers

[0148] In one aspect, provided herein is an antibody-drug conjugate comprising an antibody and a therapeutic payload, wherein the antibody and therapeutic payload are connected via a linker, and wherein the linker comprises an NLS polypeptide (i.e., a polypeptide (PP) disclosed herein). In someembodiments, the linker further comprises an alkyl or heteroalkyl portion. In some embodiments, the linker further comprises an aryl and heteroaryl portion. In some embodiments, the heteroalkyl portion comprises a polymeric section (e.g., a PEG section). In some embodiments, the linker comprises 0 to 36 amino acids. In some embodiments, the heteroalkyl portion comprises 0 to 12 polyethylene glycol (PEG) groups. In some embodiments, the heteroalkyl portion comprises an antibody-coupling group (e.g., a maleimide, a succinimide, or other lysine- or cysteine -reactive group), or an antibody-coupled linker formed therefrom. In some embodiments, the linker comprises a protein-coupling or protein-coupled group. In some embodiments, the linker comprises a homoglycine or a C1-12 alkylene group. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the linker comprises a non- cleavable linker. In some embodiments, the linker comprises a self-immolative group. In some embodiments, the linker has the structure:-(Ll)m-PP-(L2)„- wherein:LI is a first linker (e.g., a linker to an antibody or antigen-binding fragment); PP is a polypeptide (e.g., an NLS polypeptide disclosed herein); L2 is a second linker (e.g., a linker to a therapeutic payload); m is 0 or 1 ; and n is 0 or 1.

[0149] In some embodiments, LI is a cleavable linker. In some embodiments, LI is a non-cleavable linker. In some embodiments, LI comprises an alkyl portion. In some embodiments, LI comprises a heteroalkyl portion. In some embodiments, LI comprises an aryl portion. In some embodiments, LI comprises a heteroaryl portion. In some embodiments, LI comprises one or more, linear or branched Ci- 80 alkyl portions. In some embodiments, LI comprises one or more, linear or branched Ci-so heteroalkyl portions. In some embodiments, LI comprises one or more, linear or branched Ci-so heteroalkyl portions, which is optionally interrupted with one or more cycloalkyl, heterocycloalkyl, aryl, or heteroaryl rings . In some embodiments, LI comprises one or more, linear or branched Ci-so heteroalkyl portions. In some embodiments, LI is an alkyl or heteroalkyl Ci-so linker, wherein the linker further comprises one or more monocyclic or polycyclic heterocycloalkyl or heteroaryl rings. Exemplary rings include, but are not limited to, pyrroles, pyrrolidinones, imidazoles, imidazolidinones, triazoles, maleimides, succinimides, polycyclic cyclooctynes, and the like. In some embodiments, LI comprises a homoglycine or a C 2 alkylene group. In some embodiments, LI comprises a homoglycine. In some embodiments, LI comprises a C1-12 alkylene group.

[0150] In certain embodiments, LI comprises 0 to 12 (e.g., 0, 1, 2, 3, ... 12) polyethylene glycol monomers. In some embodiments, LI comprises 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) polyethylene glycol monomers. In some embodiments, LI comprises 2 to 6 (e.g., 3 or 4) polyethylene glycol monomers. In some embodiments, LI comprises 3 or 4 polyethylene glycol monomers. In some embodiments, LI comprises 4 polyethylene glycol monomers.

[0151] In some embodiments, LI comprises 0 to 12 (e.g., 0, 1, 2, 3, ... 12) amino acids. In some embodiments, LI comprises 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) amino acids. In some embodiments, LI comprises 1 to 8 amino acids. In some embodiments, LI comprises 1 to 4 amino acids. In some embodiments, LI comprises 8 to 12 amino acids. In some embodiments, LI comprises 2 to 6 (e.g., 3 or 4) amino acids. In some embodiments, LI comprises 2 to 6 amino acids. In some embodiments, LI comprises 3 or 4 amino acids.

[0152] In some embodiments, LI comprises 0-12 (e.g., 0, 1, 2, 3, ... 12) C=O groups. In some embodiments, LI comprises 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) C=O groups. In some embodiments, LI comprises 1 to 2 C=O groups. In some embodiments, LI comprises a C=O group. In some embodiments, LI comprises 2 C=O groups. In some embodiments, LI comprises 0-12 (e.g., 0, 1, 2, 3, ... 12) C(=O)NH groups. In some embodiments, LI comprises 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) C(=O)NH groups. In some embodiments, LI comprises a C(=O)NH group. In some embodiments, LI comprises 2 C(=O)NH groups.

[0153] In some embodiments, LI comprises an antibody-coupling group (e.g., amaleimide, a succinimide, or other lysine- or cysteine-reactive group), or an antibody -coupled linker formed therefrom. In some embodiments, LI comprises a protein-coupling or protein-coupled group. In certain embodiments, LI comprises a lysine-reactive / lysine-reacted group, a cysteine-reactive / cysteine-reacted group, or a glutamine -reactive / glutamine -reacted group. In certain embodiments, LI comprises a lysine- reactive / lysine-reacted group. In certain embodiments, LI comprises a cysteine -reactive / cysteine -reacted group. In certain embodiments, LI comprises a glutamine-reactive / glutamine-reacted group.

[0154] In some embodiments, LI comprises a protein-coupling group that reacts with a lysine or cysteine residue of an antibody or antigen-binding fragment thereof. Preferably, such protein-coupling groups and / or protein-coupled linkers are not susceptible to unwanted cleavage (e.g., retro-Michael elimination of a maleimide group).

[0155] In some embodiments, LI comprises the group:

[0156] In some embodiments, LI comprises the group:

[0157] In some embodiments, L2 is a cleavable linker. In certain embodiments, L2 is a non-cleavable linker. In some embodiments, L2 comprises an alkyl portion. In some embodiments, L2 comprises a heteroalkyl portion. In some embodiments, L2 comprises an aryl portion. In some embodiments, L2 comprises a heteroaryl portion. In some embodiments, L2 comprises one or more, linear or branched Ci- 8o alkyl portions. In some embodiments, L2 comprises one or more, linear or branched Ci-so heteroalkyl portions. In some embodiments, L2 comprises a self-immolative group.

[0158] In some embodiments, provided herein is a cleavable linker conjugated to a therapeutic payload via an ester, an amide, a thiophosphate, or a disulfide bond. In some embodiments, the cleavable linker is cleaved efficiently and cleanly (i.e., without leaving a post-cleavage artifact bonded to the payload at the point of attachment). In some embodiments, the protease-cleavable linker is cleaved efficiently and cleanly without the use of a self-immolative group, thereby resulting in enhanced stability.

[0159] In some embodiments, L2 comprises 0 to 12 (e.g., 0, 1, 2, 3, ... 12) amino acids. In some embodiments, L2 of the conjugate comprises 0 to 12 (e.g., 0, 1, 2, 3, ... 12) polyethylene glycol monomers. In some embodiments, L2 comprises a glycine or C1-12 alkylene group.

[0160] In some embodiments, L2 is a linker of the formula:-NH-CI.6 alkyl-[OCH2CH2]o-8-Co.6 alkyl-Z-(Ci-i2 alkyl-S)0-i-;wherein Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, -OP(=O)(SH)-, or -OP(=O)(OH)-.

[0161] In some embodiments, L2 is a linker of the formula:-NH-(CH2)2-6-(OCH2CH2)i-8-(CH2)o-3-Z-Ci-i2 alkyl-S-;-NH-(CH2)i-i2-Z-Ci-i2 alkyl-S-;-NH-(CH2)2-6-(OCH2CH2)1-8-(CH2)O-3-Z-; or -NH-(CH2)i-i2-Z-; wherein Z is -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, or a bond.

[0162] In some embodiments, L2 is a linker of the formula:

[0163] In some embodiments, L2 is a linker of the formula:

[0164] In some embodiments, L2 is a linker of the formula:

[0165] In some embodiments, L2 is a linker of the formula:

[0166] In some embodiments, L2 is a linker of the formula:

[0167] In some embodiments, n is 0 and L2 is absent. In some embodiments, n is 1, and L2 is connected to a therapeutic payload.Precursor Molecules

[0168] In another aspect, provided herein is a precursor molecule (i.e., a compound) comprising a nuclear localization signal polypeptide and a therapeutic payload, and further comprising a protein- reactive group (alternatively, a protein -coupling group). Precursor molecules provided herein, may be useful in the synthesis or manufacture of a medicament for treating diseases or disorders (e.g., cancers) ina subject. In some embodiments, provided herein is a compound for use in the manufacture of a medicament, according to Formula V or Formula VI below. In some embodiments, the precursor molecule comprises an active agent other than a therapeutic payload, such as, for example, a detectable group. In some embodiments, a precursor molecule has a structure according to Formula V, VI, or VIEAC-(Ll)m-PP-(L2)„-TP Formula VAC-(Ll)m-PP Formula VIAC-(L 1 )m-PP-(L2)n-DG Formula VII or a pharmaceutically acceptable salt thereof, wherein:AC is an antibody -coupling group configured to form a covalent bond with an antibody or antigen-binding fragment (e.g., to a sulfur atom of a cysteine side-chain of the antibody, or to a nitrogen atom of a lysine side -chain of the antibody);LI is a linker;PP is a polypeptide (e.g., an NLS polypeptide disclosed herein);L2 is a linker;TP is a therapeutic payload;DG is a detectable group; m is 0 or 1 ; and n is 0 or 1.

[0169] In some embodiments, the provided herein is a precursor molecule (which may also be referred to as a conjugate) according to Formula V or Formula VI, wherein the polypeptide (PP) comprises a sequence selected from SEQ ID NOS: 1-8. In some embodiment, the NLS polypeptide (i.e., “PP” in Formula V or VI) comprises SEQ ID NO 1: PGGKRVKLD. In another embodiment, the NLS polypeptide comprises SEQ ID NO 2: PGGKRVKLDG. In certain embodiments, the NLS polypeptide comprises SEQ ID NO 3: PAAKRVKLD. In some embodiments, the NLS polypeptide comprises SEQ ID NO 4: PAAKRVKLDG. In some embodiments, the NLS polypeptide comprises SEQ ID NO 5: PAGKRVKLDG. In some embodiments, the NLS polypeptide comprises SEQ ID NO 6: PGAKRVKLDG. In some embodiments, the NLS polypeptide comprises SEQ ID NO 7: PAAKRAKLDG. In some embodiments, the NLS polypeptide comprises SEQ ID NO 8: PAAKRVKIDG.

[0170] In some embodiments, LI is a cleavable linker. In some embodiments, LI is a non-cleavable linker. In some embodiments, LI is a stable linker. In some embodiments, LI is a peptide linker. In some embodiments, LI is a non-peptide linker. In some embodiments, LI comprises O to 12 (e.g., 0, 1, 2, 3, ... 12) amino acids. In some embodiments, LI comprises 0 amino acids. In some embodiments, LI comprises 1 to 12 amino acids. In some embodiments, LI comprises 1 to 4 amino acids. In some embodiments, LI is a PEG linker. In certain embodiments, LI comprises 0 to 12 (e.g., 0, 1, 2, 3, ... 12) polyethylene glycol monomers. In some embodiments, LI comprises 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4))polyethylene glycol monomers. In some embodiments, LI comprises 1 to 8 polyethylene glycol monomers. In some embodiments, LI comprises 2 to 6 polyethylene glycol monomers. In some embodiments, LI comprises 3 or 4 polyethylene glycol monomers. In some embodiments, LI is a linker to a protein -coupling or protein -coupled group (AC). In some embodiments, LI comprises a PEG segment and a protein-coupling segment (AC), and optionally one or more linking segments. In certain embodiments, AC comprises a lysine -reactive / ly sine -reacted group, a cysteine -reactive / cysteine -reacted group, or a glutamine -reactive / glutamine -reacted group.

[0171] In some embodiments, AC of Formula V or VI comprises the group:optionally further comprising a C1-12 alkyl or C1-12 heteroalkyl (e.g., a PEGs-e) group.

[0172] In some embodiments, AC-LI comprises one of the following structures:

[0173] In some embodiments, the detectable group of Formula VII is a dye. In some embodiments, the detectable group is a gamma emitter, beta emitter (e.g.,3H,14C,32P,35S, or90Sr), or alpha emitter. In some embodiments, the detectable group responds to a stimulus (e.g., a light, a laser, or other energy source) with an emitted response (e.g., fluorescence, phosphorescence, or other detectable light). In some embodiments, the detectable group is a donor or acceptor of a FRET pair, or a BRET pair. In some embodiments, the detectable group is a chromophore, fluorescent labels, biolumine scent label, or chemiluminescent label. In some embodiments, the detectable group is an Alexa Fluor® dye, a HiLyte™ Fluor dye, or other functional equivalent (e.g., a coumarin, rhodamine, or cyanine dye). In some embodiments, the detectable group is a coumarin dye. In some embodiments, the detectable group is a rhodamine dye. In some embodiments, the detectable group is a cyanine dye. In some embodiments, the detectable group is an Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, HiLyte™ Fluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte™ Fluor 555, Alexa Fluor® 561, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor® 647, HiLyte™ Fluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, or HiLyte™ Fluor 750, or a functional equivalent thereof. In some embodiments, the detectable group is an Alexa Fluor® 488 or HiLyte™ Fluor 647, or a functional equivalent thereof. As used herein, a functional equivalent means a chemical entity having the same essential property (e.g., excitation or emission wavelength) as a reference dye.

[0174] In some embodiments, the therapeutic payload of Formula V is an antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA- alkylating drug, a DNA-binding drug, a DNA-cleaving drug, or an RNA polymerase inhibitor. In some embodiments, the therapeutic payload is a pyrrolobenzodiazepine, duocarmycin, auristatin, maytansinoid, uncialamycin, dynemicin, thailanstatin, camptothecin, exatecan, tubulysin compound, ecubectedin, lurbinectedin, trabectedin, safracin, lenalidomide, eribulin, vincristine, vinblastine, vindesine, vinorelbine, an epothilone, a taxane (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), a cryptophy cin, a hemiasterlin, an anthracyclin, a bisnaphthylamide (e.g., elinafide), or a cytotoxic molecular glue / PROTAC compound.

[0175] In some embodiments, the therapeutic payload is an antitumor antibiotic. In some embodiments, the therapeutic payload is a microtubule inhibitor. In some embodiments, the therapeutic payload is a cytotoxic or cytostatic agent. In some embodiments, the therapeutic payload is a topoisomerase I inhibitor. In some embodiments, the therapeutic payload is or comprises a camptothecin or exatecan compound. In some embodiments, the therapeutic payload is camptothecin, 10-hydroxy camptothecin, topotecan, irinotecan, belotecan, exatecan, diflomotecan, grimatecan, lurtotecan, silatecan, rubitecan, or SN-38. In some embodiments, the therapeutic payload is an ecteinascidin. In some embodiments, the therapeutic payload is trabectedin. In some embodiments, the therapeutic payload is ecubectedin. In some embodiments, the therapeutic payload is lurbinectedin. In some embodiments, the therapeutic payload is a calicheamicin. In some embodiments, the therapeutic payload is calicheamicin yl (gamma-1). In some embodiments, the therapeutic payload is selected from any one of the following:

[0176] In another embodiment, provided herein is a precursor molecule having the structure:or a pharmaceutically acceptable salt thereof.

[0177] Also considered within the scope of the present disclosure is a method of preparing an antibodydrug conjugate, the method comprising contacting any one or more of the precursor molecules disclosed herein with an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises one or more cysteines. In some embodiments, the method comprises reacting the precursor molecule with an antibody in the presence of a base and / or a reducing agent. In some embodiments, the method comprises reacting the precursor molecule with an antibody or antigen-binding fragment thereof in a reaction mixture comprising Tris and / or TCEP. Exemplary methods of preparing an antibody-drug conjugate (e.g., of Formula I, (e.g., of Formula 1-10)) are disclosed in the examples section.Antibodies

[0178] In some embodiments, the term “antibody” is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (for example, sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, for example, bispecific, antibodies,diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgGl constant region. The antibody can comprise a human IgG4 constant region.

[0179] The terms “complementarity determining region,” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732- 745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pltickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. 2000 Dec;13(12):819-24 (“AbM” numbering scheme). The CDRs of the antibodies described herein may be defined by the Kabat, IMGT, Chothia, AbM, Aho, contact numbering scheme, or any combination thereof.

[0180] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (See for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91(2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (See for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0181] The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 ormore amino acids, and a carboxy-terminal portion includes a constant region. The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (a), delta (5), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the heavy chain constant region. The distinct heavy chains differ in size: a, 5, and y contain approximately 450 amino acids, while p and 8 contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including four subclasses of IgG, namely IgGl, IgG2, IgG3, and IgG4. A heavy chain can be a human heavy chain.

[0182] The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino -terminal portion includes a variable region of about 100 to about 110 or more amino acids, and a carboxy-terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (K) or lambda (X) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

[0183] Among the provided antibodies are antibody fragments. An “antibody fragment,” “antigenbinding fragment,” “antigen-binding domain,” “antigen-binding region,” “antigen binding fragment,” “antigen binding domain,” “antigen binding region,” and similar terms refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (for example, scFv or sFv); and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a variable heavy chain region and / or a variable light chain region, such as scFvs. Generally, an antibody fragment or antigen-binding fragment will comprise one or more CDRs from a parental antibody that are sufficient to confer binding specificity.

[0184] Generally, a humanized antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (for example, the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity. In some embodiments, a humanized antibody refers to forms of non-human (for example, murine) or not fully humanized antibodies having specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (for example, murine) sequences.

[0185] Among the provided antibodies are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, ornon-human source that utilizes human antibody repertoires or other human antibody -encoding sequences, including human antibody libraries. The term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.

[0186] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived or selected from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire. In certain embodiments, a human antibody can have sequence liabilities removed or its affinity increased by successive rounds of selection by a method such as phage display.Fc Constant Regions

[0187] Generally, the fragment crystallizable (Fc) region or domain of an antibody mediates downstream effector functions via its interaction with Fc-receptors on immune cells (for example, innate immune cells) or with complement protein Clq, the recognition molecule of the complement system. Furthermore, the interaction with Fc-receptors can lead to killing of targeted cells through a variety of immune effector mechanisms, including antibody -dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and antibody -mediated complement activation may lead to complement-dependent cytotoxicity (CDC). In addition, both Fc-receptor interactions and complement activation can exert a broad range of immunomodulatory functions.

[0188] Accordingly, in certain instances, mutations within the Fc region that reduce, inhibit, ablate, and / or abrogate Fc-mediated function are advantageous for reducing immune activation resulting from the binding of an antibody to the target. In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody, thereby generating an Fc region variant. An Fc region may comprise a C-terminal region of an immunoglobulin heavy chain that comprises a hinge region, CH2 domain, CH3 domain, or any combination thereof. As used herein, an Fc region includes native sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution, addition, or deletion) at one or more amino acid positions.Pharmaceutical Compositions

[0189] A pharmaceutical composition disclosed herein can be administered to a subject by any suitable administration route, including but not limited to, parenteral (intravenous, subcutaneous, intraperitoneal,intramuscular, intravascular, intrathecal, intravitreal, infusion, or local), topical, oral, or nasal administration.

[0190] "Pharmaceutically acceptable" may refer to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0191] "Pharmaceutically acceptable salt" may refer to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.

[0192] "Pharmaceutically acceptable excipient, carrier or adjuvant" may refer to an excipient, carrier or adjuvant that may be administered to a subject, together with at least one antibody of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[0193] "Pharmaceutically acceptable vehicle" may refer to a diluent, adjuvant, excipient, or carrier with which at least one antibody of the present disclosure is administered.Therapeutic Use

[0194] In another aspect, provided herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a nuclear localization signal polypeptide disclosed herein. In some embodiments, the NUS polypeptide is conjugated to a therapeutic payload. In some embodiments, the NUS polypeptide is further conjugated to an antibody or an antigen-binding fragment thereof. In some embodiments, the NUS polypeptide comprises SEQ ID NO 10. In some embodiments, the NUS polypeptide comprises a sequence of peptides according to one or more of SEQ ID NO 11, 12, and 13. In some embodiments, the polypeptide comprises one of SEQ ID NO 1-8.

[0195] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a therapeutic payload (e.g., a cytotoxic agent) conjugated to a NLS polypeptide. In some embodiments, the therapeutic payload is stably conjugated to the NLS polypeptide. In some embodiments, the therapeutic payload is cleavably conjugated to the NLS polypeptide. In some embodiments, the therapeutic payload is further conjugated to an antibody, or an antigen-binding fragment thereof. In some embodiments, the therapeutic payload is a cytotoxic agent. In some embodiments, the therapeutic payload is cytotoxic to a tumor cell. In some embodiments, the therapeutic payload is an inhibitor of the replication (transcription, translation, etc.) of DNA and / or RNA. In some embodiments, the therapeutic payload is an inhibitor of a nuclear target protein.

[0196] In some embodiments, the therapeutic payload is a camptothecin, an exatecan, a tubulysin, MMAE, a maytansinoid, a pyrrolobenzodiazepine, an uncialamycin, or athailanstatin. In some embodiments, the therapeutic payload is a camptothecin. In some embodiments, the therapeutic payload is an exatecan. In some embodiments, the therapeutic payload is a tubulysin. In some embodiments, the therapeutic payload is MMAE. In some embodiments, the therapeutic payload is a maytansinoid. In someembodiments, the therapeutic payload is a pyrrolobenzodiazepine. In some embodiments, the therapeutic payload is an uncialamycin. In some embodiments, the therapeutic payload is a thailanstatin. In some embodiments, the therapeutic payload is a splicing inhibitor. In some embodiments, the therapeutic payload is a topoisomerase inhibitor. In some embodiments, the disease or disorder is a hyperproliferative disease or disorder. In some embodiments, the disease or disorder is a cancer. In some embodiments, the disease or disorder is a cancer associated with overexpression of CD 19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, tissue factor. In some embodiments, the disease or disorder is a cancer associated with overexpression of AXL, BCMA, CA9, CCR7, CD123, CD166, CD19, CD20, CD205, CD22, CD25, CD276, CD30, CD33, CD37, CD46, CD70, CD74, CD79b, CEACAM5, CLDN18.2, CLDN6, CXCR4, DLL3, EFNA4, EGFR, EGFRvIII, ENPP3, EPCAM, EPHA2, F3, FOLR1, GPC3, GPNMB, GUCY2C, HER2, HERB, IGF1R, LI CAM, LIV1, LRRC15, MET, MSLN, MUC1, MUC16, NCAM1, NECTIN4, PSMA, PTK7, R0R1, R0R2, SDC1, SLC1A5, SLC34A2, TFRC, TIM1, TPBG, or TROP2.

[0197] In another aspect, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibodydrug conjugate according to Formula I.AB-[(Ll)m-PP-(L2)n-TP]p Formula I wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a NLS polypeptide;L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0198] In some embodiments, the polypeptide (PP) comprises SEQ ID NO 10. In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody -drug conjugate of Formula I to the subject. In some embodiments, the antibodydrug conjugate is of Formula I- 10.

[0199] In some embodiments, the antibody-drug conjugate is of Formula I- 10:AB-[(Ll)m-PP-(L2)n-TP]p Formula I- 10 wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide comprising SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein x1, x2,x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1;L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0200] In some embodiments, x1is G or A; x2is G or A; x3is A or V; and x4is L or I. In some embodiments, x5is G or A. In some embodiments, x5is absent. In some embodiments, the polypeptide (PP) comprises a sequence selected from SEQ ID NOs: 1-8. In some embodiments, the polypeptide (PP) of Formula is SEQ ID NO 11. In some embodiments, the polypeptide (PP) of Formula I is SEQ ID NO 12. In some embodiments, the polypeptide (PP) of Formula I is any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, or 8.

[0201] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody-drug conjugate comprising a therapeutic payload stably conjugated to an NLS polypeptide (PP) comprising the sequence PGGKRVKLD (SEQ ID NO. 1).

[0202] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody -drug conjugate comprising a therapeutic payload stably conjugated to an NLS polypeptide (PP) comprising the sequence PGGKRVKLDG (SEQ ID NO: 2).

[0203] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody -drug conjugate comprising a therapeutic payload stably conjugated to an NLS polypeptide (PP) comprising the sequence PAAKRVKLD (SEQ ID NO: 3).

[0204] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody -drug conjugate comprising a therapeutic payload stably conjugated to an NLS polypeptide (PP) comprising the sequence PAAKRVKLDG (SEQ ID NO:4).

[0205] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody -drug conjugate comprising a therapeutic payload stably conjugated to an NLS polypeptide (PP) comprising the sequence PAGKRVKLDG (SEQ ID NO:5).

[0206] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody -drug conjugate comprising a therapeutic payload stably conjugated to an NLS polypeptide (PP) comprising the sequence PGAKRVKLDG (SEQ ID NO:6)

[0207] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody -drug conjugate comprising a therapeutic payload stably conjugated to an NLS polypeptide (PP) comprising the sequence PAAKRAKLDG (SEQ ID NO:7).

[0208] In some embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody-drug conjugate comprising a therapeutic payload stably conjugated to an NLS polypeptide (PP) comprising the sequence PAAKRVKIDG (SEQ ID NO:8).

[0209] In some embodiments, the disease or disorder is a hyperproliferative disease or disorder. In some embodiments, the disease or disorder is a cancer. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the subject is a mammal. In some embodiments, the subject is a rodent. In some embodiments, the subject is a dog. In some embodiments, the subject is a primate. In some embodiments, the subject is a non -human primate. In some embodiments, the subject is a human. In some embodiments, the subject is a human having or suspected of having a hyperproliferative disease or disorder (e.g., a cancer or a tumor).In some embodiments, provided herein is a method of delivering a therapeutic payload to an intracellular target within a tumor cell, the method comprising contacting the tumor cell with a conjugate disclosed herein. In some embodiments, the conjugate is an antibody -drug conjugate of Formula I (e.g., Formula I- 10). In some embodiments, the polypeptide (PP) comprises a sequence selected from SEQ ID NOs: 1-8.

[0210] In some embodiments, the method of delivering a therapeutic payload to an intracellular target within a tumor cell comprises contacting the tumor cell with a peptide-drug conjugate of Formula III:PP-(L2)n-TP Formula III wherein:PP is a polypeptide;L2 is a linker;TP is a therapeutic payload; and n is 0 or 1.

[0211] In some embodiments, the polypeptide (PP) comprises a sequence selected from SEQ ID NOs: 1- 8.

[0212] In some embodiments, provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound or conjugate provided herein. In some embodiments, the conjugate has a structure according to Formula I and / or 1-10. In some embodiments, the subject is a mammal (e.g., a human). In some embodiments, the cancer is associated with overexpression of AXL, BCMA, CA9, CCR7, CD 123, CD166, CD19, CD20, CD205, CD22, CD25, CD276, CD30, CD33, CD37, CD46, CD70, CD74, CD79b, CEACAM5, CLDN18.2, CLDN6, CXCR4, DLL3, EFNA4, EGFR, EGFRvIII, ENPP3, EPCAM, EPHA2, F3, FOLR1, GPC3, GPNMB, GUCY2C, HER2, HER3, IGF1R, L1CAM, LIV1, LRRC15, MET, MSLN, MUC1, MUC16, NCAM1, NECTIN4, PSMA, PTK7, ROR1, ROR2, SDC1, SLC1A5, SLC34A2, TFRC, TIM1, TPBG, or TROP2. In some embodiments, the cancer is associated with overexpression of CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, tissue factor. In some embodiments, the cancer is a hematological cancer or a solid tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is non-Hodgkin’s lymphoma, breast cancer, ovarian cancer, or stomach cancer. In some embodiments, the cancer is large cell lymphoma, breast ductal carcinoma, breast adenocarcinoma, ovarian adenocarcinoma, or gastric carcinoma. Also provided herein is the use of a compound or conjugate provided herein for the treatment of a disease or a disorder, such as a cancer. Also provided herein is the conjugate of Formula I or Formula I- 10, or any compound or conjugate provided herein, for use in treating a disease or disorder. Also provided herein is a compound or conjugate disclosed herein (e.g., of Formula I, Formula I- 10, Formula III, or Formula V) for use in the manufacture of a medicament for treating a disease or disorder. In some embodiments, the disease or disorder is a cancer.Pharmacological Properties

[0213] In some embodiments, provided herein is a method for improving one or more pharmacological properties of an antibody-drug conjugate, or a component thereof (e.g., an antibody or a therapeutic payload). NLS polypeptides disclosed herein include those having SEQ ID NOS: 1-8. The method may comprise producing an antibody-drug conjugate disclosed herein. Examples of pharmacological properties may include, but are not limited to toxicity (e.g., therapeutic window) and distribution. Distribution may refer to the dispersion or dissemination of a substance (e.g., a therapeutic payload) throughout the fluids and tissues of the body. Advantageous properties of the conjugates disclosed herein include enhanced distribution (i.e., localization) of a therapeutic payload in a tumor cell (i.e., tumor enrichment).

[0214] The therapeutic window of a therapeutic payload disclosed herein may increase by at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20-fold, or more, when conjugated to an antibody-drug conjugate disclosed herein, compared to the unconjugated payload. In some embodiments, the therapeutic window of a conjugate disclosed herein (e.g., an antibody-drug conjugate, a polypeptide-drug conjugate, or a metabolite thereof) may be at least about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200 or 300-fold greater than the therapeutic window of an unconjugated therapeutic payload. In some embodiments, the therapeutic window of a conjugate disclosed herein is at least about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or greater, than the therapeutic window of an unconjugated drug. In some embodiments, the therapeutic index of a conjugate disclosed herein is at least about 50-fold, 100- fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or greater, than the therapeutic index of an unconjugated drug. As is evident from Table 6, ADCs provided herein can have over 2000-fold lower IC50 values for a cancer cell compared to a healthy cell. Calculated therapeutic indexes (IC50 neutrophil / IC50 cancer cell) are provided for a representative sample of ADCs provided herein are disclosed in Table 7.EXAMPLES

[0215] As used above, and throughout the description of the invention, the following abbreviations, unless otherwise indicated, shall be understood to have the following meanings:Abbreviations and AcronymsAb AntibodyBTFFH N'-bis(tetramethylene)formamidinium hexafluorophosphateBoc Tert-Butyloxycarbonyl protecting groupCHAPS 3 -[(3 -cholamidopropyl)dimethylammonio] - 1 -propanesulfonateCOMU l-[ l-(Cyano-2 -ethoxy-2 -oxoethylideneaminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphateDa DaltonsDAR drug-to-antibody ratioDEA DiethylamineDIPEA N-,N -diisopropylethylamineDMF DimethylformamideDMSO Dimethyl sulfoxideEDTA Ethylenediaminetetraacetic acid equiv or eq Equivalents (molar equivalents)ESI ElectrosprayFmoc Fluorenylmethoxycarbonyl protecting groupHIC Hydrophobic interaction characterizationHCC1954 HER2+ ductal carcinoma of the breast cellHRMS High-resolution mass spectraIC50 Mean concentration required to achieve 50% inhibitionLC / MS Liquid chromatography mass spectrometryLC / MSD Liquid chromatography mass selective detector mAb Monoclonal antibodyMai MaleimidoMK Megakaryote mM Milllimolar min MinutesM / Z Mass-to-chargeNAC N-acetyl cysteineNCI-N87 HER2 -positive gastric cancerNHS N-hydroxy succinimideNLS Nuclear localization sequence nm Nanometers nM NanomolarPbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl protecting groupPBS Phosphate-buffered salinePEG Polyethylene glycolRt Retention timeSEC Size exclusion chromatographySKBR3 HER2+ breast adenocarcinomaSKOV3 Human ovarian cancerSUDHL1 Large cell lymphoma tBu Tert-butylTCEP Tris(2-carboxyethyl)phosphineTFA Trifluoroacetic acidTOF Time-of-flightTris Tris(hydroxymethyl)aminomethaneUPLC Ultra-performance liquid chromatographyUV Ultraviolet v / v Volume / volumeAmino Acid abbreviationsAla (A) = Alanine Leu (L) = Leucine Arg (R) = Arginine Lys (K) = Lysine Asn (N) = Asparagine Met (M) = Methionine Asp (D) = Aspartic acid Nva = Norvaline Cys (C) = Cysteine Phe (F) = Phenylalanine Glu (E) = Glutamic acid Pro (P) = Proline Gin (Q) = Glutamine Ser (D) = Serine Gly(G) = Glycine Thr (T) = Threonine His (H) = Histidine Trp (W) = Tryptophane He (I) = Isoleucine Tyr (Y) = Tyrosine Vai (V) = Valine

[0216] The following examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.General Experimental

[0217] Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Protected polypeptide intermediates were custom synthesized by GenScript commercial vendor using standard solid phase peptide synthesis techniques. Reactions were monitored by LC / MS. High-resolution mass spectra (HRMS) were recorded on an Agilent LC / MSD TOF mass spectrometer by electrospray ionization time-of-flight (ESI-TOF) reflectron experiments.Analytical Methods

[0218] Analytical Method A.- Instrument: Agilent 1260 Infinity Lab LC / MSD- Column: Agilent Zorbax C18,5 mm, 4.6 x 50 mm- Column temperature: 45 C- Mobile Phase A: 0.1% TFA water- Mobile Phase B: 0.1% TFA acetonitrile- Gradient: 5% to 95% Mobile phase B over 3 min, hold at 95% B for 3 min, column wash for 4 min.- UV trace monitoring at 210nm and 254nm.- Positive and negative ionization modes monitoring molecular ions between 115Da-1200Da

[0219] Analytical Method B.- Instrument: Agilent 6230 TOF LC / MS- Column: Agilent Zorbax 300SB-C8,5 mm, 4.6 x 50 mm- Column temperature: 45 C- Mobile Phase A: 0.1% FA water- Mobile Phase B: 0.1% FA acetonitrile- Gradient: 5% to 95% Mobile phase B over 10 min, hold at 95% B for 5 min, column wash for 5 min.- UV trace monitoring at 210nm and 254nm.SynthesisScheme 1: General procedure for protected polypeptide coupling with payload.

[0220] Protected polypeptide COOH (1 equiv) was dissolved in dry DMF at 0.1 M concentration, followed by addition of diisopropylethylamine (DIPEA, 10 equiv) and COMU coupling reagent (5 equiv). Reaction mixture was stirred at room temperature for 10 min followed by addition of exatecan payload (2 equiv). Reaction mixture was stirred at room temperature overnight ( 16- 18h) and product formation was confirmed by LCMS. Crude product was purified using flash reverse phase chromatography using Biotage Isolera chromatography instrument equipped with Biotage Sfar Cl 8 column, gradient of 5%-95% Acetonitrile / Water with 0.1% formic acid. Purity and identity of the isolated products was determined by Analytical method A and is summarized in Table 1 below.Table 1.Scheme 2. General procedure for A-terminal Fmoc deprotection.[002211 '-tcrminal Fmoc protected polypeptide -exatecan intermediate was dissolved in 0.5-lml of 10% diethylamine in DMF and the reaction mixture was stirred for 2h at room temperature. Fmoc removal was confirmed by LCMS analytical method A as noted in the table below. The reaction mixture was diluted with distilled water at 1:25 v / v ratio, frozen and lyophilized. Resulting crude product was used for the next step.Table 2.Scheme 3. General procedure for the maleimido-PEG-carboxylic acid coupling with protected polypeptide-payload intermediate.

[0222] Maleimido-PEG4-carboxylic acid NHS ester (Quanta Biodesign) (1.5 equiv) was dissolved in dry DMF at 0.1 M concentration followed by addition of DIPEA (10 equiv) and crude N-terminal deprotected polypeptide -exatecan intermediate from Step 2. The reaction mixture was stirred at room temperature for 12-16 hr and product formation was observed by LCMS. Crude product was purified using flash reverse phase chromatography using Biotage Isolera chromatography instrument equipped with Biotage Sfar C18 column, gradient of 5%-95% Acetonitrile / Water with 0.1% formic acid. Purity and identity of the isolated products was determined by Analytical method A and is summarized in Table 3 below.Table 3.Scheme 4. General procedure for global deprotection of amino acid side chains.

[0223] Maleimido-PEG4-polypeptide-exatecan intermediate from Step 3 was treated with l-3ml of 95% TFA / Water for Ih at room temperature. The reaction mixture was diluted with distilled water at 1:25 v / v ratio, frozen and lyophilized. Crude product was taken up in DMF and purified using flash reverse phasechromatography using Biotage Isolera chromatography instrument equipped with Biotage Sfar C 18 column, gradient of 5%-95% Acetonitrile / Water with 0.1% formic acid. Purity and identity of the isolated products was determined by Analytical method B and is summarized in Table 4 below.Table 4.Scheme 5. General procedure for calicheamicin precursor molecule conjugation.

[0224] Step 1: Maleimido-PEG4-PAAKRVKLDG-carboxylic acid intermediate (1 eq) was dissolved in anhydrous DMF (0.05M) followed by addition of BTFFH (5 eq) and DIPEA (10 eq). The reaction was stirred for 30 min at room temperature followed by addition of Calicheamicin-amine ( 1.5eq) and additional DIPEA (10 eq). Reaction was stirred overnight protected from light and formation of the desired product was confirmed by LCMS. Crude product was purified using flash reverse phase chromatography using Biotage Isolera chromatography instrument equipped with Biotage Sfar C 18 column, gradient of 5%-95% Acetonitrile / Water with 0.1% formic acid. Purity and identity of the isolated products was determined by Analytical method A. HRMS Calculated Exact Mass 3620.5753, Found [M+H]+3621.5802.

[0225] Step 2: Global deprotection was done following general procedure for global deprotection of amino acid side chains as described above. The final product was purified using flash reverse phase chromatography using Biotage Isolera chromatography instrument equipped with Biotage Sfar C 18 column, gradient of 5%-95% Acetonitrile / Water with 0.1% formic acid. Purity and identity of the isolated products was determined by Analytical method A. HRMS Calculated Exact Mass 3112.3259, Found [M+H]+3113.3260.Scheme 6. General procedure for lurbinectedin precursor molecule conjugation.

[0226] Step 1: Maleimido-PEG4-PAAKRVKLDG-carboxylic acid intermediate (3 eq) was dissolved in anhydrous DMF (0.05M) followed by addition of BTFFH (6 eq) and DIPEA (5 eq). Reaction was stirred for 30 min at room temperature followed by addition of Lurbinectedin (leq) and additional DIPEA (5 eq). Reaction was stirred for 2 hours at room temperature and formation of the desired product was confirmed by LCMS. Crude product was purified using flash reverse phase chromatography using Biotage Isolera chromatography instrument equipped with Biotage Sfar Cl 8 column, gradient of 5%- 95% Acetonitrile / Water with 0.1% formic acid. Purity and identity of the isolated products was determined by Analytical method A. HRMS Calculated Exact Mass 2725.3199, [M-0H]+Calculated 2706.3093, Found [M-0H]+2706.3100.

[0227] Step 2: Global deprotection was done following general procedure for global deprotection of amino acid side chains as described above. The final product was purified using flash reverse phase chromatography using Biotage Isolera chromatography instrument equipped with Biotage Sfar C 18column, gradient of 5%-95% Acetonitrile / Water with 0.1% formic acid. Purity and identity of the isolated products was determined by Analytical method A. HRMS Calculated Exact Mass 2217.0704, [M-0H]+Calculated 2198.0598, Found [M-0H]+2198.0600.Scheme 7. General Synthesis of NLS Linker-Dye Compounds

[0228] Step 1: HiLyte™ Fluor 647 succinimidyl ester (SE) (Anaspec, AS-81256, 1 eq) was dissolved in anhydrous DMF to obtain a lOOmM solution, followed by addition of Boc-amino-PEG4-amine (Broadpharm, BP-22602, 1.5eq) and N, A-diisoprophylcthylaminc (4 eq). Reaction mixture was stirred at room temperature for 30 min protected from light and the product formation was confirmed by LCMS. Crude product was purified using reverse phase chromatography with a gradient of 5-95% water / acetonitrile with 0.1% formic acid. LCMS [M+H]+expected 1524, found [M+2FI]2+763.0.

[0229] Boc-amine-PEG4-HiLyte Fluor 647 from previous step was deprotected with 10% TFA / DCM for Ih at room temperature protected from light. Product was concentrated and dried in high vacuum overnight. Crude product was used for the next step without purification. Product identity was confirmed by LCMS, Expected [M+H]+1424, found [M+2H]2+713.0.

[0230] Step 2: Boc-amine-PEG4-HiLyte Fluor 647 from previous step was deprotected with 10% TFA / DCM for Ih at room temperature protected from light. Product was concentrated and dried in high vacuum overnight. Crude product was used for the next step without purification. Product identity was confirmed by LCMS, Expected [M+H]+1424, found [M+2H]2+713.0.

[0231] Step 3: Maleimido-PEG4-PAAKRVKLD-carboxylic acid intermediate (7-1) (2 eq) was dissolved in anhydrous DMF (100 mM) followed by addition of BTFFH (3 eq) and DIPEA (5 eq) and stirred at room temperature for 30 min. Deprotected amine -PEG4-HiLyte Fluor 647 from the previous step (1 eq) was added to the reaction mixture followed by addition of additional DIPEA (5 eq). Reaction was stirred overnight at room temperature protected from light. Product formation was confirmed by LCMS. Crude product (7-2) was purified using reverse phase chromatography with a gradient of 5-95% water / acetonitrile with 0.1% formic acid. LCMS [M+H]+expected 3311.3, found[M+3H]3+1104.4.

[0232] Step 4: Final deprotection of the Maleimido-PEG4-PAAKRVKLD-PEG4-HiLyte Fluor 647 (7-3) was carried out using 95% TFA / water for 2hrs at room temperature protected from light. Crude product was purified using reverse phase chromatography with a gradient of 5-95% water / acetonitrile with 0.1% formic acid. LCMS [M+H]+expected 2802.6, found [M+3H]3+935.

[0233] Reference Linker-Dye Compound: Synthesis of Mal-PEG2-Val-Cit-PAB-HiLyte Fluor 647

[0234] Mai -PEG2-Val-Cit-PAB-PNP (Broadpharm®, 1 eq) was dissolved in anhydrous DMF (lOOmM) followed by addition of HiLyte Fluor 647 amine (AnaSpec,AS-8256, 1 eq) and DIPEA (4 eq). Reaction mixture was stirred at room temperature protected from light for 2hrs. Product formation was confirmed by LCMS. Crude product was purified using reverse phase chromatography with a gradient of 5-95% water / acetonitrile with 0.1% formic acid. LCMS [M+H]+expected 1792.1, found[M+3H]3+896.6.

[0235] Conjugation of the Linker-Dyes

[0236] Trastuzumab antibody was first conjugated to AF488 using Abeam AF488 labeling kit (ab236553) per manufacturer’s instructions. AF488-labeled antibody was then subjected to TCEP mediated disulfide reduction (10 eq of TCEP) followed by conjugation to either Mal-Val-Cit-PAB- HiLyteFluor 647 or Mal-NLS Peptide 4-2-HiLyteFluor 647 (2 eq per each free SH) for 2h at room temperature. Excess unconjugated dye was removed using Zeba Spin Desalting columns for Biotin and Dye Removal (Pierce). Antibody conjugates were characterized using UV-VIS to determine dye / antibody ratio and results are summarized in the table below.Exemplary ADC Precursor Molecules (PM)Example PM-1 comprises SEQ ID NO. 1 (PGGKRVKLD)Example PM-2 corresponds to Int. 4-5 in Table 4 and comprises SEQ ID NO. 2 (PGGKRVKLDG)Example PM-3 corresponds to Int. 4-2 in Table 4 and comprises SEQ ID NO. 3 (PAAKRVKLD)Example PM-4 corresponds to Int. 4-1 in Table 4 and comprises SEQ ID NO. 4 (PAAKRVKLDG)Example PM-5 corresponds to Int. 4-3 in Table 4 and comprises SEQ ID NO. 5 (PAGKRVKLDG)Example PM-6 corresponds to Int. 4-4 in Table 4 and comprises SEQ ID NO. 6 (PGAKRVKLDG)Example PM-7 corresponds to Int. 4-6 in Table 4 and comprises SEQ ID NO. 7 (PAAKRAKLDG)Example PM-8 corresponds to Int. 4-7 in Table 4 and comprises SEQ ID NO. 8 (PAAKRVKIDG)Example PM-9 corresponds to Int. 4-8 in Table 4 and comprises SEQ ID NO. 9 (VAAKRVKLDG)Example PM-10 corresponds to Int. 4-9 in Table 4 and comprises SEQ ID NO. 14 (PKKKRKV)Example PM-11 corresponds to Int. 4-10 in Table 4 and comprises SEQ ID NO. 15 (PKKKRKVG)Example PM-12 corresponds to Scheme 5 in Table 4 and comprises SEQ ID NO. 4 (PAAKRVKLDG)Example PM-13 corresponds to Scheme 7 in Table 4 and comprises SEQ ID NO. 4 (PAAKRVKLDG)Scheme 8. Conjugation of a precursor molecule to an antibody or antigen-binding fragment.

[0237] The mAb was pH adjusted with 200 mM Tris, 5 mM EDTA, pH 8.5 (10% if in PBS, 30% if in proA) then reduced with TCEP 4-10 equiv at 37°C for Ih. The number of free thiols was quantified using Ellman’s test. 10% DMSO and 2 molar excess of linker-drug per mol of free thiol were added, the solution was vortexed and let sit for 2 hours at room temperature. Then, 1.2 mol A'-acctylcystcinc (NAC) per mol linker-drug was added, the solution was vortexed and let sit for 20 min at room temperature. The ADC was then buffer exchanged 3X into PBS pH7.4 to remove free linker-drug.Exemplary Antibody-Drug Conjugates (ADC)Example ADC-1:Example ADC-2:Example ADC-6:Example ADC- 10

[0238] In Tables 5, 6, and 7, reference is made to the preceding Example ADCs 2-13, each of which has the structure provided above, in combination with an antibody that is IgGl, brentuximab, or trastuzumab. Also provided for comparison is a Reference ADC, wherein the polypeptide is not an NLS polypeptide, but instead consists of the sequence GGFG.Characterization of ADC Examples

[0239] The drug-to-antibody ratio (DAR, also denoted as subscript “p” above) was determined by either hydrophobic interaction chromatography (HIC) or using mass spectrometry of intact ADC. Aggregation was determined by size exclusion chromatography (SEC).Hydrophobic Interaction Characterization of ADC.

[0240] HIC was conducted using a Poly Propyl A column (PolyLC) with 1.5 M ammonium sulfate and 25 mM potassium phosphate in water as mobile phase A, and 0.25% w / v CHAPS and 25 mM potassium phosphate in water as mobile phase B. Samples were injected directly onto the column, where a gradient of 0-100% mobile phase B was applied over 15 min. UV signal at 280 nm was collected, and the chromatogram analyzed for unconjugated antibody and higher DAR species. DAR calculations were performed by integrating the area under the HIC curve of the previously established peaks (DAR = 0, DAR = 1, DAR = 2, DAR = 4, etc) and calculating the % of each peak.Mass Spectrometry Characterization of Intact ADC.

[0241] The non-reduced masses of antibody-drug conjugates were determined on Agilent 6230 TOF LC / MS equipped with electrospray ionization (ESI) source, which was directly coupled to high- performance liquid chromatography Agilent 1260 system. Samples were first diluted to 1 mg / mL then analyzed in their non-reduced form. The proteins are separated on a reverse phase column (Zorbax 300SB-C8, 5 mm, 4.6 x 50 mm) with a denaturing mobile phase system. Mobile Phase A is 0.1% (v / v) formic acid in water. Mobile phase B is 0. 1% (v / v) formic acid in 80% (v / v) 2-propanol, 10% (v / v) acetonitrile, 10% (v / v) water (mobile phase B). The MS spectra of each protein are averaged and then deconvoluted to obtain the average mass and monoisotopic mass.Size Exclusion Chromatography Characterization of ADC Aggregation.

[0242] Size-exclusion chromatography (SEC) was used to characterize size heterogeneity of the antibody-drug conjugates. The analysis employed an Acquity 1.7 pm, 4.6 x 300 mm UPLC BEH200 SEC column with 25 mM sodium phosphate, pH 6.5, 500 mM L-arginine, and 10% isopropanol (IP A) in water as mobile phase. Samples were injected neat and the mobile phase was applied isocratically at 0.2 mL / min for 22 min. The UV signal at 280 nm was collected and the peak area was used to calculate the extent of aggregation and fragmentation of the ADC. Full characterization data is presented in Table 5 below.Table 5.Adherent Cells Cytotoxicity Assay.

[0243] Adherent cells were cultured in a T75 flask to ~ 50-80% confluency and harvested with trypsin into a single cell suspension. Three to five thousand (3,000-5,000) cells per well were seeded in tissue culture plates in 50 pL / well culture media and incubated at 37°C for 18-24 hours. Serial dilutions of ADCs in culture media were then dispensed into the plates at 50 pL / well. After plating and treatment, cells were returned to the incubator for an additional 3 to 5 days. CellTiter-Glo reagent was prepared per manufacturer’s instructions and added at 100 pL / well to the cultures. CellTiter-Glo allows for relative enumeration of metabolically active cells by quantifying intracellular ATP concentrations. After 5 minutes of incubation with CellTiter-Glo at ambient room temperature, the clear bottom black assay plates were then read in a luminometer within 30 minutes, or 125 pL / well of the Cell Titer Glo / cell lysate solution was transferred into black assay plates and read. Luminescence readings obtained from cultures that did not receive any treatment (cell culture media only) were set as 100% control and all other luminescence values were normalized to these controls (e.g., Normalized RLU, relative luminescence unit). Luminescence signals were detected using a standard plate reader and IC50s were calculated by logistic nonlinear regression using GraphPad Prism (GraphPad Software, San Diego, CA).Suspension Cells Cytotoxicity Assay.

[0244] Suspension cells were cultured in a T75 flask as a single cell suspension. Three to five thousand(3,000-5,000) cells per well were seeded in tissue culture plates in 50 pL / well culture media andincubated at 37°C for 18-24 hours. Serial dilutions of ADCs in culture media were then dispensed into the plates at 50 pL / well. After plating and treatment, cells were returned to the incubator for an additional 3-5 days. CellTiter-Glo reagent was prepared per manufacturer’s instructions and added at 100 pL / well to the cultures. CellTiter-Glo allows for relative enumeration of metabolically active cells by quantifying intracellular ATP concentrations. After 5 minutes of incubation with CellTiter-Glo at ambient room temperature, the clear bottom black assay plates were then read in a luminometer within 30 minutes, or 125 pL / well of the Cell Titer Glo / cell lysate solution was transferred into black assay plates and read. Luminescence readings obtained from cultures that did not receive any treatment (cell culture media only) were set as 100% control and all other luminescence values were normalized to these controls (e.g., Normalized RLU, relative luminescence unit). Luminescence signals were detected using a standard plate reader and IC50s were calculated by logistic nonlinear regression using GraphPad Prism (GraphPad Software, San Diego, CA).Human Neutrophils Cytotoxicity Assay.

[0245] Human CD34+ myeloid progenitor cells were seeded at 3,000 cells per well in in growth media supplemented with 10 ng / ml human recombinant IL-3 and 30 ng / ml human recombinant G-CSF (both Peprotech, Cranbury, NJ) in 96-well plates. Testing ADCs or free drugs were added to each well in duplicates or triplicate at final concentrations indicated. After 3-4 days of culture, half of the medium (100 ul) was carefully removed and CellTiter-Glo 2.0 (Promega, Madison, WI) viability assays were performed according to the manufacturer’s protocol. Luminescence signals were detected using a standard plate reader and IC50s were calculated by logistic nonlinear regression using GraphPad Prism (GraphPad Software, San Diego, CA).

[0246] Results are summarized in Table 6 below. All experiments were repeated at least three times with each datapoint in duplicate. FIGs. 1-4 show the normalized RLU, relative luminescence unit, of SKBR3 (FIG. 1), HCC1954 (FIG. 2), Neutrophil (FIG. 3), and Megakaryocyte (FIG. 4) cells following administration of Example ADC-12 and Example ADC-13, each ADC having been conjugated to either an IgGl or trastuzumab antibody. The trastuzumab-linked ADC-12 and ADC-13 significantly diminish RLU in the SKBR3 and HCC1954 breast cancer cell lines, but not in neutrophil or MK cells.Table 6. IC50 (nM) for Reference and Example ADCs vs. Various Cell TypesMK = megakaryocyte, - = no dataTable 7. Therapeutic Indexes for Example ADCs coupled to an anti-HER2 antibody (trastuzumab)The values above represent minimum therapeutic indexes, as the values were calculated based on an IC50 of 200 nM, which is the upper threshold measured by the assay. The IC50 for human neutrophils in the examples is >200 nM, unless noted otherwise. imaging Study in SKBR3 Cells

[0247] SKBR3 cells were cultured per ATCC guidelines in ATCC-formulated McCoy's 5a Medium Modified, Catalog No. 30-2007 and seeded at 2000 cells / well into the F-bottom (chimney well) mCLEAR black 96-well plates (Greiner, Catalog # 655096). Upon cell attachment the media was exchanged with the media without the phenol red and containing corresponding antibody -dye conjugates at 100 ug / ml concentration followed by th incubation on ice. The cells were then gently washed with the fresh media without phenol red in order to remove any unbound antibody-dye conjugates. Cells were incubated at 37C in the tissue culture incubator with short imaging sessions at 2h, 8h, 24h and 48h post incubation with antibody-dye conjugates. Imaging was performed using ImageXpress Micro Confocalinstrument by Molecular Devices and images were processed using manufacturer’s software with z-plane going through the center of the cell. Each antibody-dye conjugate was tested in triplicate. Representative results are summarized in FIG. 5. Cell nucleus is stained blue for clarity. AF488 is visible as green and HF647 is visible as red, colocalization of AF488 and HF647 produces yellow-green color. At 2h antibody-dye constructs both with Val-Cit and NLS linker are observed localizing to the cell surface. At 8h timepoint the internalization of the conjugates is observed and apparent accumulation in lysosomal compartments can be seen. At 8 h the conjugate prepared with NLS peptide-HF647 shows separation of the red dye from the green suggesting cleavage of the linker-dye from the antibody that remains with the AF488 green dye, the red color is seen overlapping with the blue of the nucleus. At 24h timepoint cells incubated with the val-cit-HF647 show no more of the yellow or red color suggesting full removal of the red dye from the cell, while in the cell incubated with the NLS-HF647 conjugate red color is strongly present and is overlapping with the nucleus. At 48h timepoint small amounts of the AF488 conjugated antibody remain in the cells incubated with Val-Cit conjugate and strong presence of the NLS-HF647 is evident in the cells incubated with NLS-HF647 antibody-dye conjugate. FIG. 5 shows that use of an NLS linker promotes longer retention of payload intracellularly and facilitates nuclear localization.Table 8. Nuclear Localization Sequence ID NOs.Table 9. Antibody Sequence ID NOs.Embodiments

[0248] Embodiment 1 A composition comprising a polypeptide comprising the sequence:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1; or a pharmaceutically acceptable salt thereof.

[0249] Embodiment 2 The composition of embodiment 1, or the pharmaceutically acceptable salt thereof, wherein x1is G or A.

[0250] Embodiment 3 The composition of embodiment 1 or 2, or the pharmaceutically acceptable salt thereof, wherein x2is G or A.

[0251] Embodiment 4 The composition of any one of embodiments 1-3, or the pharmaceutically acceptable salt thereof, wherein x3is A or V.

[0252] Embodiment 5 The composition of any one of embodiments 1-4, or the pharmaceutically acceptable salt thereof, wherein x4is L or I.

[0253] Embodiment 6 The composition of any one of embodiments 1-5, or the pharmaceutically acceptable salt thereof, wherein a is 0.

[0254] Embodiment 7 The composition of any one of embodiments 1-5, or the pharmaceutically acceptable salt thereof, wherein a is 1.

[0255] Embodiment 8 The composition of embodiment 7, or the pharmaceutically acceptable salt thereof, wherein x5is G or A.

[0256] Embodiment 9 The composition of any one of embodiments 1-8, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence:P-x1-x2-K-R-x3-K-x4-D SEQ ID NO 11 wherein: x1is G or A; x2is G or A; x3is A or V; and x4is L or I.

[0257] Embodiment 10 The composition of any one of embodiments 1-9, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence:P-x -x2-I<-R-x -K-x4-D-x SEQ ID NO 12 wherein: x1is G or A; x2is G or A; x3is A or V; x4is L or I; and x5is G or A.

[0258] Embodiment 11 The composition of any one of embodiments 1-10, or the pharmaceutically acceptable salt thereof, wherein x1is G.

[0259] Embodiment 12 The composition of any one of embodiments 1-10, or the pharmaceutically acceptable salt thereof, wherein x1is A.

[0260] Embodiment 13 The composition of any one of embodiments 1-12, or the pharmaceutically acceptable salt thereof, wherein x2is G.

[0261] Embodiment 14 The composition of any one of embodiments 1-12, or the pharmaceutically acceptable salt thereof, wherein x2is A.

[0262] Embodiment 15 The composition of any one of embodiments 1-14, or the pharmaceutically acceptable salt thereof, wherein x3is V.

[0263] Embodiment 16 The composition of any one of embodiments 1-15, or the pharmaceutically acceptable salt thereof, wherein x4is L.

[0264] Embodiment 17 The composition of any one of embodiments 1-16, or the pharmaceutically acceptable salt thereof, wherein x5is G.

[0265] Embodiment 18 The composition of any one of embodiments 1-6, or the pharmaceutically acceptable salt thereof, wherein x5is absent.

[0266] Embodiment 19 The composition of any one of embodiments 1-10, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence:P-x1-x2-K-R-V-K-L-D-(G)aSEQ ID NO 13 wherein x1and x2are each independently A or G; and a is 0 or 1.

[0267] Embodiment 20 The composition of embodiment 19, or the pharmaceutically acceptable salt thereof, wherein x1and x2are each A.

[0268] Embodiment 21 The composition of embodiment 19, or the pharmaceutically acceptable salt thereof, wherein x1and x2are each G.

[0269] Embodiment 22 The composition of any one of embodiments 1-6, 9, or 11, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises a sequence selected from SEQ ID NOS: 1-8.

[0270] Embodiment 23 The composition of any one of embodiments 1-9, 11, 13, 15, 16, 18, 19, 21, or 22, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 1: PGGKRVKLD.

[0271] Embodiment 24 The composition of any one of embodiments 1-5, 7-11, 13, 15, 16, 17, 19, 21 or 22, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 2: PGGKRVKLDG.

[0272] Embodiment 25 The composition of any one of embodiments 1-6, 9, 12, 14, 15, 16, 18, 19, 20 or 22, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 3: PAAKRVKLD.

[0273] Embodiment 26 The composition of any one of embodiments 1-5, 7-10, 12, 14, 15, 16, 17, 19, 20 or 22, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 4: PAAKRVKLDG.

[0274] Embodiment 27 The composition of any one of embodiments 1-5, 7-10, 12, 13, 15, 16, 17, 19, or 22, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 5: PAGKRVKLDG.

[0275] Embodiment 28 The composition of any one of embodiments 1-5, 7-11, 14, 15, 16, 17, 19, or 22, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 6: PGAKRVKLDG.

[0276] Embodiment 29 The composition of any one of embodiments 1-5, 7-10, 12, 14, 16, 17, 19, 20 or 22, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 7: PAAKRAKLDG.

[0277] Embodiment 30 The composition of any one of embodiments 1-5, 7-10, 12, 14, 15, 17, 19, 20 or 22, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 8: PAAKRVKIDG.

[0278] Embodiment 31 The composition of any one of embodiments 1-30, or the pharmaceutically acceptable salt thereof, further comprising an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment and the polypeptide are conjugated via a bond, or via a linker.

[0279] Embodiment 32 The composition of embodiment 31, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment is conjugated to the polypeptide via a bond.

[0280] Embodiment 33 The composition of embodiment 31, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment is conjugated to the polypeptide via a linker.

[0281] Embodiment 34 The composition of any one of embodiments 31-33, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment is conjugated to P of the polypeptide.

[0282] Embodiment 35 The composition of embodiment 33 or 34, or the pharmaceutically acceptable salt thereof, wherein the linker comprises one or more polyethylene glycol or polyethyleneimine groups.

[0283] Embodiment 36 The composition of embodiment 35, or the pharmaceutically acceptable salt thereof, wherein the linker comprises 1 to 12 polyethylene glycol groups.

[0284] Embodiment 37 The composition of embodiment 36, or the pharmaceutically acceptable salt thereof, wherein the linker comprises 2 to 8 polyethylene glycol groups.

[0285] Embodiment 38 The composition of embodiment 37, or the pharmaceutically acceptable salt thereof, wherein the linker comprises 3 to 6 polyethylene glycol groups.

[0286] Embodiment 39 The composition of any one of embodiments 33-38, or the pharmaceutically acceptable salt thereof, wherein the linker comprises a cleavable group (e.g., a chemically-labile group, a pH-sensitive group, a protease -cleavable group, a self-immolative group, a hydrolysable group, etc.).

[0287] Embodiment 40 The composition of any one of embodiments 33-38, or the pharmaceutically acceptable salt thereof, wherein the linker is a non-cleavable linker.

[0288] Embodiment 41 The composition of any one of embodiments 33-40, or the pharmaceutically acceptable salt thereof, wherein the linker comprises a non-cleavable heteroalkyl and / or heterocyclic group.

[0289] Embodiment 42 The composition of any one of embodiments 33-41, or the pharmaceutically acceptable salt thereof, wherein the linker comprises 1 to 12 amino acids.

[0290] Embodiment 43 The composition of any one of embodiments 31-42, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment comprises a natural or unnatural amino acid, and wherein the linker is conjugated to the antibody or antigen-binding fragment via the natural or unnatural amino acid.

[0291] Embodiment 44 The composition of any one of embodiments 31-43, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment comprises a cysteine, a lysine, or a glutamine, and wherein the linker is conjugated to the antibody or antigen-binding fragment via the cysteine, lysine, or glutamine.

[0292] Embodiment 45 The composition of any one of embodiments 31-44, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment comprises a cysteine, and wherein the linker is conjugated to the antibody or antigen-binding fragment via the cysteine.

[0293] Embodiment 46 The composition of any one of embodiments 31-45, or the pharmaceutically acceptable salt thereof, wherein the antibody comprises a constant region, and wherein the linker is conjugated to the antibody via the constant region.

[0294] Embodiment 47 The composition of any one of embodiments 31-46, or the pharmaceutically acceptable salt thereof, wherein the antibody comprises an Fc region.

[0295] Embodiment 48 The composition of embodiment 47, or the pharmaceutically acceptable salt thereof, wherein the linker is conjugated to the antibody via the Fc region.

[0296] Embodiment 49 The composition of embodiment 47, or the pharmaceutically acceptable salt thereof, wherein the Fc region has reduced effector function.

[0297] Embodiment 50 The composition of any one of embodiments 31-49, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment binds to a target antigen.

[0298] Embodiment 51 The composition of embodiment 50, or the pharmaceutically acceptable salt thereof, wherein the target antigen is expressed on a tumor cell.

[0299] Embodiment 52 The composition of embodiment 50 or 51, or the pharmaceutically acceptable salt thereof, wherein the target antigen is AXL, BCMA, CA9, CCR7, CD 19, CD20, CD22, CD25, CD30, CD33, CD37, CD46, CD70, CD74, CD79b, CD123, CD166, CD205, CD276, CEACAM5, CLDN18.2, CLDN6, CXCR4, DLL3, EFNA4, EGFR, EGFRvIII, ENPP3, EPCAM, EPHA2, F3, FOLR1, GPC3, GPNMB, GUCY2C, HER2, HER3, IGF1R, L1CAM, LIV1, LRRC15, MET, MSLN, MUC1, MUC16, NCAM1, NECTIN4, PSMA, PTK7, R0R1, ROR2, SDC1, SLC1A5, SLC34A2, TFRC, TIM1, TPBG, or TROP2.

[0300] Embodiment 53 The composition of any one of embodiments 31-52, or the pharmaceutically acceptable salt thereof, wherein the target antigen is CD 19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, or tissue factor.

[0301] Embodiment 54 The composition of any one of embodiments 1-49, or the pharmaceutically acceptable salt thereof, wherein the antibody is an IgGl subclass.

[0302] Embodiment 55 The composition of any one of embodiments 1-54, or the pharmaceutically acceptable salt thereof, further comprising a therapeutic payload, wherein the therapeutic payload and the polypeptide are conjugated via a bond, or via a second linker.

[0303] Embodiment 56 The composition of embodiment 55, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is conjugated to D or x5of the polypeptide.

[0304] Embodiment 57 The composition of embodiment 55 or 56, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is conjugated to the polypeptide via a bond.

[0305] Embodiment 58 The composition of embodiment 55 or 56, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is conjugated to the polypeptide via a second linker.

[0306] Embodiment 59 The composition of embodiment 58, or the pharmaceutically acceptable salt thereof, wherein the second linker is a C1-C12 alkyl or heteroalkyl linker.

[0307] Embodiment 60 The composition of embodiment 58, or the pharmaceutically acceptable salt thereof, wherein the second linker is a non-cleavable linker.

[0308] Embodiment 61 The composition of embodiment 58, or the pharmaceutically acceptable salt thereof, wherein the second linker is a cleavable linker.

[0309] Embodiment 62 The composition of embodiment 58, or the pharmaceutically acceptable salt thereof, wherein the second linker is a polypeptide linker.

[0310] Embodiment 63 The composition of any one of embodiments 55-62, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a cytotoxic payload.

[0311] Embodiment 64 The composition of any one of embodiments 55-63, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is cytotoxic to a tumor cell upon internalization into the tumor cell.

[0312] Embodiment 65 The composition of any one of embodiments 55-64, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload comprises a pyrrolobenzodiazepine, duocarmycin, auristatin, maytansinoid, uncialamycin, dynemicin, thailanstatin, camptothecin, exatecan, tubulysin compound, lurbinectedin, trabectedin, safracin, lenalidomide, eribulin, vincristine, vinblastine, vindesine, vinorelbine, an epothilone, ataxane (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), a cryptophycin, a hemiasterlin, an anthracyclin, a bisnaphthylamide (e.g., elinafide), or a cytotoxic molecular glue / PROTAC compound.

[0313] Embodiment 66 The composition of any one of embodiments 55-65, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload comprises a antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA-alkylating drug, a DNA-binding drug, a DNA-cleaving drug, or an RNA polymerase inhibitor.

[0314] Embodiment 67 A conjugate or a pharmaceutically acceptable salt thereof comprising an antibody and an active agent (e.g., a therapeutic payload, a detectable group, a diagnostic agent, etc.) conjugated via a linker, wherein the linker comprises a polypeptide, and wherein the polypeptide comprises a nuclear localization sequence.

[0315] Embodiment 68 The conjugate of embodiment 67, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises 9 or 10 amino acids.

[0316] Embodiment 69 The conjugate of embodiment 67 or 68, or the pharmaceutically acceptable salt thereof, wherein the active agent is a therapeutic payload (e.g., a drug).

[0317] Embodiment 70 The conjugate of embodiment 69, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is cytotoxic to a tumor cell.

[0318] Embodiment 71 The conjugate of any one of embodiments 67-70, or the pharmaceutically acceptable salt thereof, wherein the antibody is linked to the polypeptide N-terminally.

[0319] Embodiment 72 The conjugate of any one of embodiments 67-71, or the pharmaceutically acceptable salt thereof, wherein the antibody is linked to the polypeptide N-terminally via a bond or a linker.

[0320] Embodiment 73 The conjugate of any one of embodiments 67-72, or the pharmaceutically acceptable salt thereof, wherein the active agent is linked to the polypeptide C- terminally.

[0321] Embodiment 74 The conjugate of any one of embodiments 67-73, or the pharmaceutically acceptable salt thereof, wherein the active agent is linked to the polypeptide C- terminally via a bond or a linker.

[0322] Embodiment 75 The conjugate of any one of embodiments 67-74, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein x1, x2,x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0323] Embodiment 76 A conjugate (e.g, of any one of embodiments 67-75), or a pharmaceutically acceptable salt thereof, having a structure of Formula 1-10:AB-[(Ll)m-PP-(L2)n-TP]p Formula I- 10 wherein:AB is an antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide comprising SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein x1, x2,x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1;L2 is a linker;TP is a therapeutic payload; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

[0324] Embodiment 77 The conjugate of embodiment 76, or the pharmaceutically acceptable salt thereof, wherein L 1 is a cleavable linker.

[0325] Embodiment 78 The conjugate of embodiment 76, or the pharmaceutically acceptable salt thereof, wherein L 1 is a non-cleavable linker.

[0326] Embodiment 79 The conjugate of any one of embodiments 76-78, or the pharmaceutically acceptable salt thereof, wherein LI comprises 0 to 12 amino acids.

[0327] Embodiment 80 The conjugate of any one of embodiments 76-79, or the pharmaceutically acceptable salt thereof, wherein LI comprises 1 to 8 amino acids.

[0328] Embodiment 81 The conjugate of any one of embodiments 76-79, or the pharmaceutically acceptable salt thereof, wherein LI comprises 1 to 4 amino acids.

[0329] Embodiment 82 The conjugate of any one of embodiments 76-79, or the pharmaceutically acceptable salt thereof, wherein LI comprises 8 to 12 amino acids.

[0330] Embodiment 83 The conjugate of any one of embodiments 76-82, or the pharmaceutically acceptable salt thereof, wherein LI comprises 0 to 12 polyethylene glycol monomers.

[0331] Embodiment 84 The conjugate of any one of embodiments 76-83, or the pharmaceutically acceptable salt thereof, wherein LI comprises 1 to 8 polyethylene glycol monomers.

[0332] Embodiment 85 The conjugate of any one of embodiments 76-83, or the pharmaceutically acceptable salt thereof, wherein LI comprises 2 to 6 polyethylene glycol monomers.

[0333] Embodiment 86 The conjugate of any one of embodiments 76-85, or the pharmaceutically acceptable salt thereof, wherein LI comprises a protein -coupling or protein-coupled group.

[0334] Embodiment 87 The conjugate of any one of embodiments 76-86, or the pharmaceutically acceptable salt thereof, wherein LI comprises a lysine-reactive / lysine-reacted group, a cysteine-reactive / cysteine-reacted group, or a glutamine-reactive / glutamine-reacted group.

[0335] Embodiment 88 The conjugate of any one of embodiments 76-87, or the pharmaceutically acceptable salt thereof, wherein LI comprises a homoglycine or a C1-12 alkylene group.

[0336] Embodiment 89 The conjugate of any one of embodiments 76-88, or the pharmaceutically acceptable salt thereof, wherein LI comprises the group:

[0337] Embodiment 90 The conjugate of any one of embodiments 76-89, or the pharmaceutically acceptable salt thereof, wherein LI comprises the group:

[0338] Embodiment 91 The conjugate of any one of embodiments 76-90, or the pharmaceutically acceptable salt thereof, wherein x1is G or A.

[0339] Embodiment 92 The conjugate of any one of embodiments 76-91, or the pharmaceutically acceptable salt thereof, wherein x2is G or A.

[0340] Embodiment 93 The conjugate of any one of embodiments 76-92, or the pharmaceutically acceptable salt thereof, wherein x3is A or V.

[0341] Embodiment 94 The conjugate of any one of embodiments 76-93, or the pharmaceutically acceptable salt thereof, wherein x4is L or I.

[0342] Embodiment 95 The conjugate of any one of embodiments 76-94, or the pharmaceutically acceptable salt thereof, wherein a is 0.

[0343] Embodiment 96 The conjugate of any one of embodiments 76-94, or the pharmaceutically acceptable salt thereof, wherein a is 1, and x5is G or A.

[0344] Embodiment 97 The conjugate of any one of embodiments 76-94, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 11 :P-x’-x^K-R-x^K-x^D SEQ ID NO 11 wherein: x1is G or A; x2is G or A; x3is A or V; and x4is L or I. embodiments

[0345] Embodiment 98 The conjugate of any one of embodiments 76-94, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 12:P-x -x2-I<-R-x -K-x4-D-x SEQ ID NO 12 wherein: x1is G or A; x2is G or A;x3is A or V; x4is L or I; and x5is G or A.

[0346] Embodiment 99 The conjugate of any one of embodiments 76-94, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 13:P-x1-x2-K-R-V-K-L-D-(G)aSEQ ID NO 13 wherein x1and x2are each independently A or G; and a is 0 or 1.

[0347] Embodiment 100 The conjugate of embodiment 99, or the pharmaceutically acceptable salt thereof, wherein x1and x2are each A.

[0348] Embodiment 101 The conjugate of embodiment 99, or the pharmaceutically acceptable salt thereof, wherein x1and x2are each G.

[0349] Embodiment 102 The conjugate of any one of embodiments 76-94, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises a sequence selected from SEQ ID NOS: 1-8.

[0350] Embodiment 103 The conjugate of any one of embodiments 76-94 or 102, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 1: PGGKRVKLD.

[0351] Embodiment 104 The conjugate of any one of embodiments 76-94 or 102, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 2: PGGKRVKLDG.

[0352] Embodiment 105 The conjugate of any one of embodiments 76-94 or 102, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 3: PAAKRVKLD.

[0353] Embodiment 106 The conjugate of any one of embodiments 76-94 or 102, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 4: PAAKRVKLDG.

[0354] Embodiment 107 The conjugate of any one of embodiments 76-94 or 102, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 5: PAGKRVKLDG.

[0355] Embodiment 108 The conjugate of any one of embodiments 76-94 or 102, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 6: PGAKRVKLDG.

[0356] Embodiment 109 The conjugate of any one of embodiments 76-94 or 102, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 7: PAAKRAKLDG.

[0357] Embodiment 110 The conjugate of any one of embodiments 76-94 or 102, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 8: PAAKRVKIDG.

[0358] Embodiment 111 The conjugate of any one of embodiments 76-110, or the pharmaceutically acceptable salt thereof, wherein L2 is a cleavable linker.

[0359] Embodiment 112 The conjugate of any one of embodiments 76-110, or the pharmaceutically acceptable salt thereof, wherein L2 comprises a self-immolative group.

[0360] Embodiment 113 The conjugate of any one of embodiments 76-110, or the pharmaceutically acceptable salt thereof, wherein L2 is a non-cleavable linker.

[0361] Embodiment 114 The conjugate of any one of embodiments 76-110, or the pharmaceutically acceptable salt thereof, wherein L2 comprises 0 to 12 amino acids.

[0362] Embodiment 115 The conjugate of any one of embodiments 76-114, or the pharmaceutically acceptable salt thereof, wherein L2 comprises 0 to 12 polyethylene glycol monomers.

[0363] Embodiment 116 The conjugate of any one of embodiments 76-115, or the pharmaceutically acceptable salt thereof, wherein L2 comprises 1 to 8 polyethylene glycol monomers.

[0364] Embodiment 117 The conjugate of any one of embodiments 76-116, or the pharmaceutically acceptable salt thereof, wherein L2 comprises 2 to 6 polyethylene glycol monomers.

[0365] Embodiment 118 The conjugate of any one of embodiments 76-117, or the pharmaceutically acceptable salt thereof, wherein L2 comprises a glycine or C1-12 alkylene group.

[0366] Embodiment 119 The conjugate of any one of embodiments 76-110, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:-NH-CI.6 alkyl-[OCH2CH2]o-8-Co.6 alkyl-Z-(Ci-i2alkyl-S)0-i-; wherein Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, -OP(=O)(SH)-, or -OP(=O)(OH)-.

[0367] Embodiment 120 The conjugate of any one of embodiments 76-110, or 118-119, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:-NH-(CH2)2-6-(OCH2CH2)i-8-(CH2)o-3-Z-Ci-i2 alkyl-S-; -NH-(CH2)i-i2-Z-Ci-i2 alkyl-S-; -NH-(CH2)2-6-(OCH2CH2)I-8-(CH2)O-3-Z-; or -NH-(CH2)i-i2-Z-; wherein Z is -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, or a bond.

[0368] Embodiment 121 The conjugate of any one of embodiments 76-110, or 118-120, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:

[0369] Embodiment 122 The conjugate of any one of embodiments 76-110, or 118-121, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:

[0370] Embodiment 123 The conjugate of any one of embodiments 76-110, or 118-122, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:

[0371] Embodiment 124 The conjugate of any one of embodiments 76-110, or the pharmaceutically acceptable salt thereof, wherein n is 0; and L2 is absent.

[0372] Embodiment 125 The conjugate of embodiment 67, or the pharmaceutically acceptable salt thereof, wherein the active agent is a detectable group.

[0373] Embodiment 126 The conjugate of embodiment 125, or the pharmaceutically acceptable salt thereof, wherein the detectable group is a dye.

[0374] Embodiment 127 The conjugate of embodiment 125, or the pharmaceutically acceptable salt thereof, wherein the detectable group is a chromophore or fluorescent dye.

[0375] Embodiment 128 The conjugate of embodiment 125, or the pharmaceutically acceptable salt thereof, wherein the detectable group is a coumarin dye, a rhodamine dye, or a cyanine dye.

[0376] Embodiment 129 The conjugate of any one of embodiments 76-124, wherein TP is an antitumor chemotherapeutic.

[0377] Embodiment 130 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP comprises a duocarmycin, auristatin, maytansinoid, uncialamycin, dynemicin, thailanstatin, camptothecin, exatecan, or tubulysin compound.

[0378] Embodiment 131 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP comprises an antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA-alkylating drug, a DNA-binding drug, a DNA-cleaving drug, or an RNA polymerase inhibitor.

[0379] Embodiment 132 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is an antitumor antibiotic.

[0380] Embodiment 133 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is a microtubule inhibitor.

[0381] Embodiment 134 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is a cytotoxic or cytostatic agent.

[0382] Embodiment 135 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is a topoisomerase I inhibitor.

[0383] Embodiment 136 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is a camptothecin or exatecan compound.

[0384] Embodiment 137 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is camptothecin, 10-hydroxycamptothecin, topotecan, irinotecan, belotecan, exatecan, diflomotecan, grimatecan, lurtotecan, silatecan, rubitecan, or SN-38.

[0385] Embodiment 138 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is an ecteinascidin.

[0386] Embodiment 139 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is trabectedin.

[0387] Embodiment 140 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is ecubectedin.

[0388] Embodiment 141 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is lurbinectedin.

[0389] Embodiment 142 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is a calicheamicin.

[0390] Embodiment 143 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is calicheamicin yl (gamma- 1).

[0391] Embodiment 144 The conjugate of any one of embodiments 76-124, or the pharmaceutically acceptable salt thereof, wherein TP is selected from any one of the following:

[0392] Embodiment 145 The conjugate of embodiment 67 or 76, having a structure selected from the group consisting of:or a pharmaceutically acceptable salt thereof, whereinAB is the antibody or antigen-binding fragment; and p is 1-20.

[0393] Embodiment 146 The conjugate of any one of embodiments 76-145, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment thereof is an IgGl subclass.

[0394] Embodiment 147 The conjugate of any one of embodiments 76-145, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment thereof binds to a target antigen.

[0395] Embodiment 148 The conjugate of embodiment 147, or the pharmaceutically acceptable salt thereof, herein the target antigen is expressed on a tumor cell.

[0396] Embodiment 149 The conjugate of embodiment 147 or 148, or the pharmaceutically acceptable salt thereof, wherein the target antigen is CD 19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, or tissue factor.

[0397] Embodiment 150 The conjugate of any one of embodiments 76-149, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen binding fragment thereof comprises: a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 102; a heavy chain complementarity determiningregion 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 103; a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 104; and / or a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 106; a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 107; and / or a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 108; or a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 112; a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 113; a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 114; and / or a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 116; a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 117; and / or a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 118.

[0398] Embodiment 151 The conjugate of any one of embodiments 76-150, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 101; and / or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 105; or an immunoglobulin heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 111; and / or an immunoglobulin light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 115.

[0399] Embodiment 152 The conjugate of any one of embodiments 76-151, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 101; and / or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 105; or an immunoglobulin heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 111; and / or an immunoglobulin light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 115.

[0400] Embodiment 153 The conjugate of any one of embodiments 76-152, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 109; and / or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 110; or an immunoglobulin heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 119; and / or an immunoglobulin light chain comprising an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 120.

[0401] Embodiment 154 The conjugate of any one of embodiments 76-153, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen binding fragment thereof comprises: an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 109; and / or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 110; or an immunoglobulin heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 119; and / or an immunoglobulin light chain comprising the amino acid sequence set forth in SEQ ID NO: 120.

[0402] Embodiment 155 The conjugate of any one of embodiments 76-154, wherein the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, belantamab, loncastuximab, tisotumab, mirvetuximab, or a biosimilar thereof.

[0403] Embodiment 156 A pharmaceutical composition comprising the conjugate of any one of embodiments 67-155, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0404] Embodiment 157 A method of delivering a therapeutic payload to an intracellular target within a tumor cell, wherein the therapeutic payload is conjugated to a polypeptide comprising a nuclear localization sequence.

[0405] Embodiment 158 The method of embodiment 157, wherein the polypeptide comprises the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0406] Embodiment 159 The method of embodiment 157 or 158, wherein the polypeptide comprises a sequence selected from SEQ ID NOS: 1-8.

[0407] Embodiment 160 The method of any one of embodiments 157-159, wherein the polypeptide is further conjugated to an antibody or antigen-binding fragment thereof at a position that is non-adjacent (i.e., distal) to the therapeutic payload.

[0408] Embodiment 161 The method of any one of embodiments 157-160, wherein the therapeutic payload is cytotoxic.

[0409] Embodiment 162 The method of any one of embodiments 157-161, wherein the therapeutic payload is internalized into a nucleus of the tumor cell.

[0410] Embodiment 163 The method of any one of embodiments 157-162, wherein the intracellular target is within the nucleus of the tumor cell.

[0411] Embodiment 164 A method of delivering an antibody or antigen-binding fragment to an intracellular target within a tumor cell, wherein the antibody or antigen-binding fragment is conjugated to a polypeptide comprising a nuclear localization sequence.

[0412] Embodiment 165 The method of embodiments 164, wherein the polypeptide comprises the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0413] Embodiment 166 The method of embodiment 164 or 165, wherein the polypeptide comprises a sequence selected from SEQ ID NOS: 1-8.

[0414] Embodiment 167 A method of treating a disease, comprising delivering a therapeutic payload and / or an antibody to an intracellular target within a tumor cell, wherein the therapeutic payload and / or the antibody is conjugated to a polypeptide, wherein the polypeptide comprises the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

[0415] Embodiment 168 The method of any one of embodiments 158, 165, or 167, wherein: x1is G or A; x2is G or A; x3is A or V; x4is L or I; and x5is G or A.

[0416] Embodiment 169 A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of embodiments 1-66, or a conjugate according to any one of embodiments 67-155.

[0417] Embodiment 170 The method of any one of embodiments 167-169, wherein the disease or disorder is a cancer.

[0418] Embodiment 171 The method of any one of embodiments 167-169, wherein the disease or disorder is a hematological cancer.

[0419] Embodiment 172 The method of any one of embodiments 167-169, wherein the disease or disorder comprises a solid tumor.

[0420] Embodiment 173 The method of any one of embodiments 167-170, wherein the disease or disorder is non-Hodgkin’s lymphoma, breast cancer, ovarian cancer, or stomach cancer.

[0421] Embodiment 174 The method of any one of embodiments 167-170, wherein the disease or disorder is large cell lymphoma, breast ductal carcinoma, breast adenocarcinoma, ovarian adenocarcinoma, or gastric carcinoma.

[0422] Embodiment 175 The method of any one of embodiments 167-170, wherein the disease or disorder is a cancer associated with overexpression of CD 19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, tissue factor.

[0423] Embodiment 176 The method of any one of embodiments 167-170, wherein the disease or disorder is a cancer associated with overexpression of AXL, BCMA, CA9, CCR7, CD123, CD166, CD19, CD20, CD205, CD22, CD25, CD276, CD30, CD33, CD37, CD46, CD70, CD74, CD79b, CEACAM5, CLDN18.2, CLDN6, CXCR4, DLL3, EFNA4, EGFR, EGFRvIII, ENPP3, EPCAM, EPHA2, F3, FOLR1, GPC3, GPNMB, GUCY2C, HER2, HER3, IGF1R, L1CAM, LIV1, LRRC15, MET, MSLN, MUC1, MUC16, NCAM1, NECTIN4, PSMA, PTK7, ROR1, ROR2, SDC1, SLC1A5, SLC34A2, TFRC, TIM1, TPBG, or TROP2.

[0424] Embodiment 177 The method of embodiment 167, wherein the cancer is a hematological cancer or a solid tumor.

[0425] Embodiment 178 A compound or a pharmaceutically acceptable salt thereof having the structure of Formula V, Formula VI, or Formula VII:AC-(Ll)m-PP-(L2)„-TP Formula VAC-(Ll)m-PP Formula VIAC-(L 1 )m-PP-(L2)n-DG Formula VII wherein:AC is an antibody-coupling group configured to form a covalent bond with a sulfur or nitrogen atom of an amino acid side-chain of the antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide comprising the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1L2 is a linker;TP is a therapeutic payload;DG is a detectable group; m is 0 or 1 ; and n is 0 or 1.

[0426] Embodiment 179 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises any one of SEQ ID NOS: 1-8.

[0427] Embodiment 180 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises SEQ ID NO 1: PGGKRVKLD.

[0428] Embodiment 181 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises SEQ ID NO 2: PGGKRVKLDG.

[0429] Embodiment 182 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises SEQ ID NO 3: PAAKRVKLD.

[0430] Embodiment 183 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises SEQ ID NO 4: PAAKRVKLDG.

[0431] Embodiment 184 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises SEQ ID NO 5: PAGKRVKLDG

[0432] Embodiment 185 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises SEQ ID NO 6: PGAKRVKLDG.

[0433] Embodiment 186 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises SEQ ID NO 7: PAAKRAKLDG

[0434] Embodiment 187 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein PP comprises SEQ ID NO 8: PAAKRVKIDG.

[0435] Embodiment 188 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein L 1 is a non-cleavable linker.

[0436] Embodiment 189 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein LI is a PEG linker comprising 1 to 12 polyethylene glycol monomers.

[0437] Embodiment 190 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein LI is a PEG linker having the structure:

[0438] Embodiment 191 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein AC a lysine-reactive group, a cysteine-reactive group, or a glutamine-reactive group.

[0439] Embodiment 192 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein AC of Formula V or VI comprises the group:

[0440] Embodiment 193 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein AC-LI has one of the following structures:

[0441] Embodiment 194 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:-NH-CI.6 alkyl-[OCH2CH2]o-8-Co-6 alkyl-Z-(Ci-i2alkyl-S)o-i-; wherein Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, -OP(=O)(SH)-, or -OP(=O)(OH)-.

[0442] Embodiment 195 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:-NH-(CH2)2.6-(OCH2CH2)i.8-(CH2)o-3-Z-Ci.i2alkyl-S-;-NH-(CH2)M2-Z-CM2alkyl-S-;-NH-(CH2)2 6-(OCH2CH2)I.8-(CH2)O.3-Z-; or -NH-(CH2)M2-Z-; wherein Z is -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, or a bond.

[0443] Embodiment 196 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:

[0444] Embodiment 197 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the detectable group is a dye.

[0445] Embodiment 198 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the detectable group is a chromophore, fluorescent labels, bioluminescent label, or chemiluminescent label.

[0446] Embodiment 199 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the detectable group is a coumarin dye a rhodamine dye, or a cyanine dye.

[0447] Embodiment 200 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the detectable group is an Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, HiLyte™ Fluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte™ Fluor 555, Alexa Fluor® 561, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, HiLyte™ Fluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, or Alexa Fluor® 750, or HiLyte1MFluor 750, or a functional equivalent thereof.

[0448] Embodiment 201 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload of Formula V is an antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA -alkylating drug, a DNA-binding drug, a DNA-cleaving drug, or an RNA polymerase inhibitor.

[0449] Embodiment 202 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is an antitumor antibiotic.

[0450] Embodiment 203 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a microtubule inhibitor.

[0451] Embodiment 204 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a cytotoxic or cytostatic agent.

[0452] Embodiment 205 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a topoisomerase I inhibitor.

[0453] Embodiment 206 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is or comprises a camptothecin or exatecan compound.

[0454] Embodiment 207 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is camptothecin, 10 -hydroxy camptothecin, topotecan, irinotecan, belotecan, exatecan, diflomotecan, grimatecan, lurtotecan, silatecan, rubitecan, or SN-38.

[0455] Embodiment 208 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is an ecteinascidin.

[0456] Embodiment 209 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is trabectedin.

[0457] Embodiment 210 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is ecubectedin.

[0458] Embodiment 211 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is lurbinectedin.

[0459] Embodiment 212 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a calicheamicin.

[0460] Embodiment 213 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is calicheamicin yl (gamma- 1).

[0461] Embodiment 214 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is selected from any one of the following:

[0462] Embodiment 215 The compound of embodiment 178, having one of the following structures:or a pharmaceutically acceptable salt thereof.

[0463] Embodiment 216 The use of a compound of any one of embodiments 178-215, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament.

[0464] Embodiment 217 The compound of any one of embodiments 178-215, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating cancer.

[0465] Embodiment 218 The compound of any one of embodiments 178-215, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating hematological cancer.

[0466] Embodiment 219 The compound of any one of embodiments 178-215, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating solid tumor.

[0467] Embodiment 220 The compound of any one of embodiments 178-215, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating a nonHodgkin’s lymphoma, breast cancer, ovarian cancer, or stomach cancer.

[0468] Embodiment 221 The compound of any one of embodiments 178-215, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating a large cell lymphoma, breast ductal carcinoma, breast adenocarcinoma, ovarian adenocarcinoma, or gastric carcinoma.

[0469] Embodiment 222 The compound of embodiment 178, or the pharmaceutically acceptable salt thereof, having the following structure:

[0470] The preceding merely illustrates the principles of this disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of this disclosure and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present disclosure, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present disclosure is embodied by the appended claims.I l l

[0471] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

ClaimsWe claim:

1. A conjugate comprising an antibody or an antigen-binding fragment and a therapeutic payload or a detectable group, conjugated via a linker, wherein the linker comprises a polypeptide, and wherein the polypeptide comprises a nuclear localization sequence.

2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, of the formula:AB-[(Ll)m-PP-(L2)„-DG]Pwherein:AB is the antibody or antigen-binding fragment;LI is a linker;PP is the polypeptide;L2 is a linker;DG is the detectable group; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

3. The conjugate of claim 1 , or a pharmaceutically acceptable salt thereof, of Formula I:AB-[(Ll)m-PP-(L2)n-TP]p Formula I wherein:AB is the antibody or antigen-binding fragment;LI is a linker;PP is the polypeptide;L2 is a linker;TP is the therapeutic payload; m is 0 or 1; n is 0 or 1 ; and p is 1-20.

4. The conjugate of any one of claims 1-3, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein x1, x2,x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

5. The conjugate of claim 4, or the pharmaceutically acceptable salt thereof, wherein: x1is G or A; x2is G or A; x3is A or V;x4is L or I; x5is G or A; and a is 0 or 1.

6. The conjugate of any one of claims 1-5, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises a sequence selected from SEQ ID NOs. 1-8: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG.

7. The conjugate of any one of claims 1-6, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 3: PAAKRVKLD.

8. The conjugate of any one of claims 1-6, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO 4: PAAKRVKLDG.

9. The conjugate of any one of claims 1-8, or the pharmaceutically acceptable salt thereof, wherein L2 is a linker of the formula:-NH-CI.6 alkyl-[OCH2CH2]o-8-Co.6 alkyl-Z-(Ci-i2alkyl-S)o-i-; wherein Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, -OP(=O)(SH)-, or -OP(=O)(OH)-.

10. The conjugate of any one of claims 1-8, or the pharmaceutically acceptable salt thereof, wherein n is 0; and L2 is absent.

11. The conjugate of any one of claims 1-2 and 4-10, or the pharmaceutically acceptable salt thereof, wherein the detectable group is a dye.

12. The conjugate of any one of claims 1 and 3-10, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a cytotoxic or cytostatic agent.

13. The conjugate of any one of claims 1 and 3-6, having a structure selected from the group consisting of:or a pharmaceutically acceptable salt thereof, wherein:AB is the antibody or antigen-binding fragment; and p is 1-20.

14. The conjugate of any one of claims 1-13, or the pharmaceutically acceptable salt thereof, wherein the antibody or antigen -binding fragment thereof binds to a target antigen.

15. The conjugate of claim 14, or the pharmaceutically acceptable salt thereof, wherein the target antigen is CD 19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, mesothelin, BCMA, folate receptor alpha, or tissue factor.

16. The conjugate of any one of claims 1-15, wherein the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, belantamab, loncastuximab, tisotumab, mirvetuximab, or a biosimilar thereof.

17. A pharmaceutical composition comprising the conjugate of any one of claims 1-16, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

18. A method of delivering a therapeutic payload to an intracellular target within a tumor cell, wherein the therapeutic payload is conjugated to a polypeptide comprising a nuclear localization sequence.

19. The method of claim 18, wherein the polypeptide comprises the sequence SEQ ID NO 10:P-x1-x2-K-R-x -I<-x4-D-(x ) SEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1.

20. The method of claim 18 or 19, wherein the polypeptide comprises a sequence selected from SEQ ID NOs. 1-8: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG.

21. The method of any one of claims 18-20, wherein the therapeutic payload is cytotoxic.

22. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a conjugate according to any one of claims 1-16.

23. The method of claim 22, wherein the disease or disorder is a cancer.

24. The method of claim 23, wherein the cancer is a hematological cancer or a solid tumor.

25. A compound or a pharmaceutically acceptable salt thereof having the structure of Formula V, Formula VI, or Formula VIEAC-(Ll)m-PP-(L2)„-TP Formula VAC-(Ll)m-PP Formula VIAC-(Ll)m-PP-(L2)n-DG Formula VII wherein:AC is an antibody -coupling group configured to form a covalent bond with a sulfur or nitrogen atom of an amino acid side -chain of the antibody or antigen-binding fragment;LI is a linker;PP is a polypeptide comprising the sequence SEQ ID NO 10:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1L2 is a linker;TP is a therapeutic payload;DG is a detectable group; m is 0 or 1 ; and n is 0 or 1.

26. The compound of claim 25, or the pharmaceutically acceptable salt thereof, wherein PP comprises any one of SEQ ID Nos. 1-8: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG.

27. The compound of claim 25, or the pharmaceutically acceptable salt thereof, wherein the detectable group is a dye.

28. The compound of claim 25, or the pharmaceutically acceptable salt thereof, wherein the detectable group is an Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, HiLyte™ Fluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte™ Fluor 555, Alexa Fluor® 561, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, HiLyte™ Fluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, or Alexa Fluor® 750, HiLytelMFluor750, or a functional equivalent thereof.

29. The compound of claim 25, or the pharmaceutically acceptable salt thereof, wherein the therapeutic payload is an antitumor antibiotic, microtubule inhibitor, cytotoxic or cytostatic, topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA-alkylating drug, a DNA-binding drug, a DNA-cleaving drug, or an RNA polymerase inhibitor.

30. The compound of claim 25, having one of the following structures:or a pharmaceutically acceptable salt thereof.

31. The use of the compound of any one of claims 25, 26, 29, or 30, according to Formula V, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament.

32. The compound of any one of claims 25, 26, 29, or 30, according to Formula V, or the pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating cancer.

33. A composition comprising a polypeptide comprising the sequence:P-x1-x2-K-R-x3-K-x4-D-(x5)aSEQ ID NO 10 wherein: x1, x2, x3, x4, and x5are each independently any natural or unnatural amino acid; and a is 0 or 1; or a pharmaceutically acceptable salt thereof.

34. The composition of claim 33, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence:P-x’^-K-R^-K-x^D SEQ ID NO 11 wherein: x1is G or A; x2is G or A; x3is A or V; andx4is L or I.

35. The composition of claim 33, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence:P-x -x2-I<-R-x -I<-x4-D-x SEQ ID NO 12 wherein: x1is G or A; x2is G or A; x3is A or V; x4is L or I; and x5is G or A.

36. The composition of claim 33, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises the sequence:P-x1-x2-K-R-V-K-L-D-(G)aSEQ ID NO 13 wherein x1and x2are each independently A or G; and a is 0 or 1.

37. The composition of claim 33, or the pharmaceutically acceptable salt thereof, wherein the polypeptide comprises a sequence selected from SEQ ID NOs. 1-8: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG.

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