Nuclear localized polypeptides and their conjugates and their uses

Abstract

Disclosed in this invention are nuclear localized polypeptides and conjugates comprising the same. Exemplary conjugates include antibody-drug conjugates comprising an antibody or antibody fragment conjugated to a therapeutic payload via a linker containing the nuclear localized polypeptide. The conjugates provided in this invention may be useful for treating diseases or disorders such as cancer.

Description

[Technical Field]

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

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, which is incorporated into this application in its entirety by reference. The XML copy, created on March 21, 2024, is named 65657-703_601_SL.xml and has a size of 56,962 bytes.

[0003] Technical field The present invention provides nuclear localization sequence (NLS) polypeptides, conjugates containing the same, and compositions containing the same for use in delivering a payload into the interior of a cell (e.g., the nucleus). Methods using the NLS polypeptide precursor molecules and conjugates provided in the present invention for the manufacture of pharmaceuticals for treating diseases or disorders such as cancer are also provided. In one embodiment, the NLS-bound payload is retained within the cell, thereby enhancing the therapeutic window of the NLS-bound payload compared to an unbound payload. [Background technology]

[0004] Antibody-drug conjugates (ADCs) are a targeting technology that allows for the delivery of a therapeutic payload to a target cell type by conjugating the payload to a targeted antibody that binds to a cell-specific target antigen (e.g., tumor-associated antigen). Many antibodies are not currently suitable for delivering payloads to cancer cells because they are not efficiently internalized upon binding and / or are not transported to a lysosomal compartment where the payload can be released. Antibody-drug conjugates (ADCs) are often captured in endosomes, subsequently effluxed, or degraded in the endosomal-lysosomal pathway, resulting in reduced intracellular accumulation. [Overview of the project]

[0005] In one embodiment, the present invention provides a composition comprising a nuclear localization sequence (NLS), such as an antibody-drug conjugate (ADC) comprising an NLS. An NLS is, for example, an amino acid sequence that labels a protein for translocation to the nucleus of a cell by nuclear transport. An NLS can be recognized by a corresponding nuclear transporter, which can interact with nucleoporins to help the NLS-containing protein reach the nucleus via the nuclear pore complex (NPC). This NLS-dependent protein recognition is a process required for transport proteins to cross the nuclear membrane via the nuclear pore complex (NPC) and is facilitated by members of the importin superfamily. Due to the complex roles of nuclear proteins, nuclear transport via NLS is a highly controlled process.

[0006] In one embodiment, the present invention provides a nuclear localization sequence (e.g., a polypeptide) that is internalized within a cell and then transported to the cell nucleus. In some embodiments, the NLS polypeptide is conjugated (e.g., covalently) with a therapeutic payload, which is transported to the cell nucleus by conjugation to the NLS polypeptide. In some embodiments, the present invention provides a conjugate comprising an NLS polypeptide and a therapeutic payload covalently bound thereto, which can be used to deliver the therapeutic payload to a nuclear target. In some embodiments, the therapeutic payload is released from the polypeptide (e.g., within the target cell or target cell nucleus). In some embodiments, the therapeutic payload is stably bound to the polypeptide and exerts the desired effect (e.g., therapeutic effect) without release from the polypeptide. In some embodiments, the therapeutic payload is stably bound to the polypeptide, thereby reducing unwanted releases that could result in off-target toxicity or side effects that limit the effectiveness of antibody-drug conjugates in clinical settings.

[0007] In some embodiments, the therapeutic payload is transported into a cell (e.g., the cell nucleus). In some embodiments, the therapeutic payload is retained within a cell (e.g., the cell nucleus). In some embodiments, a therapeutic payload stably conjugated to an NLS polypeptide (e.g., the polypeptide (PP) disclosed in this invention) accumulates in the cell nucleus, thereby concentrating the local concentration of the payload within the nucleus. In some embodiments, a therapeutic payload conjugated to an NLS (e.g., the peptide disclosed in this invention) has a wider therapeutic window than the therapeutic payload alone because the conjugated payload is concentrated in 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 its antigen-binding fragment. In some embodiments, the antibody is an unbound antibody. In some embodiments, the antibody modulates (e.g., enhances) one or more pharmacokinetic properties (e.g., half-life, metabolism, volume of distribution, etc.) of the conjugate compared to a non-antibody conjugate. In some embodiments, the antibody is an antigen-binding fragment. For example, the antigen may be a tumor-associated antigen (i.e., a protein that is overexpressed (2x, 5x, 10x, 20x, 50x, 100x, 1000x, or more) in tumor cells compared to non-tumor cells). In some embodiments, the antibody is internalized into the target cell after binding to the target antigen.

[0009] In some embodiments, the present invention provides an antibody-drug conjugate comprising a monoclonal antibody and an NLS polypeptide conjugated to a therapeutic payload. Preferably, after the antibody binds to a target antigen (e.g., a tumor antigen), the antibody-drug conjugate is internalized into the cell. In some embodiments, the antibody is processed intracellularly. In some embodiments, upon intracellular processing, the antibody-drug conjugate releases a polypeptide-payload conjugate which has therapeutic activity (i.e., against a nuclear target). In some embodiments, the therapeutic payload is toxic to cells, resulting in, for example, cell death, growth inhibition, or growth reversal. In some embodiments, the therapeutic payload is a cytotoxic payload. In some embodiments, the antibody-drug conjugate (including an NLS polypeptide linker) increases the therapeutic window of the cytotoxic payload through a combination of tumor-specific internalization by the antibody and nuclear transport and retention of the cytotoxic payload by the NLS polypeptide conjugated to the antibody-drug conjugate.

[0010] In some embodiments, the present invention provides a conjugate comprising an antibody or antigen-binding fragment and a therapeutic payload or detection group conjugated via a linker, wherein the linker comprises a polypeptide, and the polypeptide comprises a nuclear localization sequence.

[0011] In some embodiments, the complex is given by the following formula: AB-[(L1) m -PP-(L2) n -DG] p During the ceremony: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide; L2 is a linker. DG is a detection group; m is either 0 or 1; n is either 0 or 1; and p is from 1 to 20.

[0012] In some embodiments, the conjugate is of formula I: AB-[(L1) m -PP-(L2) n -TP] p Formula I Where: AB is an antibody or an antigen-binding fragment; L1 is a linker; PP is a polypeptide; L2 is a linker TP is a therapeutic payload; m is 0 or 1; n is 0 or 1; and p is from 1 to 20.

[0013] In some embodiments, the polypeptide comprises the following sequence: P-x 1 -x 2 -K-R-x 3 -K-x 4 -D-(x 5 ) a SEQ ID NO: 10 where x 1 , x 2 , x 3 , x 4 , and x 5 is each independently any natural or non-natural amino acid; and a is 0 or 1.

[0014] In some embodiments, x 1 is G or A; x 2 is G or A; x 3 is A or V; x 4 is L or I; x 5is 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 is SEQ ID NO: 4: PAAKRVKLDG. In some embodiments, the detection group is a dye. In some embodiments, the therapeutic payload is a cytotoxic agent or a cell proliferation inhibitor. In some embodiments, the antibody or its antigen-binding fragment binds to a target antigen. In some embodiments, the target antigen is CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, or tissue factor. In some embodiments, the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, verantamab, roncastoximab, tisotumab, milbetuximab, or a biosimilar thereof. The present invention also provides a pharmaceutical composition comprising a conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0015] The present invention also provides a method for delivering a therapeutic payload to an intracellular target within a tumor cell, wherein the therapeutic payload is conjugated to a polypeptide containing a nuclear localization sequence. In some embodiments, the polypeptide includes SEQ ID NO: 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Here, x 1 , x 2 , x 3 , x 4 , and x5 Each of these is independently either a natural or non-natural amino acid; and a is either 0 or 1.

[0016] In some embodiments, the polypeptide comprises sequences selected from SEQ ID NOs: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG. In some embodiments, the therapeutic payload is cytotoxic.

[0017] The present invention also provides a method for treating a disease or disorder in a subject that requires it, the method comprising administering a therapeutically effective amount of the conjugate disclosed herein to the subject. In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is hematological cancer or a solid tumor.

[0018] In the present invention, compounds having the structure of formula V, formula VI, or formula VII, or pharmaceutically acceptable salts thereof, are also provided: AC-(L1) m -PP-(L2) n -TP formula V AC-(L1) m -PP Formula VI AC-(L1) m -PP-(L2) n -DG Formula VII During the ceremony, AC is an antibody-binding group configured to form a covalent bond with the sulfur or nitrogen atom of the amino acid side chain of an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide containing sequence number 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Here: x 1 , x 2 , x 3 , x 4 , and x 5 Each of these is independently either a natural or non-natural amino acid; and a is either 0 or 1. L2 is the linker; TP is a therapeutic payload; DG is a detection group; m is 0 or 1; and n is either 0 or 1.

[0019] In some embodiments, x 1 is G or A; x 2 is G or A; x 3 is A or V; and x 4 is L or I. In some embodiments, x 5 is G or A. In some embodiments, x 5 It does not exist. In some embodiments, the polypeptide comprises one of SEQ ID NOs: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG. In some embodiments, the detection group is a dye. In some embodiments, the detection group is Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, HiLyte TM Fluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte TM Fluor 555, Alexa Fluor® 561, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, HiLyte TMFluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, or Alexa Fluor® 750, HiLyte TM Fluor 750, or functional equivalents thereof. In some embodiments, the therapeutic payload is an antitumor antibiotic, a microtubule inhibitor, a cytotoxic or cytoactivating agent, a topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA alkylating agent, a DNA binding agent, a DNA cleaving agent, or an RNA polymerase inhibitor.

[0020] Within the scope of the present invention, the use of a compound of formula V or a pharmaceutically acceptable salt thereof for use in the manufacture of pharmaceuticals is also considered. The use of a compound of formula V or a pharmaceutically acceptable salt thereof for use in the manufacture of pharmaceuticals for treating cancer is also provided in the present invention.

[0021] In one embodiment, the present invention provides a composition comprising an NLS polypeptide (for example, a pharmaceutical or pharmaceutical composition, or a kit for preparing the same), wherein the NLS polypeptide comprises SEQ ID NO: 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Here: x 1 , x 2 , x 3 , x 4 , and x 5 Each of these is independently either a natural or non-natural amino acid; and a is either 0 or 1.

[0022] In some embodiments, the composition includes a polypeptide having Sequence ID No. 11: Px 1 -x 2 -KRx 3 -Kx 4-D Sequence Number 11 Here: x 1 is G or A; x 2 is G or A; x 3 is A or V; and x 4 is L or I.

[0023] In some embodiments, the composition comprises a polypeptide having Sequence Number 12: P-x 1 -x 2 -K-R-x 3 -K-x 4 -D-x 5 Sequence Number 12 Here: x 1 is G or A; x 2 is G or A; x 3 is A or V; x 4 is L or I; and x 5 is G or A.

[0024] In some embodiments, the composition comprises a polypeptide having Sequence Number 13: P-x 1 -x 2 -K-R-V-K-L-D-(G) a Sequence Number 13<00​​​​​​​​​​​​​​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] In the present invention, an NLS polypeptide comprising SEQ ID NO: 10 is also provided: P-x 1 -x 2 -K-R-x 3 -K-x 4 -D-(x 5 ) a SEQ ID NO: 10 where: x 1 、x 2 、x 3 、x 4 、and x 5 is each independently any natural or non-natural 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 in the present invention.

[0028] In another aspect, an antibody-drug conjugate of Formula I is also provided: AB-[(L1) m -PP-(L2) n -TP] p Equation I During the ceremony: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide; L2 is a linker. TP is a therapeutic payload; m is either 0 or 1; n is either 0 or 1; and p is between 1 and 20.

[0029] In another embodiment, the antibody-drug conjugate is of formulas I-10: AB-[(L1) m -PP-(L2) n -TP] p Formula I-10 During the ceremony: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide containing sequence number 10; Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Here: x 1 , x 2 , x 3 , x 4 , and x 5 Each of these is independently either a natural or non-natural amino acid; and a is either 0 or 1; L2 is a linker. TP is a therapeutic payload; m is either 0 or 1; n is either 0 or 1; and p is between 1 and 20.

[0030] In some embodiments, x 1is G or A; x 2 is G or A; x 3 is A or V; and x 4 is L or I. In some embodiments, x 5 It does not exist. In some embodiments, the polypeptide (PP) contains a sequence selected from sequence numbers 1 to 8.

[0031] In some embodiments, TP comprises duocalmycin, auristatin, meitansinoid, unciaramycin, dynemycin, tylanstatin, camptothecin, exatecan, or tubulicin compounds. In some embodiments, TP comprises antitumor antibiotics, microtubule inhibitors, cytotoxic or cell proliferation inhibitors, topoisomerase inhibitors, pyrrolobenzodiazepines, DNA alkylating drugs, DNA binding drugs, DNA cleavage drugs, or RNA polymerase inhibitors. In some embodiments, the therapeutic payload (TP) includes lurubinectedin, trabectedin, safracin, lenalidomide, eribulin, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, or vinorelbine); epothilon, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), cryptophycin, hemiastalin, anthracycline, bisnaphthylamide (erinafide), or cytotoxic molecular adhesive / PROTAC compounds.

[0032] In some embodiments, the antibody or its antigen-binding fragment is an unbound antibody (e.g., an unbound IgG1 antibody). In some embodiments, the antibody or its antigen-binding fragment binds to a target antigen. In some embodiments, the target antigen is expressed on tumor cells. In some embodiments, the target antigens are CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, and tissue factor. In some embodiments, the target antigens are 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, and EN. The target antigens are PP3, EPCAM, EPHA2, F3, FOLR1, GPC3, GPNMB, GUCY2C, HER2, HER3, IGF1R, L1CAM, LIV1, LRRC15, MET, MSLN, MUC1, MUC16, NCAM1, Nectin 4, PSMA, PTK7, ROR1, ROR2, SDC1, SLC1A5, SLC34A2, TFRC, TIM1, TPBG, or TROP2. In some embodiments, the target antigens correspond to genes that are TNFRSF17, ALCAM, MS4A1, LY75, B7H3, TNFRSF8, CD142, SLC39A6, CD138, CD71, or HAVCR1.

[0033] In some embodiments, the present invention provides a complex of formula I, where the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, verantamab, roncustuximab, tisotumab, mirbetuximab, or a biosimilar thereof.

[0034] In another embodiment, the present invention provides 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, the PP of formula I is selected from SEQ ID NOs: 1-8.

[0035] In yet another embodiment, the present invention provides a method for delivering a therapeutic payload to an intracellular target within a tumor cell, the method comprising contacting the tumor cell with a complex disclosed in the present invention. In some embodiments, the complex 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 in the nucleus of the tumor cell. In some embodiments, the intracellular target is located in the nucleus of the tumor cell.

[0036] In another embodiment, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate of formula I to the subject. AB-[(L1) m -PP-(L2) n -TP] p Equation I During the ceremony: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is an NLS polypeptide; L2 is a linker. TP is a therapeutic payload; m is either 0 or 1; n is either 0 or 1; and p is between 1 and 20.

[0037] In some embodiments, the polypeptide (PP) comprises SEQ ID NO: 10. In some embodiments, the PP comprises SEQ ID NO: 11 and / or SEQ ID NO: 8. In some embodiments, the method comprises administering a therapeutically effective amount of an antibody-drug conjugate of formula I, wherein the PP comprises one or more of SEQ ID NOs: 1 to 8. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is hematological cancer. In some embodiments, the disease or disorder comprises solid tumors. In some embodiments, the disease or disorder is cancer associated with the overexpression of CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, and tissue factor. [Brief explanation of the drawing]

[0038] Each patent or application file must include at least one color drawing. Copies of the color drawing in this patent or patent application publication will be provided by the Office upon request and payment of the necessary fees.

[0039] Novel features of the present invention are specifically described in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and to the appended drawings:

[0040] [Figure 1] Figure 1 shows the relative luminescence units in SKBR3 (breast cancer adenocarcinoma) cells after administration of Examples ADC12 and 13, which were conjugated with either an IgG1 antibody or an anti-HER2 antibody (trastuzumab), respectively. [Figure 2] Figure 2 shows the relative luminescence units in HCC1954 (epithelial breast cancer / ductal carcinoma) cells after administration of Examples ADC12 and 13, which were conjugated with either an IgG1 antibody or an anti-HER2 antibody (trastuzumab), respectively. [Figure 3]Figure 3 shows the relative luminescence units in human neutrophils after administration of Examples ADC12 and 13, which are conjugated with either an IgG1 antibody or an anti-HER2 antibody (trastuzumab). [Figure 4] Figure 4 shows the relative luminescence units in human megakaryocytes after administration of Examples ADC12 and 13, which were conjugated with either an IgG1 antibody or an anti-HER2 antibody (trastuzumab). [Figure 5] Figure 5 shows images taken 2, 8, 24, and 48 hours after administration of antibodies labeled with green dye (A, the top four images) or antibodies labeled with both green and red dye (B, the bottom four images), where the red dye is conjugated via an NLS linker. Detailed description of the invention

[0041] This specification describes nuclear localization peptides and compositions comprising nuclear localization peptides. Exemplary compositions consist of antibody-drug conjugates comprising nuclear localization peptides linked to an antibody or antibody fragment, for example, via one or more linker moieties. In some cases, the antibody or antibody fragment is modified to reduce or eliminate effector function. In some cases, the antibody-drug conjugate contains a therapeutic payload effective, for example, in killing cancer cells. Non-limiting examples of antibodies and therapeutic payloads are provided in the present invention.

[0042] The various antibody-drug conjugates described in this invention selectively target and kill cancer cells compared to non-cancer cells. The various drug conjugates described in this invention include a therapeutic payload (e.g., a cytotoxic moiety) that is delivered to cancer cells expressing an antigen to which the antibody-drug conjugate specifically binds. In some cases, the drug conjugates in this invention have an improved therapeutic window compared to drug conjugates lacking nuclear localization signals.

[0043] Antibody-drug conjugates containing nuclear localized peptides can be used to treat diseases or conditions of a target that require them. Generally, the treatment method involves administering a composition (e.g., a pharmaceutical composition) containing the conjugate (e.g., an antibody-drug conjugate) disclosed in the present invention to a target. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate of 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.

[0044] In various cases, the linkage of an antibody or antibody fragment to a cytotoxic site via a nuclear localization signal (NLS) as described in the present invention has the effect of increasing the therapeutic window or otherwise improving the specific delivery of the cytotoxic site to cancer cells.

[0045] Before describing the methods and compositions, it should be understood that this disclosure is not limited to the specific methods or compositions described. The scope of this disclosure is limited only by the appended claims, and the terms used are for the purpose of describing specific embodiments and are not intended to be limiting. The examples are provided to those skilled in the art to disclose and explain the methods and uses of the compositions and methods, and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent that the following experiments are all or only experiments.

[0046] definition Unless otherwise specified, the following terms used in this application have the definitions set forth below.

[0047] As used herein and in the claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, the term “sample” includes multiple samples, including mixtures thereof. As used in the present invention, the term “about” a number may refer to plus or minus 15% of that number. The term “about” a range may refer to the value obtained by subtracting 15% from the minimum value of that range and the value obtained by adding 15% from the maximum value. The use of the term “including” is not limited to other forms such as “include,” “includes,” and “included.”

[0048] The word "and / or" used in a phrase containing a list of members is intended to include all members individually, and all combinations of lists of all or some members. For example, in this invention, a word such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the word "and / or" used in a phrase such as "A, B, and / or C" is intended to include each of the following 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).

[0049] Throughout this application, various embodiments may be presented in range form. It should be understood that range form descriptions are for convenience and conciseness and should not be interpreted as limitations lacking flexibility in the scope of disclosure. Therefore, a range description should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, a range description of "1 to 6" should be considered to specifically disclose not only the individual numerical values ​​within that range (e.g., 1, 2, 3, 4, 5, 6), but also subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc. This interpretation applies regardless of the breadth of the range.

[0050] The "alkyl" group generally refers to an aliphatic hydrocarbon group. Alkyl groups may be branched or linear. In some embodiments, the "alkyl" group has 1 to 80 carbon atoms, i.e., C1 to C2. 80 It is alkyl. In some embodiments, the "alkyl" group has 1 to 12 carbon atoms, i.e., C1 to C 12 It is an alkyl group. In this invention, when a numerical range such as "1 to 12" is mentioned, it refers to each integer within the given range; for example, "1 to 12 carbon atoms" means that the alkyl group contains up to 12 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., but this definition also includes the appearance of the term "alkyl" when no numerical range is specified. In some embodiments, the alkyl group is a C1 to C6 alkyl group. In one embodiment, the alkyl group is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, or hexyl.

[0051] The term "heteroalkyl" generally refers to an alkyl group in which one or more atoms in the alkyl skeleton are atoms other than carbon, such as oxygen, nitrogen (e.g., NH, N-alkyl, N-oxide), sulfur (e.g., S, SO, SO2), or combinations thereof. In general, heteroalkyls are bonded to the rest of the molecule at the carbon atom of the heteroalkyl. In one embodiment, the heteroalkyl is a C1-C6 heteroalkyl. Heteroalkyls may include nitriles, amides, esters, ethers, amines, thioethers, thioesters, carbamates, carbonate esters, polyethers, polyamines, etc. In some embodiments, the heteroalkyl group contains one or more units or groups such as -CH2CH2O-, -CH2CH2NH-, -CH2CH2NHC(O)-, -CH2CH2C(O)NH-, -NHCH2CH2NH-, etc.

[0052] In the present invention, "conjugate" refers to a compound in which one binding molecule (e.g., an antigen-binding molecule) is covalently linked to a payload via a linker. In some embodiments, the conjugate comprises a binding molecule and one or more payloads. In some embodiments, the binding molecule is a small molecule (molecular weight < 1000 Da), a polypeptide (comprising 2 to about 20 amino acids), a protein (e.g., an antibody), or a protein fragment (e.g., the antigen-binding site of a protein). In some embodiments, the binding molecule is an antibody or an antigen-binding fragment thereof.

[0053] In the present invention, the term "target protein" generally refers to a protein that is expressed on the surface of a cell (e.g., a cancer cell) and can efficiently bind a small molecule binder. In the present invention, the term "target antigen" generally refers to an antigen that is expressed on the surface of a cell (e.g., a cancer cell) and can efficiently bind an antibody or an antigen-binding fragment thereof. In the present invention, "efficiently" generally refers to a compound having a potency of at least the micromolar level (e.g., submicromolar, nanomolar, subnanomolar, etc. used in the art).

[0054] In this invention, the term "antibody" is understood in its broadest sense and includes immunoglobulin molecules, such as complete or modified monoclonal antibodies, polyclonal antibodies, or multispecific antibodies (e.g., bispecific antibodies). The antibody preferably comprises a molecule having four peptide chains (two heavy chains (H chains) and two light chains (L chains)), which are usually linked by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region includes, for example, three regions: CH1, CH2, and CH3. Each light chain includes a variable region (abbreviated as VL) and a constant region. The light chain constant region includes a domain (abbreviated as CL). The VH domain and VL domain are further subdivided into highly variable regions (complementarity-determining regions, abbreviated as CDRs) and low-sequence-variability regions (framework regions, abbreviated as FRs). Typically, each VH and VL region consists of three CDRs and up to four FRs. For example, the order from the amino terminus to the carboxyl terminus is as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Antibodies can be obtained from any suitable species, such as rabbits, llamas, camels, mice, rats, etc. In one embodiment, the antibodies are of human or mouse origin. Antibodies may be, for example, human, humanized, or chimeric.

[0055] The terms "specifically bind," "specific binding," and similar terms, when used in the context of one molecule binding to another, mean that one molecule binds to the other molecule with significantly higher affinity than cross-reactive antigens or off-target antigens (also called non-target antigens) determined by experimental techniques such as surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS) analysis, binding equilibrium exclusion assay (KinExA), isothermal titration calorimetry (ITC), radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Typically, the specific or selective reaction will be at least twice the noise of non-target signals or non-target binding and more than 10 times that of non-target binding. An antibody or antigen-binding fragment that binds to a target of interest is useful as a therapeutic agent that targets cells or tissues expressing the target, and binds to the target with sufficient affinity so as not to cross-react significantly with other proteins. In such embodiments, the degree of binding of the antibody or antigen-binding fragment to "non-target" proteins will be less than approximately 10% of the binding of the antibody or antigen-binding fragment to specific target proteins. Specific binding of an antibody is typically at least 10%. -7 M(as the Kd value; that is, preferably the Kd value is 10) -7An antibody having an affinity for less than M, which antibody has at least twice the affinity for a given antigen / target molecule than for a non-specific antigen / target molecule (e.g., bovine serum albumin or casein) that is not the given antigen / target molecule or a related antigen / target molecule. Specific binding of an antibody or binding agent does not preclude the antibody from binding to multiple antigen / target molecules (e.g., orthologs of different species). Specific binding can be measured, for example, by determining the binding of a molecule as compared to the binding of a control molecule, which is a molecule of a similar structure that generally has no binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by the excess of unlabeled target. Thus, as used in the present invention, the terms "specific binding," "specifically binds to," "specifically inhibits," "specifically blocks," or "is specific for a particular target" mean a binding, blocking, or inhibition that binds, blocks, or inhibits a particular target without substantially binding to or inhibiting non-targets.

[0056] Percent (%) sequence identity to a reference polypeptide sequence is the percent of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps if necessary to achieve maximum percent sequence identity, and conservative substitutions are not considered as part of sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in various known ways using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared.

[0057] As used in this invention, "pharmaceutically acceptable" refers to a substance, such as a salt, solvate, carrier, or diluent, that is administered to an individual without impairing the biological activity or properties of the compound, is relatively non-toxic, that is, without causing undesirable biological effects, or without harmful interactions with any component of a composition containing it.

[0058] The term "pharmaceutically acceptable salt" refers to a form of therapeutic agent consisting of a cationic form of the therapeutic agent combined with a suitable anion, or, in alternative embodiments, an anionic form of the therapeutic agent combined with a suitable cation. In some embodiments, a pharmaceutically acceptable salt is obtained by reacting the compound described in the present invention with a base. In such cases, the acidic proton of the compound described in the present invention is substituted with a metal ion, such as a lithium ion, sodium ion, potassium ion, or magnesium ion. In some embodiments, a pharmaceutically acceptable salt is obtained by reacting the compound described in the present invention with an acid. In some embodiments, the compound described in the present invention is prepared as a chloride salt, sulfate salt, bromide salt, mesylate salt, maleate salt, citrate salt, or phosphate salt.

[0059] The term “activator” generally refers to any small molecular weight payload (<2000 Da) that can bind to a linker or polypeptide (e.g., via amide, disulfide, or thiophosphate bonds) and exert an effect from within a cell. In some embodiments, "inside the cell" refers to the cell nucleus. The activator may be a therapeutic payload (TP), indicating that it exerts a physiological effect on the invaded cell. In some embodiments, the therapeutic payload is cytotoxic, otherwise damaging to the cell, or binding to a target inside the cell (nucleus). In some embodiments, the therapeutic payload is a drug that halts proliferation, differentiation, growth, angiogenesis, or other abnormal activity, such as that seen in cancer cells or other cells with hyperproliferative disorders. Examples of therapeutic agents are provided throughout this disclosure. The activator may also be a non-therapeutic payload. An example of a non-therapeutic payload is, for example, a detection group (DG).

[0060] In some embodiments, the activator (e.g., a detection group) is a dye. In some embodiments, the activator emits a detectable signature (e.g., radioactivity). In some embodiments, the activator is a gamma emitter, beta emitter, or alpha emitter. In some embodiments, the activator responds to a stimulus (e.g., light, laser, or other energy source) with an emitted response (e.g., fluorescence, phosphorescence, or other detectable light). In some embodiments, the activator is a FRET pair, or a donor or acceptor of a BRET pair. In some embodiments, the activator is a chromophore, a fluorescent label, a bioluminescent label, or a chemiluminescent label. In some embodiments, the activator is an Alexa Fluor dye or other functional equivalent (e.g., a coumarin dye, a rhodamine dye, or a cyanine dye). In some embodiments, the activator is Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, or HiLyte TMFluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte TM Fluor 555, Alexa Fluor® 561, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, HiLyte TM Fluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, or HiLyte TM It's Fluor 750.

[0061] In another embodiment, the compounds described in the present invention are isotope-labeled (e.g., with radioactive isotopes) or labeled by other means including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, or chemiluminescent labels.

[0062] As used in the present invention, a polypeptide (e.g., PP) generally refers to a peptide consisting of at least two, preferably 20 or fewer, amino acids (also known as an oligopeptide). Unless otherwise specified, the term “polypeptide” as used in the present invention is distinguished from proteins (e.g., antibodies) which may consist of hundreds or thousands of amino acids. In some embodiments, the polypeptides (PP) provided in the present invention contain six, seven, eight, nine, ten, eleven, or twelve amino acids. In some embodiments, the polypeptides (PP) contain 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.

[0063] In the present invention, 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 halogens) that are not known to be chemically or biologically unstable. The term "non-cleavable linker" is intended to distinguish it from cleavable linkers (e.g., protease-cleavable linkers, autoimmune linkers, pH-sensitive linkers, etc.). Non-cleavable linkers may be alkyl or heteroalkyl linkers and may optionally be interrupted by one or more cyclyl or heterocyclyl groups (e.g., click partners or their artifacts). Non-cleavable linkers may consist of a polymer part (e.g., PEG), an alkyl part (e.g., C 1-12 Alkyl), and / or heteroalkyl group (for example, one or more carbon atoms are substituted or replaced by one or more heteroatoms selected from N, O, S, and P). 1-12 It may contain alkyl groups.

[0064] In this invention, "autoimmune group" is defined as a part of the linker in an antibody-drug conjugate that has the function of conditionally releasing a free drug at a site targeted by the ligand unit. An activatable autoimmune moiety includes an activatable group (AG) and an autoimmune spacer unit. Autoimmune elimination occurs when a molecular system undergoes spontaneous and irreversible degradation into constituent fragments by an electron cascade, elimination pathway, or cyclization-elimination event. Activation of an activatable group, e.g., enzymatic conversion of an amide group to an amino group, or reduction of a disulfide to a free thiol group, initiates an autoimmune reaction sequence, leading to the release of a free drug by one or more mechanisms, including the (transient) 1,6-elimination of a p-aminobenzyl group to a p-quinone methide, optionally accompanied by the release of carbon dioxide, and / or followed by a second cyclization-release mechanism. An autoimmune assembly unit can be part of a chemical spacer linking the antibody and the payload (via functional groups). Alternatively, the autoimmune group may not be part of the chemical spacer, but rather branch off from the chemical spacer that connects the antibody to the payload.

[0065] In the present invention, the term "therapeutic payload" generally refers to a chemical group, generally a low molecular weight group, that is useful for the treatment or diagnosis of a disease or disorder. For example, a therapeutic payload may include drugs, detection groups (e.g., dyes, radiolabels, contrast agents), radioisotopes (e.g., technetium-99, iodine-131), radiopharmaceuticals, chromophores, etc. A therapeutic payload may have multiple functions. For example, a radioisotope bound to a conjugate can decay and generate a specific amount of radiation that can be used for the diagnosis or treatment of a disease or disorder. In some embodiments, the therapeutic payload is a drug or contains a drug. In some embodiments, the therapeutic payload is a cytotoxic or cell proliferation inhibitory compound. In some examples, the therapeutic payload is effective in killing cancer cells or slowing their growth. In some examples, the therapeutic payload is a chemotherapeutic agent. In some examples, the therapeutic payload is a cytotoxic or cell proliferation inhibitor. In some examples, the therapeutic payload is a camptothecin compound or a derivative thereof. Specific examples of therapeutic payloads used in accordance with the present invention are provided throughout the invention.

[0066] "Chemotherapy agents" are chemical substances useful in cancer treatment. Examples of chemotherapy agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines such as altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethyloromelamamine; and TLK 286 (TELCYTA TM); acetogenins (especially bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); β-lapacon; lapachol; colchicines; betulinic acid; campotocetin (including synthetic analogues topotecan) (HYCAMTIN®, CPT-11 (irinotecan, CAMPTOSAR®), acetylcampotocetin, scopoletin, 9-aminocampotocetin); bryostatin; calistatin; CC-1065 (including its synthetic analogues adzeresin, karzeresin, and bizeresin); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin) 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodictin; spongstatin; nitrogen mustards such as chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, and uracil mustard; nitrosourea drugs such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimustine; bisphosphonate drugs such as clodronate;Enazin antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I) and anthracycline drugs such as annamycin, AD 32, Alkalobicin, Daunorubicin, Dexrazoxane, DX-52-1, Epirubicin, GPX-100, Idarubicin, KRN5500, Menogalil, Dynemycin (including Dynemycin A), Esperamycin, Neocardinostatin chromophore and related chromoprotein enazin antibiotics, Acrasinomycin, Actinomycin, Austramycin, Azaserin, Bleomycin, Kakutinomycin, Carabicin, Carminomycin, Cardinophilin, Chromomycin, Dactinomycin, Detorubicin, 6-Diazo-5-Oxo-L-Norleucine, ADRIAMYCIN® Doxorubicin (Morpholinodoxorubicin, Cyanomorpholinodoxorubicin, 2-Pyrrolinodoxorubicin, Liposomal Doxorubicin, Deoxydoxorubicin) Mitomycins such as esorubicin, marcelomycin, and mitomycin C (including cin), mycophenolic acid, nogaramycin, olibomycin, peplomycin, potoflomycin, promycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; folic acid analogs such as denopterin, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxylfluridine, enocitabine, and phloxidine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testactone; Anti-adrenal drugs (aminoglutethimide, mitotane, trilostane, etc.); folic acid supplements (folic acid (leucovorin), etc.); segraton;Antifolate antitumor agents (e.g., Alimta®, LY231514, pemetrexed), dihydrofolate reductase inhibitors (e.g., methotrexate), antimetabolites (e.g., 5-fluorouracil (5-FU) and its prodrugs (e.g., UFT), S-1 and capecitabine), thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors (e.g., larcitrexed (TOMUDEX®, TDX)); dihydropyrimidine dehydrogenase inhibitors (e.g., enyluracil), aldofosphamide Lycoside, aminolevulinic acid, amsacrin, bestlovesil, bisanthren, edatraxate, defofamin, demecolsin, diazicone; elfornithine; eriptinium acetate; epotilon; etoglucide; gallium nitrate; hydroxyurea; lentinan; ronidanin; mytansinoids such as mytansin and ansamitosin; mitogwazone; mitoxantrone; mopidammole; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, Veraculine A, Loridine A, Anguidine); Urethanes; Vindesine (Eldisine®, Fildesine®); Dacarbazine; Manomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids and taxanes, e.g., TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, New Jersey), ABRAXANE; TMCremophor-free, albumin-modified nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), TAXOTERE® docetaxel (Rhone-Poulenc-Lorraie, Antony, France), chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin, carboplatin; vinblastine (VELBAN®); etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (Oncovin®); vinca alkaloids; vinorelbine (Navierbin®); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and oxaliplatin (ELOXATIN TM Examples include FOLFOX, an abbreviation for a treatment regimen that combines 5-FU and leucovorin.

[0067] In this invention, the terms “subject,” “individual,” and “patient” are often used interchangeably. “Subject” refers to a biological entity containing expressed genetic material. A subject may be a tissue, cell, or offspring of a biological entity collected from within a living organism or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be diagnosed with or suspected of being at high risk of disease. In some cases, a subject is not necessarily diagnosed with or suspected of being at high risk of disease.

[0068] In this invention, the terms "treatment" or "to treat" refer to pharmaceuticals or other intervention regimens for obtaining beneficial or desired results in a subject. Beneficial or desired results include, but are not limited to, therapeutic effects and / or preventive effects. Therapeutic benefits may refer to the eradication or improvement of the symptoms or underlying disease being treated. In addition, therapeutic benefits may be achieved by eradicating or improving one or more physiological symptoms related to the underlying disease, even if the subject still suffers from the underlying disease, in a manner that improvement is observed in the subject. Preventive effects include delaying, preventing, or eliminating the onset of a disease or condition; delaying or eliminating the onset of symptoms of a disease or condition; delaying, stopping, or reversing the progression of a disease or condition; or a combination thereof. With respect to preventive effects, subjects at risk of developing a particular disease, or subjects reporting one or more physiological symptoms of a disease, may receive treatment even if a diagnosis of the disease has not been confirmed.

[0069] As used in this invention, the terms “therapeutic effective dose” or “effective dose” refer to a sufficient amount of the drug or compound administered that, to some extent, alleviates one or more symptoms of the disease or condition being treated. Results include reduction and / or mitigation of the signs, symptoms, or causes of the disease, or other desirable changes in the biological system. For example, “effective dose” in a therapeutic application refers to the amount of a composition containing the compound disclosed herein that is necessary to produce a clinically significant reduction in disease symptoms. The appropriate “effective” dose in individual cases may be determined, if necessary, using techniques such as dose escalation studies. The terms “therapeutic effective dose” or “effective dose” also refer to a sufficient amount of the compound to induce a biological or medical response in a biological molecule (e.g., protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, as desired by a researcher, veterinarian, physician, or clinician.

[0070] As used in this invention, the term "therapeutic range" generally refers to the concentration range of a drug that provides safe and effective treatment with minimal adverse effects. The therapeutic range typically lies between the minimum effective dose and the dose at which toxicity or adverse side effects occur. For drugs with a narrow therapeutic range, the therapeutic effective dose is similar to the toxic dose (i.e., the dose at which toxicity is observed). In some embodiments, drugs have a broad therapeutic range, which allows for the evaluation of a wider range of effective doses without causing unacceptable toxicity. In some embodiments, targeted drugs (e.g., conjugates) have a broader therapeutic range than untargeted drugs. This is because targeted agents increase the local concentration of the drug at a desired location or tissue type without causing undesirable exposure to healthy tissue, cells, or cell compartments. One indicator of the therapeutic range is the "therapeutic index," which is the LD (Low-Dose). 50 (A dose that is lethal to 50% of subjects) and ED 50 This is the ratio of the dose that is effective in 50% of the target population. In some embodiments, the conjugate provided in the present invention has a larger therapeutic range or therapeutic index than the non-conjugate.

[0071] As those skilled in the art will understand from reading this disclosure, each of the embodiments described and illustrated herein has independent components and features, and these can be readily separated or combined with features of several other embodiments without departing from the scope or spirit of the invention. The methods described may be carried out in the order described or in any other logically possible order. The section headings used in this invention are for organizational purposes only and should not be construed as limiting the subject matter described.

[0072] Each targeted peptide Intracellular protein delivery is a powerful tool for developing protein-based therapeutics, but it presents challenges due to the impermeability of the cell membrane to large biomolecules and containment into endosomes. Most delivery strategies rely on the uptake of carriers into endosomes. "Endosomal uptake" occurs when a protein transporter enters the cell via endocytosis, and the load is then taken up into the endosome. Transporters taken into endosomes generally have low escape efficiency and are eventually degraded. Proteins confined in endosomes are ultimately degraded by proteases in the acidic endolysosomal compartment without reaching the cytoplasm. Such containment and degradation can limit the effectiveness of protein delivery systems taken into cells via the endocytosis pathway. Access of delivery proteins to the cytoplasm is crucial for intracellular activity, either directly for activity or as a gateway to the nucleus or intracellular organelles. This invention provides a solution to the problem of delivering proteins (e.g., antibodies or their conjugates) to the cell nucleus via binding to NLS polypeptides.

[0073] In one embodiment, the present invention provides a polypeptide having a sequence disclosed herein that can function as a nuclear localization signal, i.e., a recognition motif that promotes the internalization of a motif and its conjugate into the cell nucleus. The polypeptide disclosed herein may be useful in promoting the intracellular (i.e., intranuclear) delivery of a second agent that can be conjugated to the polypeptide. The second agent may include, in non-limiting examples, a therapeutic payload, an antibody or antigen-binding antibody fragment, a detectable agent, a diagnostic agent, and the like.

[0074] In some embodiments, a nuclear localization signal (NLS) is a short peptide that acts as a signaling fragment mediating the transport of proteins from the cytoplasm to the nucleus. In some embodiments, a nuclear localization signal or sequence (NLS) consists of one or more short sequences of positively charged lysine or arginine exposed on the protein surface. In some embodiments, the nuclear localization signal (NLS) is a classical nuclear localization signal (cNLS). In some embodiments, the nuclear localization signal (NLS) is a non-classical nuclear localization signal (ncNLS). In some embodiments, the nuclear localization signal (NLS) is another type of nuclear localization signal. In some embodiments, the NLS is a novel nuclear localization signal.

[0075] In some embodiments, the present invention provides 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 links the therapeutic payload to the antibody or antigen-binding fragment. In some embodiments, the present invention provides an antibody-drug conjugate comprising an NLS polypeptide that is linked to an antibody or antigen-binding fragment at one end and to a therapeutic payload at the other end (of the polypeptide). In some embodiments, the NLS polypeptide links the therapeutic payload to the antibody or antigen-binding fragment (i.e., in an antibody-drug conjugate) so that the therapeutic payload or its conjugate can escape the endosomal-lysosome pathway and bypass to the nucleus, thereby increasing intracellular accumulation with high target cell selectivity. In some embodiments, the antibody-drug conjugate accumulates intracellularly while maintaining target cell selectivity, avoiding out-of-target effects due to undesirable release or localization of the payload. In some embodiments, the polypeptide is a linear polypeptide including an N-terminus and a C-terminus. In some embodiments, the present invention provides an antibody-drug conjugate comprising an antibody and a therapeutic payload, wherein the antibody and the therapeutic payload are linked via a linker, the linker being an NLS polypeptide (i.e., the polypeptide disclosed in the present invention).

[0076] In some embodiments, the NLS polypeptide comprises the sequence of SEQ ID NO: 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a (Sequence ID 10) Here: x 1 、x 2 、x 3 、x 4 、and x 5 are each independently any natural or non-natural amino acid; and a is 0 or 1.

[0077] In some embodiments, x 1 is a nonpolar amino acid. In some embodiments, x 1 is a flexible amino acid. In some embodiments, x 1 is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x 1 is G or A. In some embodiments, x 1 is G. In some embodiments, x 2 is a nonpolar amino acid. In some embodiments, x 2 is a flexible amino acid. In some embodiments, x 2 is an amino acid selected from the group consisting of G, A, L, I, and V. In certain embodiments, x 2 is G or A. In certain embodiments, x 2 is G. In some embodiments, x 3 is a flexible amino acid. In some embodiments, x 3 is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x 3 is A, L, I, or V. In some embodiments, x 3 is A or V. In some embodiments, x 4 is a flexible amino acid. In some embodiments, x 4 is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x 4 is A, L, I, or V. In particular embodiments, x 4 is L or I. In some embodiments, x 5is G or A. In some embodiments, a is 0, and in some embodiments, a is 1.

[0078] In some embodiments, the NLS polypeptide includes SEQ ID NO: 11: Px 1 -x 2 -KRx 3 -Kx 4 -D (Sequence ID 11)

[0079] In some embodiments, x 1 is G or A. In some embodiments, x 1 is G. In some embodiments, x 1 is A. In one embodiment, x 2 is G or A. In one embodiment, x 2 is G. In one embodiment, x 2 is A. In some embodiments, x 3 is A or V. In some embodiments, x 3 is A. In some embodiments, x 3 V is V. In one embodiment, x 4 is L or I. In one embodiment, x 4 L is. In one embodiment, x 4 It is I.

[0080] In some embodiments, the NLS polypeptide includes SEQ ID NO: 12: Px 1 -x 2 -KRx 3 -Kx 4 -Dx 5 (Sequence ID 12)

[0081] In some embodiments, x 1 is G or A. In some embodiments, x 1 is G. In some embodiments, x 1 is A. In one embodiment, x2 is G or A. In one embodiment, x 2 is G. In one embodiment, x 2 is A. In some embodiments, x 3 is A or V. In some embodiments, x 3 is A. In some embodiments, x 3 V is V. In one embodiment, x 4 is L or I. In one embodiment, x 4 L is. In one embodiment, x 4 is I. In some embodiments, x 5 is G or A. In some embodiments, x 5 is G. In some embodiments, x 5 It is A.

[0082] In some embodiments, the NLS polypeptide includes SEQ ID NO: 13: Px 1 -x 2 -KRVKLD-(G) a (Sequence ID 13)

[0083] In some embodiments, x 1 and x 2 These are, independently, A or G. In some embodiments, x 1 and x 2 These are A, respectively. In some embodiments, x 1 and x 2 These are G, respectively. In some embodiments, x 1 A is and x 2 is G. In some embodiments, x 1 G is G, and x 2 A is. In some embodiments, a is 0 or 1. In some embodiments, a is 0. In some embodiments, a is 1.

[0084] In some embodiments, the polypeptide includes a sequence selected from SEQ ID NOs: 1 to 8. In some embodiments, the polypeptide includes SEQ ID NO: 1: PGGKRVKLD. In some embodiments, the polypeptide includes SEQ ID NO: 2: PGGKRVKLDG. In some embodiments, the polypeptide includes SEQ ID NO: 3: PAAKRVKLD. In some embodiments, the polypeptide includes SEQ ID NO: 4: PAAKRVKLDG. In some embodiments, the polypeptide includes SEQ ID NO: 5: PAGKRVKLDG. In some embodiments, the polypeptide includes SEQ ID NO: 6: PGGRVKLDG. In some embodiments, the polypeptide includes SEQ ID NO: 7: PAAKRAKLDG. In some embodiments, the polypeptide includes SEQ ID NO: 8: PAAKRVKIDG.

[0085] Conjugate Conjugates of NLS polypeptides disclosed in the present invention are also provided. The conjugates of the present disclosure include, but are not limited to, antibody-polypeptide conjugates, polypeptide-drug conjugates, and antibody-drug conjugates (in which an antibody and a drug are linked via an NLS polypeptide). They also include their metabolites or precursor molecules. In some embodiments, the conjugate is an antibody-polypeptide conjugate. In some embodiments, the conjugate is an antibody-polypeptide conjugate, and the antibody is linked to the polypeptide via a proline residue of the polypeptide. In some embodiments, the polypeptide is linked to the antibody via a linker, and the linker forms a bond between the proline of the polypeptide and a lysine or cysteine ​​residue of the antibody. The scope of the present invention also includes conjugates comprising an NLS polypeptide described in the present invention (e.g., of formula I) and a non-antibody conjugate (e.g., a peptide conjugate, a small molecule conjugate, etc.).

[0086] In some embodiments, the antibody or antigen-binding fragment is bound to the polypeptide via a linker. In some embodiments, the antibody or antigen-binding fragment is bound to the polypeptide via a linker. In some embodiments, the antibody or antigen-binding fragment is bound to P (proline) of the polypeptide. In some embodiments, the linker comprises one or more polyethylene glycol groups 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.

[0087] In some embodiments, the linker contains a cleavable group. In some embodiments, the linker is a chemically unstable 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-degrading group. In some embodiments, the linker is a hydrolyzable 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.

[0088] In some embodiments, a conjugate having the structure of formula I: AB-[(L1) m -PP-(L2) n -TP] p Equation I Or a pharmaceutically acceptable salt thereof is provided, in the formula: AB is an antibody or antibody-binding fragment; L1 is the linker; PP is an NLS polypeptide; L2 is the linker; TP is a therapeutic payload; m is either 0 or 1; n is 0 or 1; and p is between 1 and 20.

[0089] In some embodiments, the formula is: AB-[(L1) m -PP-(L2) n -DG] p A conjugate or a pharmaceutically acceptable salt thereof is provided, in the formula: AB is an antibody or antibody-binding fragment; L1 is the linker; PP is an NLS polypeptide; L2 is the linker; DG is a detection group (e.g., coumarin dye, rhodamine dye, or cyanine dye); m is either 0 or 1; n is 0 or 1; and p is between 1 and 20.

[0090] In some embodiments, PP is a polypeptide containing (for example, consisting of) SEQ ID NO: 10 Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Or a pharmaceutically acceptable salt thereof is provided, in the formula: x 1 , x 2 , x 3 , x 4 , and x 5 Each of these is independently any natural or non-natural amino acid; and a is either 0 or 1.

[0091] In some embodiments, the conjugate has the structure of formula I-10: AB-[(L1) m -PP-(L2) n -TP] p Formula I-10 or comprising a pharmaceutically acceptable salt thereof, in the formula: AB is an antibody or antibody-binding fragment; L1 is the linker; PP is a polypeptide containing sequence number 10; Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Here, x 1 , x 2 , x 3 , x 4 , and x 5 Each of these is independently any natural or non-natural amino acid; and a is either 0 or 1; L2 is the linker; TP is a therapeutic payload; m is either 0 or 1; n is 0 or 1; and p is between 1 and 20.

[0092] In some embodiments, x of formula I-10 1 is a nonpolar amino acid. In some embodiments, x of formula I 1 is a flexible amino acid. In some embodiments, x of formula I 1 is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x 1 x is a non-charged amino acid. In some embodiments, x 1 It is either G or A.

[0093] In some embodiments, x of formula I-10 2 is a nonpolar amino acid. In some embodiments, x of formula I 2 is a flexible amino acid. In some embodiments, x of formula I 2 is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x 2x is a non-charged amino acid. In some embodiments, x 2 It is either G or A.

[0094] In some embodiments, x of formula I-10 3は It is a nonpolar amino acid. In some embodiments, x 3 is a flexible amino acid. In some embodiments, x 3 is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x 3 It is A, L, I, or V.

[0095] In some embodiments, x of formula I-10 4 x is a nonpolar amino acid. In some embodiments, x 4 is a flexible amino acid. In some embodiments, x 4 is an amino acid selected from the group consisting of G, A, L, I, and V. In some embodiments, x 4 It is A, L, I, or V.

[0096] In some embodiments, a in formula I is 0. In some embodiments of formula I, a is 1 to 10. In some embodiments, x 5 It is either G or A.

[0097] In some embodiments, x of formula I-10 1 is G or A, and in some embodiments, x 2 is G or A, and in some embodiments, x 3 is A or V, and in some embodiments, x 4 is L or I, and in some embodiments a is 0, and in some embodiments of formula I-10 a is 1, and in some embodiments x 5 It is either G or A.

[0098] In the present invention, nonpolar amino acids generally refer to amino acids having nonpolar side chains. Examples of nonpolar 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 β-alanine. In some embodiments, the flexible amino acid is glycine or alanine. In the present invention, uncharged amino acids refer to amino acids having side chains that do not form cations or anions, or salts thereof, at physiological pH. Uncharged amino acids may be polar but uncharged (e.g., serine, threonine, asparagine, glutamine, and cysteine), or nonpolar and uncharged (e.g., glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, tryptophan, and tyrosine).

[0099] In some embodiments, polypeptide (PP) of formula I is represented by sequence number 11: Px 1 -x 2 -KRx 3 -Kx 4 -D Sequence ID 11 or a pharmaceutically acceptable salt thereof, wherein: x 1 is G or A; x 2 is G or A; x 3 is A or V; and x 4 It is either L or I.

[0100] In some embodiments, polypeptide (PP) of formula I is represented by Sequence ID No. 12: Px 1 -x 2 -KRx 3 -Kx 4 -Dx 5 Sequence ID 12 or a pharmaceutically acceptable salt thereof, wherein: x 1 is G or A; x 2 is G or A; x 3 is A or V; x 4 is L or I; and x 5 It is either G or A.

[0101] In some embodiments, the polypeptide of formula I (PP) is represented by Sequence ID No. 13: Px 1 -x 2 -KRVKLD-(G) a Sequence ID 13 or a pharmaceutically acceptable salt thereof, where: x 1 and x 2 Each of them is independently either A or G; and a is either 0 or 1.

[0102] In some embodiments, the present invention provides 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 to 8. In some embodiments, PP comprises SEQ ID NO: 1: PGGKRVKLD. In another embodiment, PP comprises SEQ ID NO: 2: PGGKRVKLDG. In some 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.

[0103] In some embodiments, L1 includes a cleavable linker. In some embodiments, L1 includes a non-cleavable linker. In some embodiments, L1 includes 0 to 12 amino acids (e.g., 0, 1, 2, 3, ... 12). In some embodiments, L1 includes 0 to 12 polyethylene glycol monomers (e.g., 0, 1, 2, 3, ... 12). In some embodiments, L1 includes 1 to 8 polyethylene glycol monomers (e.g., 2 to 6 (e.g., 3 or 4)). In some embodiments, L1 includes a protein-binding group or a protein-binding group. In some embodiments, L1 includes a lysine-reactive group, a cysteine-reactive group, or a glutamine-reactive group. In some embodiments, L1 includes homoglycine or C 1~12 It contains an alkylene group. In some embodiments, L1 includes the following group: [ka]

[0104] In some embodiments, L1 includes the following group: [ka]

[0105] In some embodiments, L1 is: [ka] That is the case.

[0106] In some embodiments, AB is an antibody or its antigen-binding fragment. In some embodiments, AB is AC, where AC is an antibody-binding group (alternatively, a protein-binding group, a protein-reactive group, or an antibody-reactive group). In some embodiments, AB represents binding to an antibody. In some embodiments, AC represents a group configured to form a bond with an antibody. Specifically, the AC group may form a bond with the sulfur of a cysteine ​​side chain or the nitrogen of a lysine side chain. Alternatively, AC may form a bond with a non-natural amino acid, i.e., bioorthogonal conjugation chemistry may be used. In some embodiments, AC includes alkynes (e.g., cyclooctane), triazines, succinimides, maleimides, carbamates, esters (e.g., NHS esters), isocyanates, isothiocyanates, etc. In some embodiments, AB represents an antibody. In some embodiments, AB represents a monoclonal antibody.

[0107] In some embodiments, the antibody includes a constant region. In some embodiments, the linker binds to the antibody via the constant region. In some embodiments, the antibody includes an Fc region. In some embodiments, the linker binds to the antibody via the Fc region. In some embodiments, the Fc region has reduced effector function.

[0108] In some embodiments, the antibody or antigen-binding fragment binds to the target antigen. In some embodiments, the target antigen is expressed on tumor cells. In some embodiments, the target antigen is CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, 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.

[0109] In some embodiments, the target antigen is CD19. 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 mesoserine. In some embodiments, the target antigen is BCMA. In some embodiments, the target antigen is folate receptor α. In some embodiments, the target antigen is tissue factor.

[0110] 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, enfortuzumab, sacituzumab, verantamab, roncatuximab, tisotuzumab, mirbetuximab, or a biosimilar thereof.

[0111] In some embodiments, the antibody is trastuzumab or its biosimilar. In some embodiments, the antibody is brentuximab or its biosimilar. In some embodiments, the antibody is gemtuzumab or its biosimilar. In some embodiments, the antibody is inotuzumab or its biosimilar. In some embodiments, the antibody is moxetumomab or its biosimilar. In some embodiments, the antibody is polatuzumab or its biosimilar. In some embodiments, the antibody is enfortumab or its biosimilar. In some embodiments, the antibody is sacituzumab or its biosimilar. In some embodiments, the antibody is verantamab or its biosimilar. In some embodiments, the antibody is roncustuximab or its biosimilar. In some embodiments, the antibody is tisotumab or its biosimilar. In some embodiments, the antibody is milbetuximab or its biosimilar.

[0112] In some embodiments, the composition comprises a therapeutic payload, and the therapeutic payload and polypeptide (PP) are conjugated via a linkage or via a second linker (for example, a first linker links the polypeptide PP to an antibody or its antigen-binding fragment, if present, or to a protein-binding group (AC)). In some embodiments, the therapeutic payload is the polypeptide (PP) with D (aspartic acid) or x 5The therapeutic payload is conjugated to a polypeptide (PP) via a linker. In some embodiments, the therapeutic payload is conjugated to the polypeptide (PP) via a second linker. In some embodiments, the second linker is C1-C 12 It is an 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.

[0113] In some embodiments, L2 includes a cleavable linker. In some embodiments, L2 includes a non-cleavable linker. In some embodiments, L2 includes an autoimmune group. In some embodiments, L2 includes 0 to 12 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). In some embodiments, L2 includes 0 to 12 polyethylene glycol monomers (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12). In some embodiments, L2 includes a glycine group or C 1~12 It contains an alkylene group. In some embodiments, n is 0 and L2 is absent.

[0114] In some embodiments, the therapeutic payload is a cytotoxic payload. In some embodiments, the therapeutic payload is cytotoxic to tumor cells upon internalization into tumor cells. In some embodiments, the therapeutic payload comprises duocalmycin, auristatin, meitansinoids, unciaramycin, dynemycin, tylanstatin, camptothecin, exatecan, or tubulicin compounds. In some embodiments, the therapeutic payload comprises an antitumor antibiotic, a microtubule inhibitor, a cytotoxic agent or cell activator, a topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA alkylating agent, a DNA binding agent, a DNA cleaving agent, or an RNA polymerase inhibitor. In some embodiments, the therapeutic payload (TP) comprises equbectedin, lurubinectedin, trabectedin, safracin, lenalidomide, eribulin, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, or vinorelbine); epothilon, taxanes (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), cryptophycin, hemiastalin, anthracyclines, bisnaphthylamides (e.g., erinafide), or cytotoxic molecular adhesives / PROTAC compounds.

[0115] In some embodiments, the conjugate payload is a therapeutic payload, such as an activator or drug. In some embodiments, the therapeutic payload is cytotoxic to tumor cells. In some embodiments, the antibody in the conjugate is ligated to the N-terminus of the polypeptide (PP). In some embodiments, the antibody is ligated to the N-terminus of the polypeptide (PP) via a conjugate or linker. In some embodiments, the activator in the conjugate is ligated to the polypeptide (PP) at its C-terminus. In some embodiments, the activator in the conjugate is ligated to the C-terminus of the polypeptide (PP) via a conjugate or linker.

[0116] In some embodiments, the antibody-drug conjugate is: [ka] The structure of JPEG2026511082000006.jpg145170; or having a pharmaceutically acceptable salt thereof, AB is an antibody or its antigen-binding fragment; and p is between 1 and 20.

[0117] In some embodiments, p is 1 to 10. In some embodiments, p is 2 to 8. In some embodiments, p is 4 to 8. In some embodiments, p is 6 to 8. In some embodiments, p is 1. In each of the above-described structures, the succinimide ring may exist in one or more ring-opening isomer forms as shown below: [ka]

[0118] In some embodiments, the present invention provides a conjugate having the structure of formula II or formula III: AB-[(L1) m -PP] q Formula II PP-(L2) n -TP Formula III or a pharmaceutically acceptable salt thereof, in the formula: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide (for example, the NLS polypeptide disclosed in this invention); L2 is the linker; TP is a therapeutic payload; m is either 0 or 1; n is either 0 or 1; and q is between 1 and 20.

[0119] In some embodiments, AB, L1, PP, L2, TP, m, and n are as defined in formula I; and q is between 1 and 20. In some embodiments, q is between 1 and 10. In some embodiments, q is between 2 and 8. In some embodiments, q is between 3 and 6.

[0120] In some embodiments, the present invention provides an antibody-polypeptide conjugate of formula II: AB-[(L1) m -PP] q Formula II or a pharmaceutically acceptable salt thereof, in the formula: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide (for example, the NLS polypeptide disclosed in this invention); m is 0 or 1; and q is between 1 and 20.

[0121] In some embodiments, AB is an antibody. In some embodiments, L1 is a polymer linker. In some embodiments, L1 comprises a PEG group and an antibody-binding group. In some embodiments, L1 is the linker disclosed in the present invention. In some embodiments, L1 is a PEG 3~6It is a linker. In some embodiments, L1 is a PEG3 or PEG4 linker. In some embodiments, PP includes sequence number 10. In some embodiments, PP includes sequence number 11. In some embodiments, PP includes sequence number 12. In some embodiments, PP includes sequence number 13. In some embodiments, PP includes any one of sequence numbers 1 through 8. In some embodiments, PP includes sequence number 1. In some embodiments, PP includes sequence number 2. In some embodiments, PP includes sequence number 3. In some embodiments, PP includes sequence number 4. In some embodiments, PP includes sequence number 5. In some embodiments, PP includes sequence number 6. In some embodiments, PP includes sequence number 7. In some embodiments, PP includes sequence number 8. In some embodiments, m is 1. In some embodiments, q is an integer from 1 to 20. In some embodiments, q is from 1 to 10. In some embodiments, q is from 1 to 8. In some embodiments, q is from 2 to 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.

[0122] In some embodiments, the conjugate of formula (II) is an antibody-polypeptide PP conjugate. In some embodiments, the conjugate of formula (II) is useful for the delivery of antibodies to intracellular (i.e., nuclear) targets. In some embodiments, the antibody-polypeptide conjugate binds to a target antigen specifically expressed on cancer cells, and the antibody-polypeptide conjugate is then endocytized or internalized by the cell to form an early endosome, which subsequently matures into a late endosome and finally fuses with a lysosome. In some embodiments, in the fused lysosome, under specific conditions (pH, presence of degrading enzymes), enzymatic cleavage of the linker or degradation of the antibody occurs, forming metabolites. In some embodiments, inclusion and degradation in the endosomal-lysosomal pathway on which AC relies to deposit the molecular payload into the target cell results in reduced intracellular accumulation. In some embodiments, NLS polypeptide (PP) can facilitate the transport of the antibody-polypeptide conjugate to the nucleus, escaping endosomal containment, resulting in increased intracellular accumulation with high target cell selectivity.

[0123] In some embodiments, the present invention provides a polypeptide-drug conjugate of formula III: PP-(L2) n -TP Formula III or a pharmaceutically acceptable salt thereof, in the formula: PP is a polypeptide (for example, the NLS polypeptide disclosed in this invention); L2 is the linker; TP is a therapeutic payload; and n is either 0 or 1.

[0124] In some embodiments, PP is an NLS polypeptide. In some embodiments, PP is the polypeptide of SEQ ID NO: 10. In some embodiments, PP contains the sequence of SEQ ID NO: 11 and / or SEQ ID NO: 12. In a specific embodiment, PP contains the sequence of SEQ ID NO: 13. In a more specific embodiment, PP is one of SEQ ID NOs: 1 to 8. In some embodiments, L2 is a stable (non-degradable) linker. In some embodiments, L2 is C 1~6 It is an alkyl or heteroalkyl linker. In some embodiments, L2 is a bond (alternatively, L2 is absent or n is expressed as 0). In some embodiments, the therapeutic payload is a cytotoxic agent. In some embodiments, the cytotoxic agent inhibits the proliferation of cells (e.g., tumor cells). In some embodiments, the therapeutic payload is a DNA transcription or translation inhibitor. In some embodiments, the therapeutic payload is an RNA transcription or translation inhibitor. In some embodiments, the therapeutic payload is a nuclear enzyme inhibitor (e.g., a nuclear enzyme involved in DNA or RNA processing). In some embodiments, the therapeutic payload inhibits DNA or RNA synthesis or replication. In some embodiments, the therapeutic payload is a topoisomerase inhibitor.

[0125] In some embodiments, the conjugate of formula (III) is a polypeptide-drug conjugate (e.g., an NLS polypeptide-drug conjugate). In some embodiments, the conjugate of formula (III) is useful for delivering a therapeutic payload to an intracellular (i.e., nuclear) target. In some embodiments, the therapeutic payload inhibits or blocks DNA transcription, translation, or replication. In some embodiments, the therapeutic payload is cytotoxic to cancer cells. 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 intracellularly cytotoxic to cancer cells (e.g., cells expressing cancer-associated antigens). In some embodiments, the conjugate of formula (III) produces less toxicity (e.g., extratarget cytotoxicity) than the therapeutic payload alone. In some embodiments, the conjugate of formula (I) has a broader therapeutic range (i.e., less extratarget toxicity and / or higher on-target toxicity) than a reference conjugate lacking the polypeptide disclosed in the present invention (e.g., an NLS polypeptide). In some embodiments, the conjugate of formula (III) has a broader therapeutic range (i.e., less in-target toxicity and / or higher on-target toxicity) than a reference payload (e.g., any one of SEQ ID NOs: 1 to 13) that lacks the polypeptide disclosed in the present invention (e.g., NLS polypeptide).

[0126] metabolite In some embodiments, the polypeptide (PP) comprises a linker adduct. As used in the present invention, the linker adduct refers to a linker fragment produced by cleavage (i.e., metabolic cleavage) of the conjugate or linker provided in the present invention. In some embodiments, the linker adduct comprises a PEG group (i.e., polyethylene glycol monomer 1, 2, 3, 4, 5, or 6). In some embodiments, the linker adduct further comprises a heteroalkyl group or a heterocycloalkyl group. In some embodiments, the linker adduct further comprises an amino acid. In some embodiments, the compound provided in the present invention has the structure of formula IV: (LA) r -PP-(L2) n -TP Formula IV During the ceremony: LA is a linker adduct; PP is a polypeptide; L2 is the linker; TP is a therapeutic payload; r is 0 or 1; and n is either 0 or 1.

[0127] In some embodiments, PP in formula IV is an NLS polypeptide. In some embodiments, PP is the polypeptide of sequence number 10. In some embodiments, PP contains the sequence of sequence number 11 and / or sequence number 12. In a specific embodiment, PP contains the sequence of sequence number 13. In a more specific embodiment, PP is one of sequence numbers 1 to 8. In some embodiments, LA is: [ka] Or a fragment thereof.

[0128] In some embodiments, the therapeutic payload (TP) is a warhead (preferably a cytotoxic agent) as described in the present invention.

[0129] 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 in a cell (e.g., a tumor cell) following the intracellular processing of an antibody (AB) of formula I. Provided in one embodiment is a method for inhibiting the transcription and / or translation of genetic information (e.g., DNA, RNA, etc.) in a cell, comprising contacting a target protein with the compound of formula IV. In some embodiments, the target protein is expressed in a cell. 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, the present invention provides a method for killing tumor cells or inhibiting the proliferation of tumor cells in a subject, comprising administering an antibody-drug conjugate according to formula I to the subject. In some embodiments, the present invention provides a compound of formula IV for use in a method for treating a disease or disorder in a subject. In some embodiments, the disease or disorder is cancer. In some embodiments, the method involves administering a therapeutically effective dose of an antibody-drug conjugate of formula I to a target. In some embodiments, the compounds provided in the present invention are those of formulas IV-10: (LA) r -PP-(L2) n -TP Formula IV-10 During the ceremony: LA is a linker adduct; PP is a polypeptide containing sequence number 10; Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Here, x 1 , x 2 , x 3 , x 4 , and x 5Each is independently a natural or non-natural amino acid; and is either 0 or 1; L2 is the linker; TP is a therapeutic payload; r is 0 or 1; and n is either 0 or 1.

[0130] In some embodiments, x 1 is G or A; x 2 is G or A; x 3 is A or V; and x 4 , L or I. 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 to 8. In some embodiments, the polypeptide (PP) of formula IV-10 is any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the present invention provides a compound of formula IV-10 for use in a method of treating a disease or disorder (e.g., cancer) in a subject requiring it.

[0131] Therapeutic payload The term therapeutic payload generally refers to any payload useful for the treatment, diagnosis, or detection of a disease, and includes, but is not limited to, therapeutically active compounds, drugs, diagnostic agents, detection agents, radioligands, etc. In some embodiments, the therapeutic payload (TP) is a warhead in which an antibody polypeptide-drug conjugate exerts cytotoxicity after being taken up into cancer cells. In some embodiments, the therapeutic payload has at least acceptable efficacy in destroying tumor cells. In some embodiments, the therapeutic payload is stable under physiological conditions and is bound to the antibody via a linker. The therapeutic payload can be, for example, a cytotoxic agent or an immunostimulant. Preferably, the therapeutic payload is permeable to the cell membrane (e.g., tumor cell membrane). More preferably, the therapeutic payload penetrates tumor cells and exerts cytotoxic or antiproliferative effects within the cells.

[0132] In some embodiments, the therapeutic payload (TP) is an anticancer agent. In some embodiments, the therapeutic payload (TP) is an antitumor antibiotic, a microtubule inhibitor, a cytotoxic or cell proliferation inhibitor, a topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA alkylating agent, a DNA binding agent, a DNA cleavage agent, or an RNA polymerase inhibitor. In some embodiments, the therapeutic payload inhibits the synthesis or replication of DNA or RNA. 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.

[0133] In some embodiments, the therapeutic payload (TP) includes an antitumor antibiotic. In some embodiments, the therapeutic payload (TP) includes a microtubule inhibitor. In some embodiments, the therapeutic payload (TP) includes a cytotoxic agent or a cell proliferation inhibitor. In some embodiments, the therapeutic payload (TP) includes a topoisomerase inhibitor. In some embodiments, the therapeutic payload (TP) includes a pyrrolobenzodiazepine. In some embodiments, the therapeutic payload (TP) includes a DNA alkylating drug. In some embodiments, the therapeutic payload (TP) is a DNA-binding drug. In some embodiments, the therapeutic payload (TP) is a DNA-cleaving drug. In some embodiments, the therapeutic payload (TP) includes an RNA polymerase inhibitor.

[0134] 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 an unciaramycin compound. In some embodiments, the therapeutic payload (TP) is a dynemycin compound. In some embodiments, the therapeutic payload (TP) is a tylanstatin compound. In some embodiments, the therapeutic payload (TP) is a tubulisin compound. In some embodiments, the therapeutic payload (TP) is a vinca alkaloid compound. In some embodiments, the therapeutic payload is camptothecin, exatecan, tubulidine, MMAE, maytansinoid, pyrrolobenzodiazepine, unciaramycin, or tylanstatin. In some embodiments, the therapeutic payload (TP) is duocalmycin, auristatin, maytansinoid, unciaramycin, dynemycin, tylanstatin, camptothecin, exatecan, or tubulisin compounds. In some embodiments, the therapeutic payload (TP) is camptothecin, exatecan, tubulizine, MMAE, maytansinoid, pyrrolobenzodiazepine, unciaramycin, tylanstatin, lurubinectedin, trabectedin, safracin, lenalidomide, eribulin, vinca alkaloid (e.g., vincristine, vinblastine, vindesine, or vinorelbine); epothyron, taxane (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), cryptophycin, hemiastarin, anthracycline, bisnaphthylamide (e.g., erinafide), or cytotoxic molecular adhesive / PROTAC compounds.

[0135] In some embodiments, the therapeutic payload (TP) comprises an antiproliferative agent. In some embodiments, the therapeutic payload (TP) comprises a cytotoxic agent and / or a cell proliferation inhibitor. In some embodiments, the therapeutic payload (TP) comprises angiogenic inhibitor, a cell cycle progression inhibitor, an RNA polymerase inhibitor, a MAPK inhibitor, a PI3K inhibitor, an mTOR inhibitor, an EGFR inhibitor, a VEGFR inhibitor, an HDAC inhibitor, a PARP inhibitor, a Wnt hedgehog inhibitor, a tubulin polymerization inhibitor, a topoisomerase inhibitor, an antitumor antibiotic, an oxidative phosphorylation inhibitor, or a vinca alkaloid. In some embodiments, the therapeutic payload (TP) comprises a DNA conjugate, a DNA intercalator, a DNA alkylating agent (e.g., calicheamicin, dactinomycin, 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.

[0136] 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.

[0137] In some embodiments, the therapeutic payload (TP) is a duocalmycin compound. In some embodiments, the therapeutic payload (TP) is duocalmycin A. In some embodiments, the therapeutic payload (TP) is duocalmycin B. In some embodiments, the therapeutic payload (TP) is duocalmycin CI. In some embodiments, the therapeutic payload (TP) is duocalmycin C2. In some embodiments, the therapeutic payload (TP) is duocalmycin D. In some embodiments, the therapeutic payload (TP) is duocalmycin SA. In some embodiments, the therapeutic payload (TP) is CC-1065. In some embodiments, the therapeutic payload (TP) is adzeresin. In some embodiments, the therapeutic payload (TP) is bizeresin. In some embodiments, the therapeutic payload (TP) is karzeresin. In some embodiments, the therapeutic payload (TP) is duocalmycin A, duocalmycin B, duocalmycin B2, duocalmycin CI, duocalmycin C2, duocalmycin D, duocalmycin SA, CC-1065, adzeresin, bizeresin, or karzeresin.

[0138] 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 hydroxypropylamide (MMAFHPA). In some embodiments, the therapeutic payload (TP) is auristatin F hydroxypropylamide (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 a drastatin compound. In some embodiments, the therapeutic payload (TP) is drastatin 10. In some embodiments, the therapeutic payload (TP) is drastatin 15. In some embodiments, the therapeutic payload (TP) is drastatin 10 or drastatin 15.

[0139] In some embodiments, the therapeutic payload (TP) is an antitumor chemotherapy agent. In some embodiments, the therapeutic payload (TP) is a cytotoxic agent or a cell proliferation inhibitor. 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, berotecan, exatecan, diflomotecan, glimotecan, lulutotecan, 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-11 (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) includes the following components: [ka] During the ceremony, [ka] The symbol indicates a binding point 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.

[0140] In some embodiments, the therapeutic payload (TP) is an antitumor antibiotic. In some embodiments, the therapeutic payload (TP) is calichemycin. In some embodiments, TP is a calichemycin residue conjugated to a sulfur atom at L2. In some embodiments, the therapeutic payload (TP) is a calichemycin having the following structure: [ka] During the ceremony, [ka] The symbol indicates a binding point 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.

[0141] In some embodiments, the therapeutic payload (TP) is a DNA alkylating agent. In some embodiments, the therapeutic payload (TP) is a DNA groove binding agent. In some embodiments, the therapeutic payload (TP) is an RNA polymerase II inhibitor. In some embodiments, the therapeutic payload (TP) is ectenacidin. In some embodiments, the therapeutic payload (TP) is trabectedin, ecbectedin, or lurubinectedin. In some embodiments, the therapeutic payload (TP) is: [ka] and; During the ceremony, [ka] The symbol indicates a binding point 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.

[0142] Linker In one embodiment, the present invention provides an antibody-drug conjugate comprising an antibody and a therapeutic payload, wherein the antibody and the therapeutic payload are linked via a linker, the linker comprising an NLS polypeptide (i.e., a polypeptide (PP) disclosed herein). In some embodiments, the linker further comprises an alkyl or heteroalkyl moiety. In some embodiments, the linker further comprises an aryl moiety and a heteroaryl moiety. In some embodiments, the heteroalkyl moiety comprises a polymer moiety (e.g., a PEG moiety). In some embodiments, the linker comprises 0 to 36 amino acids. In some embodiments, the heteroalkyl moiety comprises 0 to 12 polyethylene glycol (PEG) groups. In some embodiments, the heteroalkyl moiety comprises an antibody-binding group (e.g., maleimide, succinimide, or other lysine- or cysteine-reactive group), or an antibody-binding linker formed therefrom. In some embodiments, the linker comprises a protein-binding group or a protein-binding group. In some embodiments, the linker comprises homoglycine or C 1-12 It contains an alkylene group. In some embodiments, the linker contains a cleavable linker. In some embodiments, the linker contains a non-cleavable linker. In some embodiments, the linker contains an autoimmune group. In some embodiments, the linker has the following structure: -(L1) m -PP-(L2) n - Here: L1 is the first linker (e.g., a linker to an antibody or antigen-binding fragment); PP is a polypeptide (for example, the NLS polypeptide disclosed in this invention); L2 is the second linker (for example, the linker to the therapeutic payload); m is 0 or 1; and n is either 0 or 1.

[0143] In some embodiments, L1 is a cleavable linker. In some embodiments, L1 is a non-cleavable linker. In some embodiments, L1 includes an alkyl moiety. In some embodiments, L1 includes a heteroalkyl moiety. In some embodiments, L1 includes an aryl moiety. In some embodiments, L1 includes a heteroaryl moiety. In some embodiments, L1 includes one or more linear or branched C 1-80 It contains an alkyl moiety. In some embodiments, L1 is one or more linear or branched C 1-80 It contains a heteroalkyl moiety. In some embodiments, L1 is one or more linear or branched C atoms suspended by one or more cycloalkyl, heterocycloalkyl, aryl, or heteroaryl rings. 1-80 It contains a heteroalkyl moiety. In some embodiments, L1 is one or more linear or branched C 1-80 It contains a heteroalkyl moiety. In some embodiments, L1 is alkyl or heteroalkyl C 1-80 The linker further comprises one or more monocyclic or polycyclic heterocycloalkyl or heteroaryl rings. Exemplary rings include, but are not limited to, pyrrole, pyrrolidinone, imidazole, imidazolidinone, triazole, maleimide, succinimide, and polycyclic cyclooctyne. In some embodiments, L1 is homoglycine or C 1-12 It contains an alkylene group. In some embodiments, L1 consists of homoglycine. In some embodiments, L1 is C 1-12 Contains alkylene groups.

[0144] In some embodiments, L1 contains 0 to 12 (e.g., 0, 1, 2, 3, ... 12) polyethylene glycol monomers. In some embodiments, L1 contains 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) polyethylene glycol monomers. In some embodiments, L1 contains 2 to 6 (e.g., 3 or 4) polyethylene glycol monomers. In some embodiments, L1 contains 3 or 4 polyethylene glycol monomers. In some embodiments, L1 contains 4 polyethylene glycol monomers.

[0145] In some embodiments, L1 contains 0 to 12 (e.g., 0, 1, 2, 3, ... 12) amino acids. In some embodiments, L1 contains 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) amino acids. In some embodiments, L1 contains 1 to 8 amino acids. In some embodiments, L1 contains 1 to 4 amino acids. In some embodiments, L1 contains 8 to 12 amino acids. In some embodiments, L1 contains 2 to 6 (e.g., 3 or 4) amino acids. In some embodiments, L1 contains 2 to 6 amino acids. In some embodiments, L1 contains 3 or 4 amino acids.

[0146] In some embodiments, L1 contains 0 to 12 (e.g., 0, 1, 2, 3, ... 12) C=O groups. In some embodiments, L1 contains 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) C=O groups. In some embodiments, L1 contains 1 to 2 C=O groups. In some embodiments, L1 contains 1 C=O group. In some embodiments, L1 contains 2 C=O groups. In some embodiments, L1 contains 0 to 12 (e.g., 0, 1, 2, 3, ... 12) C(=O)NH groups. In some embodiments, L1 contains 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) C(=O)NH groups. In some embodiments, L1 contains 1 C(=O)NH group. In some embodiments, L1 contains 2 C(=O)NH groups.

[0147] In some embodiments, L1 comprises an antibody-binding group (e.g., maleimide, succinimide, or other lysine-reactive or cysteine-reactive group), or an antibody-binding linker formed therefrom. In some embodiments, L1 comprises a protein-binding group or a protein-binding group. In some embodiments, L1 comprises a lysine-reactive group, a cysteine-reactive group, or a glutamine-reactive group. In some embodiments, L1 comprises a lysine-reactive group. In some embodiments, L1 comprises a cysteine-reactive group. In some embodiments, L1 comprises a glutamine-reactive group.

[0148] In some embodiments, L1 includes a protein-binding group that reacts with a lysine or cysteine ​​residue of the antibody or its antigen-binding fragment. Preferably, such a protein-binding group and / or protein-binding linker is less susceptible to undesirable cleavage (e.g., retromichael removal of the maleimide group).

[0149] In some embodiments, L1 includes the following groups: [ka]

[0150] In some embodiments, L1 includes the following groups: [ka]

[0151] In some embodiments, L2 is a cleavable linker. In some embodiments, L2 is a non-cleavable linker. In some embodiments, L2 includes an alkyl moiety. In some embodiments, L2 includes a heteroalkyl moiety. In some embodiments, L2 includes an aryl moiety. In some embodiments, L2 includes a heteroaryl moiety. In some embodiments, L2 includes one or more linear or branched C1~80 It contains an alkyl moiety. In some embodiments, L2 is one or more linear or branched C 1~80 It contains a heteroalkyl group. In some embodiments, L2 contains an autoimmune group.

[0152] In some embodiments, the present invention provides a cleavable linker bound to a therapeutic payload via an ester, amide, thiophosphate, or disulfide bond. In some embodiments, the cleavable linker is efficiently and cleanly cleaved (i.e., without leaving any post-cleavage artifacts bound to the payload at the attachment site). In some embodiments, the protease-cleavable linker is efficiently and cleanly cleaved without the use of autoimmune groups, thereby resulting in enhanced stability.

[0153] In some embodiments, L2 contains 0 to 12 (e.g., 0, 1, 2, 3, ..., 12) amino acids. In some embodiments, the conjugate L2 contains 0 to 12 (e.g., 0, 1, 2, 3, ..., 12) polyethylene glycol monomers. In some embodiments, L2 contains glycine or C 1~12 Contains alkylene groups.

[0154] In some embodiments, L2 is a linker of the following formula: -NH-C 1-6 Alkyl-[OCH2CH2] 0-8 -C 0-6 Alkyl-Z-(C 1-12 Alkyl-S) 0-1 -; In the formula, Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, -OP(=O)(SH)-, or -OP(=O)(OH)-.

[0155] In some embodiments, L2 is a linker of the following formula: -NH-(CH2) 2-6-(OCH2CH2) 1-8 -(CH2) 0-3 -ZC 1-12 Alkyl-S-; -NH-(CH2) 1-12 -ZC 1-12 Alkyl-S-; -NH-(CH2) 2-6 -(OCH2CH2) 1-8 -(CH2) 0-3 -Z-; or -NH-(CH2) 1-12 -Z-; In the formula, Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, or a bond.

[0156] In some embodiments, L2 is a linker of the following formula: [ka]

[0157] In some embodiments, L2 is a linker of the following formula: [ka]

[0158] In some embodiments, L2 is a linker of the following formula: [ka]

[0159] In some embodiments, L2 is a linker of the following formula: [ka]

[0160] In some embodiments, L2 is a linker of the following formula: [ka]

[0161] In some embodiments, n is 0 and L2 is absent. In some embodiments, n is 1 and L2 is coupled to a therapeutic payload.

[0162] Precursor molecule In another embodiment, the present invention provides a precursor molecule (i.e., a compound) comprising a nuclear localization signal polypeptide and a therapeutic payload, further comprising a protein-reactive group (alternatively, a protein-binding group). The precursor molecule provided in the present invention may be useful in the synthesis or manufacture of pharmaceuticals for treating diseases or disorders in a subject (e.g., cancer). In some embodiments, the present invention provides a compound for use in the manufacture of pharmaceuticals according to the following formula V or formula VI. In some embodiments, the precursor molecule includes an activator other than the therapeutic payload, such as a detection group. In some embodiments, the precursor molecule has the structure of formula V, VI, or VII: AC-(L1) m -PP-(L2) n -TP formula V AC-(L1) m -PP Formula VI AC-(L1) m -PP-(L2) n -DG Formula VII or having pharmaceutically acceptable salts thereof, in formula: AC is an antibody-binding group configured to form a covalent bond with an antibody or antigen-binding fragment (e.g., a sulfur atom in the cysteine ​​side chain of an antibody, or a nitrogen atom in the lysine side chain of an antibody); L1 is the linker; PP is a polypeptide (for example, the NLS polypeptide disclosed in this invention); L2 is the linker; TP is a therapeutic payload; DG is a detection group; m is 0 or 1; and n is either 0 or 1.

[0163] In some embodiments, the present invention provides a precursor molecule (sometimes called a conjugate) of formula V or formula VI, wherein the polypeptide (PP) comprises a sequence selected from SEQ ID NOs: 1 to 8. In some embodiments, 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 yet another embodiment, 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.

[0164] In some embodiments, L1 is a cleavable linker. In some embodiments, L1 is a non-cleavable linker. In some embodiments, L1 is a stable linker. In some embodiments, L1 is a peptide linker. In some embodiments, L1 is a non-peptide linker. In some embodiments, L1 contains 0 to 12 (e.g., 0, 1, 2, 3, ... 12) amino acids. In some embodiments, L1 contains 0 amino acids. In some embodiments, L1 contains 1 to 12 amino acids. In some embodiments, L1 contains 1 to 4 amino acids. In some embodiments, L1 is a PEG linker. In certain embodiments, L1 contains 0 to 12 (e.g., 0, 1, 2, 3, ... 12) polyethylene glycol monomers. In some embodiments, L1 contains 1 to 8 (e.g., 2 to 6 (e.g., 3 or 4)) polyethylene glycol monomers. In some embodiments, L1 contains 1 to 8 polyethylene glycol monomers. In some embodiments, L1 contains 2 to 6 polyethylene glycol monomers. In some embodiments, L1 comprises three or four polyethylene glycol monomers. In some embodiments, L1 is a linker to a protein-binding group or a protein-conjugated group (AC). In some embodiments, L1 comprises a PEG segment and a protein-binding segment (AC), and optionally one or more linking segments. In certain embodiments, the AC comprises a lysine-reactive group, a cysteine-reactive group, or a glutamine-reactive group.

[0165] In some embodiments, AC of formula V or VI comprises the following bases: [ka] Optionally, C 1-12 Alkyl alkyl group or C 1-12 Heteroalkyl (e.g., PEG) 3-6 It further contains the group.

[0166] In some embodiments, AC-L1 includes one of the following structures: [ka]

[0167] In some embodiments, the detection group of formula VII is a dye. In some embodiments, the detection group is a gamma emitter, a beta emitter (for example, 3 H, 14 C, 32 P, 35 S, or 90 The detection group is Sr) or an alpha emitter. In some embodiments, the detection group responds to a stimulus (e.g., light, laser, or other energy source) with an emitted response (e.g., fluorescence, phosphorescence, or other detectable light). In some embodiments, the detection group is a FRET pair, or a donor or acceptor of a BRET pair. In some embodiments, the detection group is a chromophore, a fluorescent label, a bioluminescent label, or a chemiluminescent label. In some embodiments, the detection group is an Alexa Fluor® dye, HiLyte TM The detection group is a fluorine dye or other functional equivalent (e.g., coumarin dye, rhodamine dye, or cyanine dye). In some embodiments, the detection group is a coumarin dye. In some embodiments, the detection group is a rhodamine dye. In some embodiments, the detection group is a cyanine dye. In some embodiments, the detectable group is Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, HiLyte TM Fluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte TM Fluor 555, Alexa Fluor® 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor® 647, HiLyte TMFluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, or HiLyte TM Fluor 750, or a functional equivalent thereof. In some embodiments, the detection group is Alexa Fluor® 488 or HiLyte TM Fluorine 647, or a functional equivalent thereof. In this invention, a functional equivalent means a chemical entity having the same essential properties (e.g., excitation or emission wavelength) as the reference dye.

[0168] In some embodiments, the therapeutic payload of formula V is an antitumor antibiotic, a microtubule inhibitor, a cytotoxic or cell proliferation inhibitor, a topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA alkylating drug, a DNA binding drug, a DNA cleavage drug, or an RNA polymerase inhibitor. In some embodiments, the therapeutic payload is pyrrolobenzodiazepine, duocalmycin, auristatin, meitansinoid, unciaramycin, dynemycin, tylanstatin, camptothecin, exatecan, tubulsin compounds, ekhbectedin, lurubinectedin, trabectedin, safracin, lenalidomide, eribulin, vincristine, vinblastine, vindesine, vinorelbine, epothilon, taxane (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), cryptophycin, hemiastalin, anthracycline, bisnaphthylamide (e.g., erinafide), or cytotoxic molecular adhesive / PROTAC compounds.

[0169] 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 agent or a cell proliferation inhibitor. In some embodiments, the therapeutic payload is a topoisomerase I inhibitor. In some embodiments, the therapeutic payload is or consists of camptothecin or exatecan compounds. In some embodiments, the therapeutic payload is camptothecin, 10-hydroxycamptothecin, topotecan, irinotecan, berotecan, exatecan, diflomothecan, glimatecan, lulutotecan, silatecan, rubitecan, or SN-38. In some embodiments, the therapeutic payload is ectenacydin. In some embodiments, the therapeutic payload is trabectedin. In some embodiments, the therapeutic payload is ecubectedin. In some embodiments, the therapeutic payload is lurubinectedin. In some embodiments, the therapeutic payload is calichemycin. In some embodiments, the therapeutic payload is calichemycin γ1(γ-1). In some embodiments, the therapeutic payload is selected from one of the following: [ka]

[0170] In another embodiment, the present invention provides a precursor molecule having the following structure: [ka] JPEG2026511082000026.jpg234170 JPEG2026511082000027.jpg167170 or a pharmaceutically acceptable salt thereof.

[0171] Also considered within the scope of this disclosure are methods for preparing antibody-drug conjugates, which include contacting one or more of the precursor molecules disclosed in the present invention with an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment comprises one or more cysteine ​​molecules. In some embodiments, the method includes reacting the precursor molecule with the antibody in the presence of a base and / or a reducing agent. In some embodiments, the method includes reacting the precursor molecule with the antibody or its antigen-binding fragment in a reaction mixture comprising Tris and / or TCEP. Exemplary methods for preparing antibody-drug conjugates (e.g., of formula I, (e.g., of formula I-10)) are disclosed in the Examples section.

[0172] antibody In some embodiments, the term “antibody” is used in its broadest sense and includes intact antibodies, polyclonal and monoclonal antibodies containing their functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, single-chain antibody fragments containing F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. This term encompasses intrabody, peptide body, chimeric antibody, fully human antibody, humanized antibody, heteroconjugate antibody, multispecific antibody, e.g., bispecific antibody, diabody, triabody, and tetrabody, tandemdi scFv, tandemtri scFv, etc., and genetically modified and / or otherwise altered forms of immunoglobulins. Unless otherwise specified, the term “antibody” should be understood to encompass its functional antibody fragment. Furthermore, this term also encompasses intact antibodies or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. Antibodies may contain the human IgG1 constant region. Antibodies may contain the human IgG4 constant region.

[0173] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR," and are known in the art to refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) along its entire length, and each light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) along its entire length. The precise amino acid sequence boundaries of a given CDR or FR are as follows: 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.This can be easily determined using one of many well-known schemes, including those described by “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plueckthun 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 modeling antibodies on the WEB,” Protein Eng. 2000 Dec;13(12):819-24 (“AbM” numbering scheme). In this invention, the CDR of the antibody described may be defined by Kabat, IMGT, Chothia, AbM, Aho, contact numbering scheme, or any combination thereof.

[0174] The term "variable region" or "variable domain" refers to the domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain consists of four conserved framework regions (FRs) and three CDRs (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). In some cases, a single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain from an antibody that binds to the antigen (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0175] When used in relation to antibodies, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, with a variable region at the amino terminus containing approximately 120-130 or more amino acids, and a constant region at the carboxy terminus. The constant region can be one of five different types (e.g., isotypes) called α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), based on the amino acid sequence of the heavy chain constant region. α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When bound to a light chain, these different types of heavy chains give rise to five well-known antibody classes (e.g., isotypes): IgA, IgD, IgE, IgG, and IgM, respectively, including the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0176] When used in relation to antibodies, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, with a variable region of approximately 100 to 110 or more amino acids in the amino-terminal region and a constant region in the carboxy-terminal region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, called κ (kappa) or λ (lambda), based on the amino acid sequence of the constant domain. The amino acid sequences of light chains are well known in the art. The light chain may be a human light chain.

[0177] The antibodies provided may include antibody fragments. The terms "antibody fragment," "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to molecules other than the intact antibody that constitute part of the intact antibody that binds to 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 (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. Generally, an antibody fragment or antigen-binding fragment consists of one or more CDRs from the parent antibody sufficient to confer binding specificity.

[0178] 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 may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has been typically humanized to reduce immunogenicity against humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues of a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived) to restore or improve the specificity or affinity of the antibody, for example. In some embodiments, a humanized antibody refers to a form of antibody that is not a fully humanized antibody but a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human (e.g., mouse) sequences.

[0179] The antibodies offered include human antibodies. “Human antibody” means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, human cell, or non-human source utilizing a human antibody repertoire, including a human antibody library, or other human antibody coding sequences. This term excludes humanized forms of non-human antibodies that include a non-human antigen-binding region, such as those in which all or substantially all CDRs are non-human.

[0180] Human antibodies can be prepared by administering an immunogen to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such transgenic animals typically contain, or have in all or part of, human immunoglobulin loci replacing endogenous immunoglobulin loci, either extrachromosomally or randomly incorporated into the animal's chromosomes. In such transgenic animals, endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived or selected from human antibody libraries, including phage display libraries and cell-free libraries, containing antibody coding sequences derived from the human repertoire. In some embodiments, human antibodies can be de-degraded or have increased affinity through a series of selections, such as by phage display.

[0181] Fc constant region Generally, the fragment crystallizable (Fc) region or domain of an antibody mediates downstream effector functions through interaction with Fc receptors on immune cells (e.g., innate immune cells) or with complement protein C1q, a complement system recognition molecule. Furthermore, interaction with Fc receptors can lead to target cell killing through various immunoeffector mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP), while antibody-mediated complement activation can lead to complement-dependent cell-mediated cytotoxicity (CDC). In addition, both Fc-receptor interactions and complement α activation may exert a wide range of immunomodulatory functions.

[0182] Therefore, in some embodiments, mutations within the Fc region that reduce, inhibit, excise, and / or eliminate Fc-mediated function are advantageous in reducing the immune activation resulting from antibody binding to a 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. The Fc region may consist of the C-terminal region of an immunoglobulin heavy chain, comprising a hinge region, a CH2 domain, a CH3 domain, or any combination thereof. As used in the present invention, the Fc region includes a native sequence Fc region and a variant Fc region. The Fc region variant may consist of a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitution, addition, or deletion) at one or more amino acid positions.

[0183] Pharmaceutical composition The pharmaceutical compositions disclosed in the present invention can be administered to a subject by any suitable route of administration, including but not limited to parenteral administration (intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravenous infusion, or topical), topical administration, oral administration, or nasal administration.

[0184] "Pharmacologically acceptable" may mean that it is approved or eligible for approval by a federal or state regulatory agency, or that it is listed in the United States Pharmacopeia or other generally accepted pharmacopoeia for use in animals, including humans.

[0185] The term "pharmaceutically acceptable salt" may refer to a salt of a compound that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound.

[0186] "Pharmacologically acceptable excipients, carriers, or adjuvants" may mean excipients, carriers, or adjuvants that can be administered to a subject together with at least one antibody of the present disclosure, which do not disrupt the pharmacological activity and are non-toxic when administered in a dose sufficient to deliver a therapeutic dose of the compound.

[0187] "Pharmacologically acceptable vehicle" may mean a diluent, adjuvant, excipient, or carrier to which at least one of the antibodies of this disclosure is administered.

[0188] therapeutic use In another embodiment, the present invention provides a method for treating a disease or disorder in a subject requiring treatment, comprising administering a therapeutically effective amount of a composition comprising the nuclear localization signal polypeptide disclosed herein to the subject. In some embodiments, the NLS polypeptide is conjugated to a therapeutic payload. In some embodiments, the NLS polypeptide is further conjugated to an antibody or its antigen-binding fragment. In some embodiments, the NLS polypeptide comprises SEQ ID NO: 10. In some embodiments, the NLS polypeptide comprises the sequence of one or more peptides from SEQ ID NOs: 11, 12, and 13. In some embodiments, the polypeptide comprises one of SEQ ID NOs: 1 to 8.

[0189] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring treatment, comprising administering a therapeutically effective amount of a therapeutic payload (e.g., a cytotoxic agent) conjugated to an NLS polypeptide to the subject. In some embodiments, the therapeutic payload is stably bound 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 its antigen-binding fragment. In some embodiments, the therapeutic payload is a cytotoxic agent. In some embodiments, the therapeutic payload is cytotoxic to tumor cells. In some embodiments, the therapeutic payload is an inhibitor of DNA and / or RNA replication (transcription, translation, etc.). In some embodiments, the therapeutic payload is an inhibitor of a nuclear target protein.

[0190] In some embodiments, the therapeutic payload is camptothecin, exatecan, tubulcin, MMAE, maytansinoid, pyrrolobenzodiazepine, unciaramycin, or tylanstatin. In some embodiments, the therapeutic payload is camptothecin. In some embodiments, the therapeutic payload is exatecan. In some embodiments, the therapeutic payload is tubulcin. In some embodiments, the therapeutic payload is MMAE. In some embodiments, the therapeutic payload is maytansinoid. In some embodiments, the therapeutic payload is pyrrolobenzodiazepine. In some embodiments, the therapeutic payload is unciaramycin. In some embodiments, the therapeutic payload is tylanstatin. 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 cancer. In some embodiments, the disease or disorder is a cancer associated with the overexpression of CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, Nectin-4, Mesoserine, BCMA, Folate Receptor α, and Tissue Factor. In some embodiments, the disease or disorder 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, E These are NPP3, 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.

[0191] In another embodiment, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate of formula I to the subject. AB-[(L1) m -PP-(L2) n -TP] p Equation I During the ceremony: AB is an antibody or its antigen-binding fragment; L1 is the linker; PP is an NLS polypeptide; L2 is the linker; TP is a therapeutic payload; m is either 0 or 1; n is either 0 or 1; and p is between 1 and 20.

[0192] In some embodiments, the polypeptide (PP) comprises SEQ ID NO: 10. In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an antibody-drug conjugate of formula I to the subject. In some embodiments, the antibody-drug conjugate is of formula I-10.

[0193] In some embodiments, the antibody-drug conjugate is of formula I-10: AB-[(L1) m -PP-(L2) n -TP] p Formula I-10 AB is an antibody or its antigen-binding fragment; L1 is the linker; PP is a polypeptide containing sequence number 10; Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 L2 is the linker; TP is a therapeutic payload; m is either 0 or 1; n is either 0 or 1; and p is between 1 and 20.

[0194] In some embodiments, x 1 is G or A; x 2 is G or A; x 3 is A or V; and x 4 is L or I. In some embodiments, the polypeptide (PP) comprises a sequence selected from SEQ ID NOs: 1 to 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 one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.

[0195] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to a subject, which includes a therapeutic payload stably bound to an NLS polypeptide (PP) containing the sequence PGGKRVKLD (SEQ ID NO: 1).

[0196] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to a subject, which includes a therapeutic payload stably bound to an NLS polypeptide (PP) containing the sequence PGGKRVKLDG (SEQ ID NO: 2).

[0197] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to a subject, which includes a therapeutic payload stably bound to an NLS polypeptide (PP) containing the sequence PAAKRVKLD (SEQ ID NO: 3).

[0198] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to a subject, which includes a therapeutic payload stably bound to an NLS polypeptide (PP) containing the sequence PAAKRVKLDG (SEQ ID NO: 4).

[0199] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to a subject, which includes a therapeutic payload stably bound to an NLS polypeptide (PP) containing the sequence PAGKRVKLDG (SEQ ID NO: 5).

[0200] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to a subject, which includes a therapeutic payload stably bound to an NLS polypeptide (PP) containing the sequence PGAKRVKLDG (SEQ ID NO: 6).

[0201] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to a subject, which includes a therapeutic payload stably bound to an NLS polypeptide (PP) containing the sequence PAAKRAKLDG (SEQ ID NO: 7).

[0202] In some embodiments, the present invention provides a method for treating a disease or disorder in a subject requiring such treatment, comprising administering a therapeutically effective amount of an antibody-drug conjugate to a subject, which includes a therapeutic payload stably bound to an NLS polypeptide (PP) containing the sequence PAAKRVKIDG (SEQ ID NO: 8).

[0203] In some embodiments, the disease or disorder is a hyperproliferative disease or disorder. In some embodiments, the disease or disorder is 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 having a hyperproliferative disease or disorder (e.g., cancer or tumor). In some embodiments, the present invention provides a method for delivering a therapeutic payload to an intracellular target within tumor cells, comprising contacting tumor cells 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.

[0204] In some embodiments, a method for delivering a therapeutic payload to an intracellular target within tumor cells includes contacting tumor cells with a peptide-drug conjugate of formula III: PP-(L2) n -TP Formula III PP is a polypeptide; L2 is the linker; TP is a therapeutic payload; and n is either 0 or 1.

[0205] In some embodiments, the polypeptide (PP) comprises an sequence selected from sequence numbers 1 to 8.

[0206] In some embodiments, the present invention provides a method for treating cancer in a subject requiring such treatment, comprising administering a therapeutically effective amount of a compound or conjugate provided in the present invention to the subject. In some embodiments, the conjugate has the structure of formula I and / or I-10. In some embodiments, the subject is a mammal (e.g., human). In some embodiments, the cancer 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. In some embodiments, cancer is associated with the overexpression of CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, and tissue factor. In some embodiments, cancer is a hematological cancer or a solid tumor. In some embodiments, cancer is a solid tumor. In some embodiments, cancer is non-Hodgkin lymphoma, breast cancer, ovarian cancer, or gastric cancer. In some embodiments, cancer is large cell lymphoma, ductal carcinoma, mammary gland cancer, ovarian adenocarcinoma, or gastric cancer. Also provided in the present invention is the use of compounds or conjugates provided in the present invention for the treatment of diseases or disorders such as cancer. Also provided in the present invention is a conjugate of formula I or formula I-10, or a compound or conjugate provided in the present invention, for use in the treatment of diseases or disorders.Furthermore, the present invention provides compounds or conjugates (e.g., of formula I, formula I-10, formula III, or formula V) disclosed herein for use in the manufacture of pharmaceuticals for treating diseases or disorders. In some embodiments, the disease or disorder is cancer.

[0207] Pharmaceutical properties In some embodiments, the present invention provides a method for improving one or more pharmaceutically acceptable properties of an antibody-drug conjugate or its components (e.g., an antibody or a therapeutic payload). The NLS polypeptides disclosed in the present invention include those having SEQ ID NOs: 1 to 8. The method may also include producing the antibody-drug conjugates disclosed in the present invention. Examples of pharmaceutically acceptable properties include, but are not limited to, toxicity (e.g., therapeutic window) and distribution. Distribution may refer to the dispersion or dispersal of a substance (e.g., a therapeutic payload) throughout body fluids and tissues. Advantageous properties of the conjugates disclosed in the present invention include enhanced distribution (i.e., localization) of the therapeutic payload in tumor cells (i.e., tumor enrichment).

[0208] The therapeutic window of a therapeutic payload disclosed in the present invention, when conjugated with an antibody-drug conjugate disclosed in the present invention, can increase by at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 times or more compared to an unconjugated payload. In some embodiments, the therapeutic window of a conjugate disclosed in the present invention (e.g., an antibody-drug conjugate, a polypeptide-drug conjugate, or their metabolites) can be at least about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or 300 times compared to the therapeutic window of an unconjugated therapeutic payload. In some embodiments, the therapeutic window of a conjugate disclosed in the present invention is at least about 100, 200, 300, 400, 500, 1000, or more than the therapeutic window of an unconjugated drug. In some embodiments, the therapeutic index of the conjugates disclosed in the present invention is at least about 50 times, 100 times, 200 times, 300 times, 400 times, 500 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, or more, greater than the therapeutic index of the non-conjugate drugs. As is clear from Table 6, the ADCs provided in the present invention have an IC2000 times lower than that of cancer cells compared to healthy cells. 50 It may have a value. For a representative sample of ADC provided in the present invention, the calculated therapeutic index (IC) 50 Neutrophil / IC 50 Cancer cells are disclosed in Table 7. [Examples]

[0209] Abbreviations and acronyms Ab antibody BTFFH N'-bis(tetramethylene)formamidinium hexafluorophosphate Boc Tert-butyloxycarbonyl protecting group CHAPS 3-[(3-Colamidopropyl)dimethylammonio]-1-propanesulfonate COMU 1-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate Dalton DAR drug-antibody ratio DEA (Diethylamine) DIPEA N-,N-diisopropylethylamine DMF Dimethylformamide DMSO (Dimethyl Sulfoxide) EDTA (Ethylenediaminetetraacetic acid) equiv or eq equivalent (molar equivalent) ESI Electrospray Fmoc fluorenylmethoxycarbonyl protecting group HIC Hydrophobic Interaction Properties HCC1954 HER2+ ductal carcinoma cells HRMS High-Resolution Mass Spectrometry I C 50 Average concentration required to achieve 50% inhibition LC / MS Liquid Chromatography Mass Spectrometry LC / MSD liquid chromatography mass-selective detector mAb monoclonal antibody Mal Maleimide MK megakaryocyte mM millimoles min M / Z mass-to-charge ratio NAC (N-acetylcysteine) NCI-N87 HER2-positive gastric cancer NHS N-hydroxysuccinimide NLS nuclear localization sequence nm (nanometer) nM nanomoles Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl protecting group PBS (phosphate-buffered saline) PEG polyethylene glycol Rt Retention Time SEC size exclusion chromatography SKBR3 HER2+ Breast Cancer SKOV3 Human Ovarian Cancer SUDHL1 Large cell lymphoma tBu Tert-butyl TCEP Tris(2-carboxyethyl)phosphine TFA (Trifluoroacetic Acid) TOF Flight Time Tris (Hydroxymethyl)aminomethane UPLC (Ultra High Performance Liquid Chromatography) UV ultraviolet light v / v volume / volume

[0210] [Table 1]

[0211] The following examples are provided for illustrative purposes only and do not limit the scope of the claims provided in the present invention.

[0212] General experiment Analysis method A - Equipment: Agilent 1260 Infinity Lab LC / MSD - Column: Agilent Zorbax C18, 5mm, 4.6x50mm - Column temperature: 45°C - Mobile phase A: 0.1% TFA water - Mobile phase B: 0.1% TFA acetonitrile - Gradient: Hold at 5% to 95% mobile phase B for 3 minutes, hold at 95% B for 3 minutes, then wash column for 4 minutes. - UV trace monitor at 210nm and 254nm. - Positive and negative ionization modes for monitoring molecular ions in the range of 115 Da to 1200 Da

[0213] Analysis method B - Equipment: Agilent 6230 TOF LC / MS - Column: Agilent Zorbax 300SB-C8, 5mm, 4.6x50mm - Column temperature: 45°C - Mobile phase A: 0.1% FA water - Mobile phase B: 0.1% FA acetonitrile - Gradient: Hold at 5% to 95% mobile phase B for 10 minutes, hold at 95% B for 5 minutes, then wash column for 5 minutes. - UV trace monitor at 210nm and 254nm.

[0214] synthesis Scheme 1: General procedure for coupling a protected polypeptide with a payload. [ka]

[0215] Protected polypeptide COOH (1 equivalent) was dissolved in dry DMF at a concentration of 0.1 M, followed by the addition of diisopropylethylamine (DIPEA, 10 equivalents) and COMU coupling reagent (5 equivalents). The reaction mixture was stirred at room temperature for 10 minutes, after which the exatecan payload (2 equivalents) was added. The reaction mixture was stirred at room temperature overnight (16-18 hours), and product formation was confirmed by LC-MS. The crude product was purified using flash reverse-phase chromatography with a gradient of 5%-95% acetonitrile / water containing 0.1% formic acid, using a Biotage Isolera chromatograph equipped with a Biotage Sfar C18 column. The purity and identity of the isolated product were determined by analytical method A and summarized in Table 1 below.

[0216] [Table 2]

[0217] Scheme 2. General procedure for N-terminal Fmoc deprotection. [ka]

[0218] The N-terminal Fmoc-protected polypeptide-exatecan intermediate was dissolved in 0.5-1 ml of 10% diethylamine in DMF, and the reaction mixture was stirred at room temperature for 2 hours. Removal of Fmoc was confirmed by LC-MS analysis A as shown in the table below. The reaction mixture was diluted with distilled water in a 1:25 v / v ratio and lyophilized. The resulting crude product was used in the next step.

[0219] [Table 3]

[0220] Scheme 3. General procedure for coupling a protected polypeptide-payload intermediate with maleimide-PEG-carboxylic acid. [ka]

[0221] Maleimide-PEG4-carboxylic acid NHS ester (Quanta Biodesign) (1.5 equivalents) was dissolved in dry DMF at a concentration of 0.1 M, followed by the addition of DIPEA (10 equivalents) and the crude N-terminal deprotected polypeptide-exatecan intermediate from step 2. The reaction mixture was stirred at room temperature for 12-16 hours, and product formation was observed by LC-MS. The crude product was purified by flash reverse-phase chromatography using a Biotage Isolera chromatograph equipped with a Biotage Sfar C18 column, with a gradient of 5%-95% acetonitrile / water and 0.1% formic acid. The purity and identity of the isolated product were determined by analytical method A and summarized in Table 3 below.

[0222] [Table 4]

[0223] Scheme 4. General procedure for global deprotection of amino acid side chains. [ka]

[0224] The maleimide-PEG4-polypeptide-exatecan intermediate from Step 3 was treated with 1-3 ml of 95% TFA / water at room temperature for 1 hour. The reaction mixture was diluted with distilled water in a 1:25 v / v ratio and lyophilized. The crude product was incorporated into DMF and purified by flash reverse-phase chromatography using a Biotage Isolera chromatography apparatus equipped with a Biotage Sfar C18 column with a gradient of 5%-95% acetonitrile / water and 0.1% formic acid. The purity and identity of the isolated product were identified by analytical method B and summarized in Table 4 below.

[0225] [Table 5]

[0226] Scheme 5. General procedure for conjugation of calichemycin precursor molecules. [ka]

[0227] Step 1: Maleimide-PEG4-PAAKRVKLDG-carboxylic acid intermediate (1 eq) was dissolved in anhydrous DMF (0.05 M), and BTFFH (5 eq) and DIPEA (10 eq) were added. The reaction mixture was stirred at room temperature for 30 minutes, after which calichemycinamine (1.5 eq) and additional DIPEA (10 eq) were added. The reaction was stirred overnight in the dark, and the formation of the target product was confirmed by LC-MS. The crude product was purified by flash reverse-phase chromatography using a Biotage Isolera chromatography system equipped with a Biotage Sfar C18 column, with a gradient of 5%-95% acetonitrile / water containing 0.1% formic acid. The purity and identity of the isolated product were identified by analytical method A. HRMS calculated accurate mass 3620.5753, measured [M+H] + 3621.5802.

[0228] Step 2: Global deprotection was performed following the general procedure for global deprotection of amino acid side chains described above. The final product was purified by flash reverse-phase chromatography using a Biotage Isolera chromatography system equipped with a Biotage Sfar C18 column, with a gradient of 5%–95% acetonitrile / water and 0.1% formic acid. The purity and identity of the isolated product were identified by analytical method A. HRMS calculated accurate mass 3112.3259, measured [M+H] + 3113.3260.

[0229] Scheme 6: General procedure for lurubinectedin precursor molecular conjugation. [ka]

[0230] Step 1: Maleimide-PEG4-PAAKRVKLDG-carboxylic acid intermediate (3 eq) was dissolved in anhydrous DMF (0.05 M), and BTFFH (6 eq) and DIPEA (5 eq) were added. The reaction mixture was stirred at room temperature for 30 minutes, after which lurubinectedin (1 eq) and additional DIPEA (5 eq) were added. The reaction mixture was stirred at room temperature for 2 hours, and the formation of the desired product was confirmed by LC-MS. The crude product was purified by flash reverse-phase chromatography using a Biotage Isolera chromatography apparatus equipped with a Biotage Sfar C18 column with a gradient of 5%-95% acetonitrile / water and 0.1% formic acid. The purity and identity of the isolated product were identified by analytical method A. HRMS calculated accurate mass 2725.3199, [M-OH] + Calculated value: 2706.3093, measured value: [M-OH] + 2706.3100.

[0231] Step 2: Global deprotection was performed following the general procedure for global deprotection of amino acid side chains described above. The final product was purified by flash reverse-phase chromatography using a Biotage Isolera chromatography system equipped with a Biotage Sfar C18 column and a gradient of 5% to 95% acetonitrile / water containing 0.1% formic acid. The purity and identity of the isolated product were identified by analytical method A. HRMS calculated accurate mass: 2217.0704, [M-OH] + Calculated value: 2198.0598, measured value: [M-OH] + 2198.0600.

[0232] Scheme 7. General synthesis of NLS linker-dye compounds [ka]

[0233] Step 1: HiLyte TM Fluorine 647 succinimidyl ester (SE) (Anaspec, AS-81256, 1 eq) was dissolved in anhydrous DMF to a 100 mM solution, followed by the addition of Boc-amino-PEG4-amine (Broadpharm, BP-22602, 1.5 eq) and N,N-diisopropylethylamine (4 eq). The reaction mixture was stirred at room temperature for 30 minutes under light shielding, and the formation of the product was confirmed by LC-MS. The crude product was purified by reverse-phase chromatography using a gradient of 5-95% water / acetonitrile containing 0.1% formic acid.

[0234] The Boc-amine-PEG4-HiLyte Fluor 647 from the previous step was deprotected in 10% TFA / DCM for 1 hour at room temperature, shielded from light. The product was concentrated and dried overnight under high vacuum. The crude product was used in the next step without purification. The identity of the product was confirmed by LC-MS, and the expected [M+H] + 1424, measured [M+2H] 2+ The value was 713.0.

[0235] Step 2: Boc-amine-PEG4-HiLyte Fluor 647 from the previous step was deprotected in 10% TFA / DCM for 1 hour, shielded from light, at room temperature. The product was concentrated and dried overnight under high vacuum. The crude product was used in the next step without purification. The identity of the product was confirmed by LC-MS, and the expected [M+H] + 1424, actual measurement [M+2H] 2+ The value was 713.0.

[0236] Step 3: Maleimide-PEG4-PAAKRVKLD-carboxylic acid intermediate (7-1) (2 eq) was dissolved in anhydrous DMF (100 mM), followed by the addition of BTFFH (3 eq) and DIPEA (5 eq), and the mixture was stirred at room temperature for 30 minutes. The amine-PEG4-HiLyte Fluor 647 (1 eq), which had been deprotected in the previous step, was added to the reaction mixture, and then DIPEA (5 eq) was added. The reaction mixture was stirred overnight at room temperature in the dark. Product formation was confirmed by LC-MS. The crude product (7-2) was purified by reverse-phase chromatography using a gradient of 5-95% water / acetonitrile containing 0.1% formic acid. LC-MS [M+H] + Predicted 3311.3, actual measurement [M+3H] 3+ 1104.4.

[0237] Step 4: Final deprotection of maleimide-PEG4-PAAKRVKLD-PEG4-HiLyte Fluor 647(7-3) was performed using 95% TFA / water at room temperature in the dark for 2 hours. The crude product was purified by reverse-phase chromatography using a gradient of 5-95% water / acetonitrile containing 0.1% formic acid. LCMS[M+H] + Predicted 2802.6, actual measurement [M+3H] 3+ 935.

[0238] Reference linker-dye compound: Synthesis of Mal-PEG2-Val-Cit-PAB-HiLyte Fluor 647

[0239] Mal-PEG2-Val-Cit-PAB-PNP (Broadpharm®, 1 eq) was dissolved in anhydrous DMF (100 mM), and HiLyte Fluor 647 amine (AnaSpec, AS-8256, 1 eq) and DIPEA (4 eq) were added. The reaction mixture was stirred at room temperature in the dark for 2 hours. Product formation was confirmed by LC-MS. The crude product was purified by reverse-phase chromatography using a gradient of 5-95% water / acetonitrile containing 0.1% formic acid. LC-MS[M+H] + Predicted 1792.1, actual measurement [M+3H] 3+ 896.6.

[0240] Linker dye conjugation

[0241] First, trastuzumab antibodies were conjugated to AF488 using the Abcam AF488 labeling kit (ab236553) according to the manufacturer's instructions. Next, the AF488-labeled antibodies were subjected to disulfide reduction via TCEP (10 eq of TCEP), and then conjugated to Mal-Val-Cit-PAB-HiLyteFluor 647 or Mal-NLS peptide 4-2-HiLyteFluor 647 (2 eq of each per free SH) at room temperature for 2 hours. Excess unconjugated dye was removed using biotin and a Zeba Spin Desalting column (Pierce). The dye / antibody ratio was measured using UV-VIS to characterize the antibody conjugates, and the results are summarized in the table below.

[0242] [Table 6]

[0243] Exemplary ADC precursor molecule (PM) For example, PM-1 contains sequence number 1 (PGGKRVKLD). [ka]

[0244] Example PM-2 corresponds to intermediates 4-5 in Table 4 and contains sequence number 2 (PGGKRVKLDG). [ka]

[0245] Example PM-3 corresponds to intermediate 4-2 in Table 4 and contains sequence number 3 (PAAKRVKLD). [ka]

[0246] Example PM-4 corresponds to intermediate 4-1 in Table 4 and contains sequence number 4 (PAAKRVKLDG). [ka]

[0247] Example PM-5 corresponds to intermediate 4-3 in Table 4 and contains sequence number 5 (PAGKRVKLDG). [ka]

[0248] Example PM-6 corresponds to intermediate 4-4 in Table 4 and contains sequence number 6 (PGAKRVKLDG). [ka]

[0249] Example PM-7 corresponds to intermediates 4-6 in Table 4 and contains sequence number 7 (PAAKRAKLDG). [ka]

[0250] Example PM-8 corresponds to intermediates 4-7 in Table 4 and contains sequence number 8 (PAAKRVKIDG). [ka]

[0251] Example PM-9 corresponds to intermediate 4-8 in Table 4 and contains sequence number 9 (VAAKRVKLDG). [ka]

[0252] Example PM-10 corresponds to intermediates 4-9 in Table 4 and contains sequence number 14 (PKKKRKV). [ka]

[0253] Example PM-11 corresponds to intermediate 4-10 in Table 4 and contains sequence number 15 (PKKKRKVG). [ka]

[0254] Example PM-12 corresponds to scheme 5 in Table 4 and includes sequence number 4 (PAAKRVKLDG). [ka]

[0255] Example PM-13 corresponds to scheme 7 in Table 4 and includes sequence number 4 (PAAKRVKLDG). [ka]

[0256] Scheme 8. Conjugation of a precursor molecule to an antibody or antigen-binding fragment. [ka]

[0257] The mAb was pH adjusted with 200 mM Tris, 5 mM EDTA, pH 8.5 (10% in PBS, 30% in proA), and reduced with TCEP 4-10 equiv at 37°C for 1 hour. The number of free thiols was quantified using Ellman's test. 10% DMSO and 2 moles of excess linker-agent per mole of free thiols were added, the solution was vortexed, and left at room temperature for 2 hours. Next, 1.2 moles of N-acetylcysteine ​​(NAC) per mole of linker-agent were added, the solution was vortexed, and left at room temperature for 20 minutes. Subsequently, the ADC was replaced with 3x buffer in PBS pH 7.4 to remove the free linker-agent.

[0258] Exemplary antibody-drug conjugates (ADCs) [ka]

[0259] [ka]

[0260] [ka]

[0261] [ka]

[0262] [ka]

[0263] [ka]

[0264] [ka]

[0265] [ka]

[0266] [ka]

[0267] [ka]

[0268] [ka]

[0269] [ka]

[0270] [ka]

[0271] Tables 5, 6, and 7 refer to prior examples ADCs 2-13 having the structures provided above, combined with antibodies that are IgG1, brentuximab, or trastuzumab, respectively. For comparison, reference ADCs are also provided in which the polypeptide is not an NLS polypeptide but instead consists of the sequence GGFG.

[0272] ADC Implementation (Cryptography) The drug-antibody ratio (DAR, also denoted as the subscript "p") was determined by either hydrophobic interaction chromatography (HIC) or mass spectrometry of intact ADCs. Aggregation was determined by size exclusion chromatography (SEC).

[0273] Characterization of hydrophobic interactions in ADCs. HIC was performed using a polypropyl A column (PolyLC) with 1.5 M ammonium sulfate and 25 mM potassium phosphate aqueous solution as mobile phase A and 0.25% w / v CHAPS and 25 mM potassium phosphate aqueous solution as mobile phase B. The sample was injected directly into the column and a 0-100% gradient of mobile phase B was applied over 15 minutes. UV signals at 280 nm were collected, and unconjugated antibodies and higher-order DAR species were analyzed from the chromatogram. DAR was calculated by integrating the area under the HIC curve of pre-defined peaks (DAR=0, DAR=1, DAR=2, DAR=4, etc.) and calculating the percentage of each peak.

[0274] Characterization of inactive ADCs by mass spectrometry. The unreduced mass of antibody-drug conjugates was measured using an Agilent 6230 TOF LC / MS equipped with an electrospray ionization (ESI) source directly coupled to an Agilent 1260 high-performance liquid chromatography system. Samples were first diluted to 1 mg / mL and analyzed in their unreduced state. Proteins were separated using a reversed-phase column (Zorbax 300SB-C8, 5 mm, 4.6 x 50 mm) with a denaturing mobile phase system. Mobile phase A was 0.1% (v / v) formic acid in water. Mobile phase B was 0.1% (v / v) formic acid in 80% (v / v) 2-propanol, 10% (v / v) acetonitrile, and 10% (v / v) water (mobile phase B). The MS spectra of each protein were averaged and deconvoluted to obtain the average mass and monoisotopic mass.

[0275] Characterization of ADC aggregation by size exclusion chromatography. Size exclusion chromatography (SEC) was used to evaluate the size heterogeneity of antibody-drug conjugates. An Acquity 1.7 μm, 4.6 x 300 mm UPLC BEH200SEC column was used for the analysis, with 25 mM sodium phosphate, pH 6.5, 500 mM L-arginine, and 10% isopropanol (IPA) in water as the mobile phase. Samples were injected neatly, and the mobile phase was applied isocratically at 0.2 mL / min for 22 minutes. UV signals at 280 nm were collected, and the degree of ADC aggregation and fragmentation was calculated from the peak area. Complete characterization data is shown in Table 5 below.

[0276] [Table 7] JPEG2026511082000069.jpg57170

[0277] Cytotoxic assay of adherent cells. Adherent cells were cultured in T75 flasks until ~50-80% confluence and harvested as single-cell suspensions using trypsin. 3,000-5,000 cells per well were seeded into tissue culture plates in 50 mL / well of culture medium and incubated at 37°C for 18-24 hours. Subsequently, ADC, serially diluted with culture medium, was dispensed into plates at 50 mL / well. After plating and processing, the cells were returned to the incubator and cultured for a further 3-5 days. CellTiter-Glo reagent was prepared according to the manufacturer's instructions and added to the culture medium at a rate of 100 mL / well. CellTiter-Glo allows for the relative enumeration of cells with high metabolic activity by quantifying intracellular ATP concentration. After incubation with CellTiter-Glo at room temperature for 5 minutes, the black assay plate with a clear bottom was read with a luminometer within 30 minutes, or 125 mL / well of CellTiter Glo / cell lysate was transferred to a black assay plate and read. Luminescence measurements obtained from untreated cultures (cell culture medium only) were used as the 100% control, and all other luminescence values ​​were normalized against these controls (e.g., normalized RLU, relative luminescence units). Luminescence signals were detected using a standard plate reader, and IC50 was calculated using logistic nonlinear regression with GraphPad Prism (GraphPad Software, San Diego, CA).

[0278] Suspension cell cytotoxicity assay. Suspended cells were cultured as single-cell suspensions in T75 flasks. 3,000–5,000 cells per well were seeded onto tissue culture plates with 50 mL / well of culture medium and incubated at 37°C for 18–24 hours. Subsequently, ADC, serially diluted with culture medium, was dispensed into plates at 50 mL / well. After plating and processing, the cells were returned to the incubator and cultured for a further 3–5 days. CellTiter-Glo reagent was prepared according to the manufacturer's instructions, and 100 mL / well was added to the culture medium. CellTiter-Glo allows for the relative enumeration of cells with high metabolic activity by quantifying intracellular ATP concentration. After incubation with CellTiter-Glo at room temperature for 5 minutes, the black assay plate with a clear bottom was read with a luminometer within 30 minutes, or 125 mL / well of CellTiter Glo / cell lysate was transferred to a black assay plate and read. Luminescence measurements obtained from untreated cultures (cell culture medium only) were used as the 100% control, and all other luminescence values ​​were normalized against these controls (e.g., normalized RLU, relative luminescence units). Luminescence signals were detected using a standard plate reader, and IC50 was calculated using logistic nonlinear regression with GraphPad Prism (GraphPad Software, San Diego, CA).

[0279] Human neutrophil cytotoxicity assay. Human CD34+ myeloid progenitor cells were seeded at a rate of 3,000 cells per well in growth medium supplemented with 10 ng / ml human recombinant IL-3 and 30 ng / ml human recombinant G-CSF (both from Peprotech, Cranbury, NJ) in 96-well plates. Test ADCs or free drugs were added in double or triple doses to the final concentrations indicated on each well. After 3-4 days of culture, half of the medium (100 μl) was carefully removed, and a viability assay using CellTiter-Glo 2.0 (Promega, Madison, WI) was performed according to the manufacturer's protocol. Luminescence signals were detected using a standard plate reader, and IC50 was calculated by logistic nonlinear regression using GraphPad Prism (GraphPad Software, San Diego, CA).

[0280] The results are summarized in Table 6 below. All experiments were repeated at least three times with each data point duplicated. Figures 1-4 show the normalized RLU (relative luminescence units) of SKBR3 (Figure 1), HCC1954 (Figure 2), neutrophils (Figure 3), and megakaryocytes (Figure 4) cells after administration of Example ADC-12 and Example ADC-13, with each ADC bound to either IgG1 antibody or trastuzumab antibody. Trastuzumab-conjugated ADC-12 and ADC-13 significantly reduced RLU in SKBR3 and HCC1954 breast cancer cell lines, but not in neutrophils or MK cells.

[0281] [Table 8] JPEG2026511082000071.jpg253170JPEG2026511082000072.jpg149170

[0282] [Table 9]

[0283] Imaging research using SKBR3 cells Following ATCC guidelines, SKBR3 cells were cultured in ATCC-prepared McCoy's 5a Medium Modified (catalog no. 30-2007) and seeded at 2000 cells / well in F-bottom (chimney-well) mCLEAR black 96-well plates (Greiner, catalog no. 655096). After cell adhesion, the medium was replaced with a medium free of phenol red and containing the corresponding antibody-dye conjugate at a concentration of 100 ug / ml, and incubated on ice for 1 hour. Subsequently, the cells were gently washed with fresh medium free of phenol red to remove unbound antibody-dye conjugates. The cells were incubated in a tissue culture incubator at 37°C, and short-duration imaging was performed after 2 hours, 8 hours, 24 hours, and 48 hours of antibody-dye conjugate incubation. Imaging was performed using a Molecular Devices ImageXpress Micro Confocal instrument, with the z-plane aligned to the center of the cells, and images were processed using the manufacturer's software. Each antibody-dye conjugate was tested in triplicates. Representative results are summarized in Figure 5. Cell nuclei were stained blue for clarity. AF488 appeared green, HF647 appeared red, and co-localization of AF488 and HF647 produced a yellowish-green color. After 2 hours, antibody-dye constructs containing Val-Cit and NLS linkers were observed localized on the cell surface. After 8 hours, internalization of the conjugates was observed, with clear accumulation in the lysosomal compartment. After 8 hours, the conjugate prepared with NLS peptide-HF647 showed separation of red and green pigments, suggesting cleavage of the linker pigment from the antibody. After 24 hours, cells incubated with val-cit-HF647 showed no yellow or red color, suggesting complete removal of the red pigment from the cells, while cells incubated with the NLS-HF647 conjugate showed a strong red color overlapping the nucleus. At 48 hours, cells incubated with Val-Cit antibody still contained small amounts of AF488 antibody, while cells incubated with NLS-HF647 antibody-dye conjugate showed a strong presence of NLS-HF647.Figure 5 shows that using an NLS linker increases the intracellular residence time of the payload and promotes nuclear localization.

[0284] [Table 10]

[0285] [Table 11] JPEG2026511082000076.jpg249170JPEG2026511082000077.jpg156170

[0286] manner Aspect 1: Array Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 A composition comprising a polypeptide containing, or a pharmaceutically acceptable salt thereof: During the ceremony: x 1 , x 2 , x 3 , x 4 , and x 5 Each of these is independently either a natural or non-natural amino acid; and a is either 0 or 1.

[0287] Appearance 2 x 1 The composition according to Embodiment 1, or a pharmaceutically acceptable salt thereof, wherein the compound is G or A.

[0288] Embodiment 3 x 2 The composition according to embodiment 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the composition is G or A.

[0289] Appearance 4 x 3 The composition according to any one of embodiments 1 to 3, wherein A or V, or a pharmaceutically acceptable salt thereof.

[0290] Appearance 5 x 4 A composition according to any one of embodiments 1 to 4, wherein L or I, or a pharmaceutically acceptable salt thereof.

[0291] A composition according to any one of embodiments 1 to 5, wherein a is 0, or a pharmaceutically acceptable salt thereof.

[0292] A composition according to any one of embodiments 1 to 5, wherein embodiment 7a is 1, or a pharmaceutically acceptable salt thereof.

[0293] Appearance 8 x 5 The composition according to Embodiment 7, or a pharmaceutically acceptable salt thereof, wherein the composition is G or A.

[0294] Apparatus 9 Polypeptide, sequence Px 1 -x 2 -KRx 3 -Kx 4 -D Sequence ID 11 Includes: During the ceremony: x 1 However, it is G or A; x 2 However, it is G or A; x 3 However, it is A or V; x 4 However, it is L or I; x 5 However, it is G or A. A composition according to any one of embodiments 1 to 8, or a pharmaceutically acceptable salt thereof.

[0295] Embodiment 10 Polypeptide, sequence Px 1 -x 2 -KRx 3 -Kx 4 -Dx 5 Sequence ID 12 Includes: During the ceremony: x 1 is G or A; x2 is G or A; x 3 is either A or V; x 4 is L or I; x 5 is G or A. A composition according to any one of embodiments 1 to 9, or a pharmaceutically acceptable salt thereof.

[0296] Embodiment 11 x 1 A composition according to any one of embodiments 1 to 10, wherein is G, or a pharmaceutically acceptable salt thereof.

[0297] Appearance 12 x 1 A composition according to any one of embodiments 1 to 10, wherein is A, or a pharmaceutically acceptable salt thereof.

[0298] Appearance 13 x 2 A composition according to any one of embodiments 1 to 12, wherein is G, or a pharmaceutically acceptable salt thereof.

[0299] Appearance 14 x 2 A composition according to any one of embodiments 1 to 12, wherein is A, or a pharmaceutically acceptable salt thereof.

[0300] Appearance 15 x 3 A composition according to any one of embodiments 1 to 14, wherein is V, or a pharmaceutically acceptable salt thereof.

[0301] Embodiment 16 x 4 A composition according to any one of embodiments 1 to 15, wherein L is present, or a pharmaceutically acceptable salt thereof.

[0302] Appearance 17 x 5 A composition according to any one of embodiments 1 to 16, wherein is G, or a pharmaceutically acceptable salt thereof.

[0303] Appearance 18 x 5 A composition according to any one of embodiments 1 to 6, or a pharmaceutically acceptable salt thereof, in which the above is absent.

[0304] Apparatus 19 Polypeptide, sequence Px 1 -x 2 -KRVKLD-(G) a Sequence ID 13 Includes: In the formula, x 1 and x 2 A is independently A or G; a is 0 or 1, the composition according to any one of embodiments 1 to 10, or a pharmaceutically acceptable salt thereof.

[0305] Appearance 20 x 1 and x 2 The composition described in Embodiment 19, or a pharmaceutically acceptable salt thereof, wherein each is A.

[0306] Appearance 21 x 1 and x 2 The composition according to embodiment 19, or a pharmaceutically acceptable salt thereof, wherein each is G.

[0307] Embodiment 22 A composition according to any one of Embodiments 1 to 6, 9, or 11, wherein the polypeptide comprises a sequence selected from SEQ ID NOs: 1 to 8, or a pharmaceutically acceptable salt thereof.

[0308] Embodiment 23 A composition according to any one of Embodiments 1 to 9, 11, 13, 15, 16, 18, 19, 21, or 22, wherein the polypeptide comprises SEQ ID NO: PGGKRVKLD, or a pharmaceutically acceptable salt thereof.

[0309] Embodiment 24 A composition according to any one of Embodiments 1 to 5, 7 to 11, 13, 15, 16, 17, 19, 21 or 22, wherein the polypeptide comprises SEQ ID NO: PGGKRVKLDG, or a pharmaceutically acceptable salt thereof.

[0310] Embodiment 25 A composition according to any one of Embodiments 1 to 6, 9, 12, 14, 15, 16, 18, 19, 20, or 22, wherein the polypeptide comprises SEQ ID NO: PAAKRVKLD, or a pharmaceutically acceptable salt thereof.

[0311] Embodiment 26 A composition according to any one of Embodiments 1-5, 7-10, 12, 14, 15, 16, 17, 19, 20, or 22, wherein the polypeptide comprises SEQ ID NO: PAAKRVKLDG, or a pharmaceutically acceptable salt thereof.

[0312] Embodiment 27 A composition according to any one of Embodiments 1 to 5, 7 to 10, 12, 13, 15, 16, 17, 19, or 22, wherein the polypeptide comprises SEQ ID NO: PAGKRVKLDG, or a pharmaceutically acceptable salt thereof.

[0313] Embodiment 28 A composition according to any one of Embodiments 1 to 5, 7 to 11, 14, 15, 16, 17, 19, or 22, wherein the polypeptide comprises SEQ ID NO: 6: PGAKRVKLDG, or a pharmaceutically acceptable salt thereof.

[0314] Embodiment 29 A composition according to any one of Embodiments 1-5, 7-10, 12, 14, 16, 17, 19, 20, or 22, wherein the polypeptide comprises SEQ ID NO: PAAKRAKLDG, or a pharmaceutically acceptable salt thereof.

[0315] Embodiment 30 A composition according to any one of Embodiments 1 to 5, 7 to 10, 12, 14, 15, 17, 19, 20, or 22, wherein the polypeptide comprises SEQ ID NO: PAAKRVKIDG, or a pharmaceutically acceptable salt thereof.

[0316] Embodiment 31 A composition according to any one of Embodiments 1 to 30, further comprising an antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment and the polypeptide are conjugated via a linkage or via a linker, or a pharmaceutically acceptable salt thereof.

[0317] Embodiment 32 The composition according to Embodiment 31, or a pharmaceutically acceptable salt thereof, wherein an antibody or antigen-binding fragment is conjugated to a polypeptide via binding.

[0318] Embodiment 33 The composition according to Embodiment 31, or a pharmaceutically acceptable salt thereof, wherein an antibody or antigen-binding fragment is conjugated to a polypeptide via a linker.

[0319] Embodiment 34 A composition according to any one of Embodiments 31 to 33, wherein an antibody or antigen-binding fragment is bound to P of a polypeptide, or a pharmaceutically acceptable salt thereof.

[0320] Embodiment 35 The composition according to Embodiment 33 or 34, or a pharmaceutically acceptable salt thereof, wherein the linker comprises one or more polyethylene glycol groups or polyethyleneimine groups.

[0321] Embodiment 36 The composition according to Embodiment 35, or a pharmaceutically acceptable salt thereof, wherein the linker comprises 1 to 12 polyethylene glycol groups.

[0322] Embodiment 37 The composition according to Embodiment 36, or a pharmaceutically acceptable salt thereof, wherein the linker comprises 2 to 8 polyethylene glycol groups.

[0323] Embodiment 38 The composition according to Embodiment 37, or a pharmaceutically acceptable salt thereof, wherein the linker comprises 3 to 6 polyethylene glycol groups.

[0324] Embodiment 39 A composition according to any one of Embodiments 33 to 38, wherein the linker comprises a cleavable group (e.g., a chemically labelable group, a pH-sensitive group, a protease-cleavable group, an autoimmune group, a hydrolyzable group, etc.), or a pharmaceutically acceptable salt thereof.

[0325] Embodiment 40 The composition according to any one of Embodiments 33 to 38, or a pharmaceutically acceptable salt thereof, wherein the linker is a non-cleaving linker.

[0326] Embodiment 41 A composition according to any one of Embodiments 33 to 40, or a pharmaceutically acceptable salt thereof, wherein the linker comprises a non-cleavable heteroalkyl group and / or a heterocyclic group.

[0327] Embodiment 42 The composition according to any one of Embodiments 33 to 41, wherein the linker comprises 1 to 12 amino acids, or a pharmaceutically acceptable salt thereof.

[0328] Embodiment 43 A composition according to any one of Embodiments 31 to 42, wherein the antibody or antigen-binding fragment comprises a natural amino acid or a non-natural amino acid, and the linker is bound to the antibody or antigen-binding fragment via the natural amino acid or non-natural amino acid, or a pharmaceutically acceptable salt thereof.

[0329] Embodiment 44 A composition according to any one of Embodiments 31 to 43, wherein the antibody or antigen-binding fragment comprises cysteine, lysine, or glutamine, and the linker is bound to the antibody or antigen-binding fragment via cysteine, lysine, or glutamine, or a pharmaceutically acceptable salt thereof.

[0330] Embodiment 45 A composition according to any one of Embodiments 31 to 44, wherein the antibody or antigen-binding fragment contains cysteine, and the linker is bound to the antibody or antigen-binding fragment via cysteine, or a pharmaceutically acceptable salt thereof.

[0331] Embodiment 46 A composition according to any one of Embodiments 31 to 45, wherein the antibody includes a constant region and a linker is bound to the antibody via the constant region, or a pharmaceutically acceptable salt thereof.

[0332] Embodiment 47 A composition according to any one of Embodiments 31 to 46, wherein the antibody comprises an Fc region, or a pharmaceutically acceptable salt thereof.

[0333] Embodiment 48 The composition according to Embodiment 47, or a pharmaceutically acceptable salt thereof, wherein the linker is conjugated to an antibody via an Fc region.

[0334] Embodiment 49 The composition according to Embodiment 47, or a pharmaceutically acceptable salt thereof, wherein the Fc region has reduced effector function.

[0335] Embodiment 50 A composition according to any one of Embodiments 31 to 49, wherein an antibody or antigen-binding fragment binds to a target antigen, or a pharmaceutically acceptable salt thereof.

[0336] Embodiment 51 The composition according to Embodiment 51, or a pharmaceutically acceptable salt thereof, wherein the target antigen is expressed on tumor cells.

[0337] Apparatus 52 The target antigen is AXL, BCMA, CA9, CCR7, CD19, 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, FOLR 1. A composition according to embodiment 50 or 51, or a pharmaceutically acceptable salt thereof, comprising 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.

[0338] Embodiment 53 A composition according to any one of Embodiments 31 to 52, wherein the target antigen is CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, or tissue factor, or a pharmaceutically acceptable salt thereof.

[0339] Embodiment 54 A composition according to any one of Embodiments 1 to 49, wherein the antibody is of the IgG1 subclass, or a pharmaceutically acceptable salt thereof.

[0340] Embodiment 55 A composition according to any one of Embodiments 1 to 54, further comprising a therapeutic payload, wherein the therapeutic payload and the polypeptide are conjugated via a bond or via a second linker, or a pharmaceutically acceptable salt thereof.

[0341] Embodiment 56 The therapeutic payload is polypeptide D or x 5 A composition according to embodiment 55, or a pharmaceutically acceptable salt thereof, conjugated therein.

[0342] Embodiment 57 The composition according to Embodiment 55 or 56, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is conjugated to a polypeptide via binding.

[0343] Embodiment 58 The composition according to Embodiment 55 or 56, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is conjugated to a polypeptide via a second linker.

[0344] Appearance 59 The second linker is C1~C 12 A composition according to embodiment 58, which is an alkyl or heteroalkyl linker, or a pharmaceutically acceptable salt thereof.

[0345] Embodiment 60 The composition according to Embodiment 58, or a pharmaceutically acceptable salt thereof, wherein the second linker is a non-cleaving linker.

[0346] Embodiment 60 The composition according to Embodiment 58, or a pharmaceutically acceptable salt thereof, wherein the second linker is a cleavable linker.

[0347] Embodiment 62 The composition according to Embodiment 58, or a pharmaceutically acceptable salt thereof, wherein the second linker is a polypeptide linker.

[0348] Embodiment 63 A composition according to any one of Embodiments 55 to 62, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a cytotoxic payload.

[0349] Embodiment 64 A composition according to any one of Embodiments 55 to 63, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload, when internalized within tumor cells, is cytotoxic to tumor cells.

[0350] Embodiment 65 A composition according to any one of Embodiments 55 to 64, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is pyrrolobenzodiazepine, duocalmycin, auristatin, meitansinoid unciaramycin, dynemicin, tylanstatin, camptothecin, exatecan, tubulicin compounds, lurubinectedin, trabectedin, safracin, lenalidomide, eribulin, vincristine, vinblastine, vindesine, vinorelbine, epothilon, taxane (e.g., paclitaxel, docetaxel, cabazitaxel, etc.), cryptophycin, hemiastalin, anthracycline, bisnaphthylamide (e.g., erinafide), or a cytotoxic molecule adhesion / PROTAC compound.

[0351] Embodiment 66 A composition according to any one of Embodiments 55 to 65, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload comprises an antitumor antibiotic, a microtubule inhibitor, a cytotoxic or cell proliferation inhibitor, a topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA alkylating agent, a DNA binding agent, a DNA cleavage agent, or an RNA polymerase inhibitor.

[0352] Embodiment 67 A conjugate or a pharmaceutically acceptable salt thereof comprising an antibody conjugated via a linker and an activator (e.g., a therapeutic payload, a detection group, a diagnostic agent, etc.), wherein the linker comprises a polypeptide and the polypeptide comprises a nuclear localization sequence.

[0353] Embodiment 68 A conjugate according to Embodiment 67, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises nine or ten amino acids.

[0354] Embodiment 69 A conjugate according to Embodiment 67 or 68, or a pharmaceutically acceptable salt thereof, wherein the activator is a therapeutic payload (e.g., a drug).

[0355] Embodiment 70 The conjugate according to Embodiment 69, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is cytotoxic to tumor cells.

[0356] Embodiment 71 A conjugate according to any one of Embodiments 67 to 70, wherein an antibody is linked to a polypeptide at its N-terminus, or a pharmaceutically acceptable salt thereof.

[0357] Embodiment 72 A conjugate according to any one of Embodiments 67 to 71, or a pharmaceutically acceptable salt thereof, wherein the antibody is linked to a polypeptide at its N-terminus via a bond or linker.

[0358] Embodiment 73 A conjugate according to any one of Embodiments 67 to 72, wherein the activator is linked to a polypeptide at its C-terminus, or a pharmaceutically acceptable salt thereof.

[0359] Embodiment 74 A conjugate according to any one of Embodiments 67 to 73, or a pharmaceutically acceptable salt thereof, wherein the activator is polypeptide-linked at the C-terminus via a bond or linker.

[0360] Apparatus 75 Polypeptide in sequence: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Includes: Here, x 1 , x 2 , x 3 , x 4 , and x 5 A conjugate according to any one of embodiments 67 to 74, or a pharmaceutically acceptable salt thereof, wherein each of the above is independently either a natural or non-natural amino acid, and a is either 0 or 1.

[0361] Appearance 76, Type I-10: AB-[(L1) m -PP-(L2) n -TP] p Formula I-10 Having a structure, During the ceremony: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide containing sequence number 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Here, x 1 , x 2 , x 3 , x 4 , and x 5 Each of these is independently either a natural or non-natural amino acid; and a is either 0 or 1; L2 is the linker; TP is a therapeutic payload; m is either 0 or 1; n is 0 or 1; and p is 1 to 20, a conjugate (e.g., as described in any one of embodiments 67 to 75), or a pharmaceutically acceptable salt thereof.

[0362] Embodiment 77: The conjugate according to Embodiment 76, or a pharmaceutically acceptable salt thereof, wherein L1 is a cleavable linker.

[0363] Embodiment 78: The conjugate according to Embodiment 76, or a pharmaceutically acceptable salt thereof, wherein L1 is a non-cleavable linker.

[0364] Embodiment 79: A conjugate according to any one of Embodiments 76 to 78, wherein L1 contains 0 to 12 amino acids, or a pharmaceutically acceptable salt thereof.

[0365] Embodiment 80: A conjugate according to any one of Embodiments 76 to 79, wherein L1 contains 1 to 8 amino acids, or a pharmaceutically acceptable salt thereof.

[0366] Embodiment 81: A conjugate according to any one of Embodiments 76 to 79, wherein L1 contains 1 to 4 amino acids, or a pharmaceutically acceptable salt thereof.

[0367] Embodiment 82: A conjugate according to any one of Embodiments 76 to 79, wherein L1 contains 8 to 12 amino acids, or a pharmaceutically acceptable salt thereof.

[0368] Embodiment 83: A conjugate according to any one of Embodiments 76 to 82, or a pharmaceutically acceptable salt thereof, wherein L1 contains 0 to 12 polyethylene glycol monomers.

[0369] Embodiment 84 A conjugate according to any one of Embodiments 76 to 83, or a pharmaceutically acceptable salt thereof, wherein L1 contains 1 to 8 polyethylene glycol monomers.

[0370] Embodiment 85: A conjugate according to any one of Embodiments 76 to 83, or a pharmaceutically acceptable salt thereof, wherein L1 contains 2 to 6 polyethylene glycol monomers.

[0371] Embodiment 86: A conjugate according to any one of Embodiments 76 to 85, wherein L1 contains a protein-binding group or a group to which a protein is bound, or a pharmaceutically acceptable salt thereof.

[0372] Embodiment 87 A conjugate according to any one of Embodiments 76 to 86, wherein L1 comprises a lysine-reactive group, a cysteine-reactive group, or a glutamine-reactive group, or a pharmaceutically acceptable salt thereof.

[0373] Appearance 88 L1 is homoglycine or C 1-12 A conjugate containing an alkylene group, according to any one of embodiments 76 to 87, or a pharmaceutically acceptable salt thereof.

[0374] Appearance 89 L1: [ka] A conjugate according to any one of embodiments 76 to 88, or a pharmaceutically acceptable salt thereof.

[0375] In aspect 90, L1 is: [ka] A conjugate according to any one of embodiments 76 to 89, or a pharmaceutically acceptable salt thereof.

[0376] Appearance 91 x 1 A conjugate according to any one of embodiments 76 to 90, wherein the conjugate is G or A, or a pharmaceutically acceptable salt thereof.

[0377] Appearance 92 x 2 A conjugate according to any one of embodiments 76 to 91, wherein the conjugate is G or A, or a pharmaceutically acceptable salt thereof.

[0378] Appearance 93 x 3 A conjugate according to any one of embodiments 76 to 92, or a pharmaceutically acceptable salt thereof, wherein the conjugate is A or V.

[0379] Appearance 94 x 4 A conjugate according to any one of embodiments 76 to 93, wherein L or I, or a pharmaceutically acceptable salt thereof.

[0380] A conjugate according to any one of embodiments 76 to 94, or a pharmaceutically acceptable salt thereof, wherein embodiment 95a is 0.

[0381] Appearance 96 a is 1, and x 5 A conjugate according to any one of embodiments 76 to 94, wherein is G or A, or a pharmaceutically acceptable salt thereof.

[0382] Embodiment 97 Polypeptide, Sequence ID 11: Px 1 -x 2 -KRx 3 -Kx 4 -D Sequence ID 11 Includes; Here, x 1 However, it is G or A; x 2However, it is G or A; x 3 However, A or V; and x 4 A conjugate according to any one of embodiments 76 to 94, wherein L or I, or a pharmaceutically acceptable salt thereof.

[0383] Embodiment 98 Polypeptide, SEQ ID NO: 12: Px 1 -x 2 -KRx 3 -Kx 4 -Dx 5 Sequence ID 12 Includes; Here, x 1 However, it is G or A; x 2 However, it is G or A; x 3 However, A or V; and x 4 However, it is L or I; and x 5 A conjugate according to any one of embodiments 76 to 94, wherein the conjugate is G or A, or a pharmaceutically acceptable salt thereof.

[0384] Embodiment 99 Polypeptide, Sequence ID 13: Px 1 -x 2 -KRVKLD-(G) a Sequence ID 13 Includes; Here, x 1 and x 2 A conjugate according to any one of embodiments 76 to 94, or a pharmaceutically acceptable salt thereof, wherein each is independently G or A; and a is 0 or 1.

[0385] Appearance 100 x 1 and x 2 The conjugate described in Embodiment 99, or a pharmaceutically acceptable salt thereof, wherein each is A.

[0386] Appearance 101 x1 and x 2 The conjugate described in embodiment 99, or a pharmaceutically acceptable salt thereof, wherein each is G.

[0387] Embodiment 102 A conjugate according to any one of Embodiments 76 to 94, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises a sequence selected from SEQ ID NOs: 1 to 8.

[0388] Embodiment 103 A conjugate according to any one of Embodiments 76 to 94 or 102, wherein the polypeptide comprises SEQ ID NO: PGGKRVKLD, or a pharmaceutically acceptable salt thereof.

[0389] Embodiment 104 A conjugate according to any one of Embodiments 76 to 94 or 102, wherein the polypeptide comprises SEQ ID NO: PGGKRVKLDG, or a pharmaceutically acceptable salt thereof.

[0390] Embodiment 105 A conjugate according to any one of Embodiments 76 to 94 or 102, wherein the polypeptide comprises SEQ ID NO: 3:PAAKRVKLD, or a pharmaceutically acceptable salt thereof.

[0391] Embodiment 106 A conjugate according to any one of Embodiments 76 to 94 or 102, wherein the polypeptide comprises SEQ ID NO: 4:PAAKRVKLDG, or a pharmaceutically acceptable salt thereof.

[0392] Embodiment 107 A conjugate according to any one of Embodiments 76 to 94 or 102, wherein the polypeptide comprises SEQ ID NO: PAGKRVKLDG, or a pharmaceutically acceptable salt thereof.

[0393] Embodiment 108 A conjugate according to any one of Embodiments 76 to 94 or 102, wherein the polypeptide comprises SEQ ID NO: 6:PGAKRVKLDG, or a pharmaceutically acceptable salt thereof.

[0394] Embodiment 109 A conjugate according to any one of Embodiments 76 to 94 or 102, wherein the polypeptide comprises SEQ ID NO: PAAKRAKLDG, or a pharmaceutically acceptable salt thereof.

[0395] Embodiment 110 A conjugate according to any one of Embodiments 76 to 94 or 102, wherein the polypeptide comprises SEQ ID NO: 8:PAAKRVKIDG, or a pharmaceutically acceptable salt thereof.

[0396] Embodiment 111: A conjugate according to any one of Embodiments 76 to 110, or a pharmaceutically acceptable salt thereof, wherein L2 is a cleavable linker.

[0397] Embodiment 112: A conjugate according to any one of Embodiments 76 to 110, or a pharmaceutically acceptable salt thereof, wherein L2 contains a self-degrading group.

[0398] Embodiment 113: A conjugate according to any one of Embodiments 76 to 110, or a pharmaceutically acceptable salt thereof, wherein L2 is a non-cleavable linker.

[0399] Embodiment 114: A conjugate according to any one of Embodiments 76 to 110, wherein L2 contains 0 to 12 amino acids, or a pharmaceutically acceptable salt thereof.

[0400] Embodiment 115: A conjugate according to any one of Embodiments 76 to 114, or a pharmaceutically acceptable salt thereof, wherein L2 contains 0 to 12 polyethylene glycol monomers.

[0401] Embodiment 116: A conjugate according to any one of Embodiments 76 to 115, or a pharmaceutically acceptable salt thereof, wherein L2 contains 1 to 8 polyethylene glycol monomers.

[0402] Embodiment 117: A conjugate according to any one of Embodiments 76 to 116, or a pharmaceutically acceptable salt thereof, wherein L2 contains 2 to 6 polyethylene glycol monomers.

[0403] Embodiment 118 L2 is glycine or C 1-12 A conjugate containing an alkylene group, according to any one of embodiments 76 to 117, or a pharmaceutically acceptable salt thereof.

[0404] In aspect 119, L2 is given by the formula: -NH-C 1-6 Alkyl-[OCH2CH2] 0-8 -C 0-6 Alkyl-Z-(C 1-12 Alkyl-S) 0-1 - It is the linker; A conjugate according to any one of embodiments 76 to 110, or a pharmaceutically acceptable salt thereof, wherein Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, -OP(=O)(SH)-, or -OP(=O)(OH)-.

[0405] In aspect 120, L2 is given by the formula: -NH-(CH2) 2-6 -(OCH2CH2) 1-8 -(CH2) 0-3 -ZC 1-12 Alkyl-S-;-NH-(CH2) 1-12 -ZC 1-12 Alkyl-S-;-NH-(CH2) 2-6 -(OCH2CH2) 1-8 -(CH2) 0-3 -Z-; or -NH-(CH2) 1-12 -Z- It is the linker; A conjugate according to any one of embodiments 76 to 110 or 118 to 119, wherein Z is -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, or a bond.

[0406] Embodiment 121 L2 is given by formula: [ka] A conjugate according to any one of embodiments 76-110 or 118-120, or a pharmaceutically acceptable salt thereof.

[0407] Embodiment 122 L2 is given by the formula: [ka] A conjugate according to any one of embodiments 76-110 or 118-121, or a pharmaceutically acceptable salt thereof.

[0408] Embodiment 123 L2 is given by the formula: [ka] A conjugate according to any one of embodiments 76-110 or 118-122, or a pharmaceutically acceptable salt thereof.

[0409] Embodiment 124: A conjugate according to any one of Embodiments 76 to 110, or a pharmaceutically acceptable salt thereof, wherein n is 0 and L2 is absent.

[0410] Embodiment 125 The conjugate described in Embodiment 67, or a pharmaceutically acceptable salt thereof, wherein the activator is a detection group.

[0411] Embodiment 126 The conjugate according to Embodiment 125, or a pharmaceutically acceptable salt thereof, wherein the detection group is a dye.

[0412] Embodiment 127 The conjugate according to Embodiment 125, or a pharmaceutically acceptable salt thereof, wherein the detection group is a chromogenic dye or a fluorescent dye.

[0413] Embodiment 128 The conjugate according to Embodiment 125, or a pharmaceutically acceptable salt thereof, wherein the detection group is a coumarin dye, a rhodamine dye, or a cyanine dye.

[0414] Embodiment 129 A conjugate according to any one of Embodiments 76 to 124, or a pharmaceutically acceptable salt thereof, wherein TP is an antitumor chemotherapy agent.

[0415] Embodiment 130: A conjugate according to any one of Embodiments 76 to 124, or a pharmaceutically acceptable salt thereof, wherein TP comprises duocalmycin, auristatin, meitansinoid, unciaramycin, dynemycin, tylanstatin, camptothecin, exatecan, or tubulisin compound.

[0416] Embodiment 131 A conjugate according to any one of Embodiments 76 to 124, wherein TP comprises an antitumor antibiotic, a microtubule inhibitor, a cytotoxic or cytoactivating agent, a topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA alkylating agent, a DNA binding agent, a DNA cleavage agent, or an RNA polymerase inhibitor, or a pharmaceutically acceptable salt thereof.

[0417] Embodiment 132 A conjugate according to any one of Embodiments 76 to 124, or a pharmaceutically acceptable salt thereof, wherein TP is an antitumor antibiotic.

[0418] Embodiment 133 A conjugate according to any one of Embodiments 76 to 124, wherein TP is a microtubule inhibitor, or a pharmaceutically acceptable salt thereof.

[0419] Embodiment 134 A conjugate according to any one of Embodiments 76 to 124, or a pharmaceutically acceptable salt thereof, wherein TP is a cytotoxic agent or a cell proliferation inhibitor.

[0420] Embodiment 135 A conjugate according to any one of Embodiments 76 to 124, wherein TP is a topoisomerase I inhibitor, or a pharmaceutically acceptable salt thereof.

[0421] Embodiment 136 A conjugate according to any one of Embodiments 76 to 124, wherein TP is a camptothecin compound or an exatecan compound, or a pharmaceutically acceptable salt thereof.

[0422] Embodiment 137 A conjugate according to any one of Embodiments 76 to 124, or a pharmaceutically acceptable salt thereof, wherein TP is camptothecin, 10-hydroxycamptothecin, topotecan, irinotecan, berotecan, exatecan, diflomothecan, glimatecan, lulutotecan, silatecan, rubitecan, or SN-38.

[0423] Embodiment 138 A conjugate according to any one of Embodiments 76 to 124, wherein TP is ectenacydin, or a pharmaceutically acceptable salt thereof.

[0424] Embodiment 139 A conjugate according to any one of Embodiments 76 to 124, wherein TP is trabectedin, or a pharmaceutically acceptable salt thereof.

[0425] Embodiment 140: A conjugate according to any one of Embodiments 76 to 124, wherein TP is ecubectedin, or a pharmaceutically acceptable salt thereof.

[0426] Embodiment 141 A conjugate according to any one of Embodiments 76 to 124, wherein TP is lurubinectedin, or a pharmaceutically acceptable salt thereof.

[0427] Embodiment 142 A conjugate according to any one of Embodiments 76 to 124, wherein TP is calicacea myocardine, or a pharmaceutically acceptable salt thereof.

[0428] Embodiment 143 A conjugate according to any one of Embodiments 76 to 124, wherein TP is calichemycin γ1 (gamma-1), or a pharmaceutically acceptable salt thereof.

[0429] Embodiment 144 TP is a conjugate according to any one of Embodiments 76 to 124, or a pharmaceutically acceptable salt thereof, selected from any one of the following: [ka]

[0430] Appearance 145 [ka] A conjugate according to embodiment 67 or 76, or a pharmaceutically acceptable salt thereof, selected from the group consisting of JPEG2026511082000085.jpg207170JPEG2026511082000086.jpg225170JPEG2026511082000087.jpg53170: During the ceremony, AB is an antibody or antigen-binding fragment; and p is between 1 and 20.

[0431] Embodiment 146 A conjugate according to any one of Embodiments 76 to 145, wherein the antibody or its antigen-binding fragment is an IgG1 subclass, or a pharmaceutically acceptable salt thereof.

[0432] Embodiment 147 A conjugate according to any one of Embodiments 76 to 145, wherein an antibody or its antigen-binding fragment binds to a target antigen, or a pharmaceutically acceptable salt thereof.

[0433] Embodiment 148 A conjugate according to Embodiment 147, or a pharmaceutically acceptable salt thereof, wherein the target antigen is expressed on tumor cells.

[0434] Embodiment 149 A conjugate according to Embodiment 147 or 148, or a pharmaceutically acceptable salt thereof, wherein the target antigen is CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, or tissue factor.

[0435] Embodiment 150 An antibody or its antigen-binding fragment: Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 102; heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 103; heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 104; and / or light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 106; light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence described in SEQ ID NO: 107; and / or light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in SEQ ID NO: 108; or Heavy chain complementarity determination region 1 (HCDR1) containing the amino acid sequence described in SEQ ID NO: 112; heavy chain complementarity determination region 2 (HCDR2) containing the amino acid sequence described in SEQ ID NO: 113; heavy chain complementarity determination region 3 (HCDR3) containing the amino acid sequence described in SEQ ID NO: 114; and / or light chain complementarity determination region 1 (LCDR1) containing the amino acid sequence described in SEQ ID NO: 116; light chain complementarity determination region 2 (LCDR2) containing the amino acid sequence described in SEQ ID NO: 117; and / or light chain complementarity determination region 3 (LCDR3) containing the amino acid sequence described in SEQ ID NO: 118. A conjugate according to any one of embodiments 76 to 149, or a pharmaceutically acceptable salt thereof, including the above.

[0436] Embodiment 151 An antibody or its antigen-binding fragment: An immunoglobulin heavy chain variable region containing an amino acid sequence identical to at least 85%, 90%, 95%, 97%, 98%, or 99% of SEQ ID NO: 101; and / or an immunoglobulin light chain variable region containing an amino acid sequence identical to at least 85%, 90%, 95%, 97%, 98%, or 99% of SEQ ID NO: 105; or An immunoglobulin heavy chain variable region containing 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 containing an amino acid sequence at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 115. A conjugate according to any one of embodiments 76 to 150, or a pharmaceutically acceptable salt thereof, including the above.

[0437] Embodiment 152 An antibody or its antigen-binding fragment: An immunoglobulin heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 101; and / or an immunoglobulin light chain variable region containing the amino acid sequence described in SEQ ID NO: 105; or The immunoglobulin heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 111; and / or the immunoglobulin light chain variable region containing the amino acid sequence described in SEQ ID NO: 115. A conjugate according to any one of embodiments 76 to 151, or a pharmaceutically acceptable salt thereof, including the above.

[0438] Embodiment 153 An antibody or its antigen-binding fragment: An immunoglobulin heavy chain containing an amino acid sequence identical to at least 85%, 90%, 95%, 97%, 98%, or 99% of SEQ ID NO: 109; and / or an immunoglobulin light chain containing an amino acid sequence identical to at least 85%, 90%, 95%, 97%, 98%, or 99% of SEQ ID NO: 110; or Immunoglobulin heavy chains containing an amino acid sequence identical to at least 85%, 90%, 95%, 97%, 98%, or 99% of SEQ ID NO: 119; and / or immunoglobulin light chains containing an amino acid sequence identical to at least 85%, 90%, 95%, 97%, 98%, or 99% of SEQ ID NO: 120. A conjugate according to any one of embodiments 76 to 152, or a pharmaceutically acceptable salt thereof, including the above.

[0439] Apparatus 154 An antibody or its antigen-binding fragment: An antibody or its antigen-binding fragment: An immunoglobulin heavy chain containing the amino acid sequence described in SEQ ID NO: 109; and / or an immunoglobulin light chain containing the amino acid sequence described in SEQ ID NO: 110; or Immunoglobulin heavy chains containing the amino acid sequence described in SEQ ID NO: 119; and / or immunoglobulin light chains containing the amino acid sequence described in SEQ ID NO: 120. A conjugate according to any one of embodiments 76 to 153, or a pharmaceutically acceptable salt thereof, including the above.

[0440] Embodiment 155 A conjugate according to any one of Embodiments 76 to 154, or a pharmaceutically acceptable salt thereof, wherein the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, verantamab, roncastoximab, tisotumab, mirbetuximab, or a biosimilar thereof.

[0441] Aspect 156 A pharmaceutical composition comprising a conjugate according to any one of aspects 67 to 155, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0442] Embodiment 157 A method for delivering a therapeutic payload to an intracellular target within a tumor cell, wherein the therapeutic payload is conjugated to a polypeptide containing a nuclear localization sequence.

[0443] Embodiment 158 ​​Polypeptide, SEQ ID NO: 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Includes an array of, Here: x 1 , x 2 , x 3 , x 4 , and x 5 The method according to embodiment 157, wherein each is independently either a natural or non-natural amino acid; and a is 0 or 1.

[0444] Embodiment 159 The method according to Embodiment 157 or 158, wherein the polypeptide comprises a sequence selected from SEQ ID NOs: 1 to 8.

[0445] Embodiment 160 The method according to any one of Embodiments 157 to 159, wherein the polypeptide is further conjugated to an antibody or its antigen-binding fragment at a position not adjacent to (i.e., distal to) the therapeutic payload.

[0446] Embodiment 161 The method according to any one of Embodiments 157 to 160, wherein the therapeutic payload is cytotoxic.

[0447] Embodiment 162 The method according to any one of Embodiments 157 to 161, wherein the therapeutic payload is internalized within the nucleus of tumor cells.

[0448] Embodiment 163 The method according to any one of Embodiments 157 to 162, wherein the intracellular target is located in the nucleus of a tumor cell.

[0449] Embodiment 164 A method for 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 containing a nuclear localization sequence.

[0450] Embodiment 165 Polypeptide, SEQ ID NO: 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Includes an array of, Here: x 1 , x 2 , x 3 , x 4 , and x 5 The method according to embodiment 164, wherein each is independently either a natural or non-natural amino acid; and a is 0 or 1.

[0451] Embodiment 166 The method according to Embodiment 164 or 165, wherein the polypeptide comprises an sequence selected from SEQ ID NOs: 1 to 8.

[0452] Embodiment 167 A method for treating a disease comprising delivering a therapeutic payload and / or antibody to an intracellular target within a tumor cell, wherein the therapeutic payload and / or antibody is conjugated to a polypeptide, the polypeptide being Sequence ID No. 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 Includes an array of, Here: x 1 , x 2 , x 3 , x 4 , and x 5 A method wherein each of the following is independently either a natural or non-natural amino acid; and a is either 0 or 1.

[0453] Appearance 168 x 1 However, it is G or A; x 2 However, it is G or A; x 3 However, it is A or V; x 4 However, it is L or I; and x 5 The method according to embodiment 158, 165, or 167, wherein G or A.

[0454] Aspect 169: A method for treating a disease or disorder in a subject requiring therapeutic action, comprising administering a therapeutically effective amount of the composition described in any one of Aspects 1 to 66, or the conjugate described in any one of Aspects 67 to 155, to the subject.

[0455] Embodiment 170 The method according to any one of Embodiments 167 to 169, wherein the disease or disorder is cancer.

[0456] Embodiment 171 The method according to any one of Embodiments 167 to 169, wherein the disease or disorder is blood cancer.

[0457] Embodiment 172 The method according to any one of Embodiments 167 to 169, wherein the disease or disorder includes a solid tumor.

[0458] Embodiment 173 The method according to any one of Embodiments 167 to 170, wherein the disease or disorder is non-Hodgkin lymphoma, breast cancer, ovarian cancer, or gastric cancer.

[0459] Embodiment 174 The method according to any one of Embodiments 167 to 170, wherein the disease or disorder is large cell lymphoma, ductal carcinoma, mammary adenocarcinoma, ovarian adenocarcinoma, or gastric cancer.

[0460] Embodiment 175 The method according to any one of Embodiments 167 to 170, wherein the disease or disorder is a cancer associated with the overexpression of CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, or tissue factor.

[0461] Appearance 176 The disease or disorder 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 The method according to any one of embodiments 167 to 170, wherein the cancer is associated with the overexpression of GPC3, GPNMB, GUCY2C, HER2, HER3, IGF1R, L1CAM, LIV1, LRRC15, MET, MSLN, MUC1, MUC16, NCAM1, Nectin 4, PSMA, PTK7, ROR1, ROR2, SDC1, SLC1A5, SLC34A2, TFRC, TIM1, TPBG, or TROP2.

[0462] Embodiment 177 The method according to Embodiment 167, wherein the cancer is a blood cancer or a solid tumor.

[0463] Embodiment 178 Compounds having the structure of formula V, formula VI, or formula VII, or pharmaceutically acceptable salts thereof: AC-(L1) m -PP-(L2) n -TP formula V AC-(L1) m -PP Formula VI AC-(L1) m -PP-(L2) n -DG Formula VII During the ceremony: AC is an antibody-binding group configured to form a covalent bond with the sulfur or nitrogen atom of the amino acid side chain of an antibody or antigen-binding fragment; L1 is the linker; PP is sequence number 10: Px 1 -x 2 -KRx 3 -Kx 4 -D-(x 5 ) a Sequence ID 10 It is a polypeptide containing the sequence, Here, x 1 , x 2 , x 3 , x 4 , and x 5 Each of these is independently either a natural or non-natural amino acid; and a is either 0 or 1. L2 is the linker; TP is a therapeutic payload; DG is a detection group; m is 0 or 1; and n is either 0 or 1.

[0464] Embodiment 179: The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein PP contains any one of SEQ ID NOs: 1 to 8.

[0465] Embodiment 180: The compound according to Embodiment 178, wherein PP contains sequence number PGGKRVKLD, or a pharmaceutically acceptable salt thereof.

[0466] Embodiment 181 PP is the compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein PP contains the sequence number PGGKRVKLDG.

[0467] Embodiment 182 PP is the compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, comprising sequence number PAAKRVKLD.

[0468] Embodiment 183 PP is the compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein PP contains SEQ ID NO: PAAKRVKLDG.

[0469] Embodiment 184 PP is the compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein PP contains Sequence ID PAGKRVKLDG.

[0470] Embodiment 185 PP is a compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein PP contains sequence number PGAKRVKLDG.

[0471] Embodiment 186 PP is a compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, comprising the sequence number PAAKRAKLDG.

[0472] Embodiment 187 PP is a compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, comprising sequence number PAAKRVKIDG.

[0473] Embodiment 188: The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein L1 is a non-cleavable linker.

[0474] Embodiment 189: The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein L1 is a PEG linker containing 1 to 12 polyethylene glycol monomers.

[0475] A compound according to Aspect 178, or a pharmaceutically acceptable salt thereof, wherein L1 is a PEG linker having the following structure. [ka]

[0476] Embodiment 191 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein AC is a lysine-reactive group, a cysteine-reactive group, or a glutamine-reactive group.

[0477] Embodiment 192 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein AC of formula V or formula VI comprises the following groups. [ka]

[0478] Embodiment 193 AC-L1 is a compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, having one of the following structures. [ka]

[0479] Embodiment 194 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein L2 is a linker of the following formula: -NH-C 1-6 Alkyl-[OCH2CH2] 0-8 -C 0-6 Alkyl-Z-(C 1-12 Alkyl-S) 0-1 -; In the formula, Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, -OP(=O)(SH)-, or -OP(=O)(OH)-.

[0480] Embodiment 195 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein L2 is a linker of the following formula: -NH-(CH2) 2-6 -(OCH2CH2) 1-8 -(CH2) 0-3 -ZC 1-12 Alkyl-S-;-NH-(CH2) 1-12 -ZC 1-12 Alkyl-S-;-NH-(CH2) 2-6-(OCH2CH2) 1-8 -(CH2) 0-3 -Z-; or -NH-(CH2) 1-12 -Z-; In the formula, Z is -NH-, -O-, -S-, -CONH-, -CON(CH3)-, -NHCO-, -N(CH3)CO-, -CO-, or a bond.

[0481] Embodiment 196 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein L2 is a linker of the following formula. [ka]

[0482] Embodiment 197 The compound according to Embodiment 178, wherein the detection group is a dye, or a pharmaceutically acceptable salt thereof.

[0483] Embodiment 198 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the detection group is a colorimetric label, a fluorescent label, a bioluminescent label, or a chemiluminescent label.

[0484] Embodiment 199 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the detection group is a coumarin dye, a rhodamine dye, or a cyanine dye.

[0485] Appearance 200 The detection group is Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, HiLyte TM Fluor 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, HiLyte TM Fluor 555, Alexa Fluor® 561, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, HiLyte TMFluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, or Alexa Fluor® 750, or HiLyte TM Fluor 750, or a functional equivalent thereof, the compound described in Embodiment 178, or a pharmaceutically acceptable salt thereof.

[0486] Aspect 201: The compound according to Aspect 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload of Formula V is an antitumor antibiotic, a microtubule inhibitor, a cytotoxic or cell proliferation inhibitor, a topoisomerase inhibitor, a pyrrolobenzodiazepine, a DNA alkylating agent, a DNA binding agent, a DNA cleavage agent, or an RNA polymerase inhibitor.

[0487] Embodiment 202 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is an antitumor antibiotic.

[0488] Embodiment 203 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a microtubule inhibitor.

[0489] Embodiment 204 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a cytotoxic agent or a cell proliferation inhibitor.

[0490] Embodiment 205 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a topoisomerase I inhibitor.

[0491] Embodiment 206 The therapeutic payload is a camptothecin or exatecan compound, or a compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof.

[0492] Embodiment 207 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is camptothecin, 10-hydroxycamptothecin, topotecan, irinotecan, berotecan, exatecan, diflomothecan, glimethecan, lulutotecan, silatecan, rubitecan, or SN-38.

[0493] Embodiment 208 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is ectenacydin.

[0494] Embodiment 209 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is trabectedin.

[0495] Embodiment 210 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is expectedin.

[0496] Embodiment 211 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is lurubinectedine.

[0497] Embodiment 212 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is calicheamycin.

[0498] Embodiment 213 The compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is calicheamicin γ1 (gamma-1).

[0499] Embodiment 214 The therapeutic payload is a compound according to Embodiment 178, or a pharmaceutically acceptable salt thereof, selected from any one of the following: [ka]

[0500] Embodiment 215 A compound according to Embodiment 178 having one of the following structures, or a pharmaceutically acceptable salt thereof. [ka] JPEG2026511082000094.jpg213170JPEG2026511082000095.jpg51170

[0501] Applicable aspect 216: Use of a compound according to any one of Applicable aspects 178 to 215, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a pharmaceutical product.

[0502] Apparatus 217 A compound according to any one of Apparatus 178 to 215, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a pharmaceutical for treating cancer.

[0503] Apparatus 218 A compound according to any one of Apparatus 178 to 215, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a pharmaceutical for treating blood cancer.

[0504] Apparatus 219 A compound according to any one of Apparatus 178 to 215, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a pharmaceutical for treating solid tumors.

[0505] Apparatus 220 A compound according to any one of Apparatus 178 to 215, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for treating non-Hodgkin lymphoma, breast cancer, ovarian cancer, or gastric cancer.

[0506] Apparatus 221 A compound according to any one of Apparatus 178 to 215, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a pharmaceutical for treating large cell lymphoma, ductal carcinoma, mammary adenocarcinoma, ovarian adenocarcinoma, or gastric cancer.

[0507] Embodiment 222 A compound according to Embodiment 178 having the following structure, or a pharmaceutically acceptable salt thereof. [ka]

[0508] The foregoing merely illustrates the principles of the present disclosure. Those skilled in the art will understand that various arrangements embodying the principles of the present invention and falling within its spirit and scope can be devised, even if not explicitly described or illustrated herein. Furthermore, all examples and conditional statements incorporated herein are intended primarily to assist the reader in understanding the principles of the present disclosure and the concepts to which the inventors have contributed to further the art, and are not limited to such specifically incorporated examples and conditions. Moreover, all descriptions herein illustrating the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both their structural and functional equivalents. Furthermore, such equivalents are intended to include both currently known and future-developed equivalents, i.e., any element that performs the same function regardless of its structure. Therefore, the scope of the present disclosure is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present disclosure are embodied in the appended claims.

[0509] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous variations, modifications, and substitutions will arise for those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be employed when carrying out the present invention. The following claims define the scope of the present disclosure, and methods and structures within the scope of these claims and their equivalents are intended to be covered thereby.

Claims

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

2. formula: AB-[(L1) ｍ -PP-(L2) ｎ -DG] ｐ The conjugate according to claim 1, or a pharmaceutically acceptable salt thereof: During the ceremony: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide; L2 is the linker; DG is a detection group; m is either 0 or 1; n is 0 or 1; and p is between 1 and 20.

3. Formula I: AB - [(L1) ｍ - PP - (L2) ｎ - TP] ｐ Formula I The conjugate according to claim 1, or a pharmaceutically acceptable salt thereof: During the ceremony: AB is an antibody or antigen-binding fragment; L1 is the linker; PP is a polypeptide; L2 is the linker; TP is a therapeutic payload; m is either 0 or 1; n is 0 or 1; and p is between 1 and 20.

4. Polypeptide, in sequence: P-x １ -x ２ -K-R-x ３ -K-x ４ -D-(x ５ ) ａ Sequence No. 10 Includes, x １ , x ２ , x ３ , x ４ , and x ５ Each of these is independently either a natural or non-natural amino acid; and a is 0 or 1, the conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.

5. x １ However, it is G or A; x ２ However, it is G or A; x ３ However, it is A or V; x ４ However, it is L or I; x ５ However, it is G or A; and The conjugate according to claim 4, or a pharmaceutically acceptable salt thereof, wherein a is 0 or 1.

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

7. A conjugate according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO: PAAKRVKLD.

8. A conjugate according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises SEQ ID NO: PAAKRVKLDG.

9. L2 is the formula: -NH-C １－６ Alkyl-[OCH] ２ CH ２ ] ０－８ -C ０－６ Alkyl-Z-(C １－１２ Alkyl-S) ０－１ - It is the linker, In the formula, Z is a bond, -NH-, -O-, -S-, -CONH-, -CON(CH ３ )-, -NHCO-, -N(CH ３ A conjugate according to any one of claims 1 to 8, which is CO-, -CO-, -OP(=O)(SH)-, or -OP(=O)(OH)-, or a pharmaceutically acceptable salt thereof.

10. A conjugate according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein n is 0 and L2 is absent.

11. A conjugate according to any one of claims 1 to 2 and 4 to 10, wherein the detection group is a dye, or a pharmaceutically acceptable salt thereof.

12. A conjugate according to any one of claims 1 and 3 to 10, or a pharmaceutically acceptable salt thereof, wherein the therapeutic payload is a cytotoxic agent or a cell proliferation inhibitor. 【Request Item 13】 【Chemistry 1】 【change】 【change】 A conjugate according to any one of claims 1 and 3 to 6, having a structure selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof: Here, AB is an antibody or antigen-binding fragment; and p is between 1 and 20.

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

15. The conjugate according to claim 14, or a pharmaceutically acceptable salt thereof, wherein the target antigen is CD19, CD22, CD30, CD33, CD79b, HER2, TROP2, EGFR, nectin-4, mesoserine, BCMA, folate receptor α, or tissue factor.

16. A conjugate according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein the antibody is trastuzumab, brentuximab, gemtuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, sacituzumab, verantamab, roncastoximab, tisotumab, mirbetuximab, or a biosimilar thereof.

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

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

19. Polypeptide, SEQ ID NO: 10: P-x １ -x ２ -K-R-x ３ -K-x ４ -D-(x ５ ) ａ Sequence ID 10 Includes an array of Here: x １ , x ２ , x ３ , x ４ , and x ５ Each of these is independently either a natural or non-natural amino acid; and The method according to claim 18, wherein a is 0 or 1.

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

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

22. A method for treating a disease or disorder in a subject requiring treatment, comprising administering a therapeutically effective amount of the conjugate described in any one of claims 1 to 16 to the subject.

23. The method according to claim 22, wherein the disease or disorder is cancer.

24. The method according to claim 23, wherein the cancer is a blood cancer or a solid tumor.

25. Formula V, Formula VI, or Formula VII: AC-(L1) ｍ -PP-(L2) ｎ -TP type V AC-(L1) ｍ -PP type VI AC-(L1) ｍ -PP-(L2) ｎ -DG Formula VII Compounds having the structure of or pharmaceutically acceptable salts thereof: During the ceremony: AC is an antibody-binding group configured to form a covalent bond with the sulfur or nitrogen atom of the amino acid side chain of an antibody or antigen-binding fragment; L1 is the linker; PP is sequence number 10: P-x １ -x ２ -K-R-x ３ -K-x ４ -D-(x ５ ) ａ Sequence ID 10 It is a polypeptide containing the sequence, Here: L2 is the linker; TP is a therapeutic payload; DG is a detection group; m is 0 or 1; and n is either 0 or 1.

26. The compound according to claim 25, or a pharmaceutically acceptable salt thereof, wherein PP comprises a sequence selected from SEQ ID NOs: PAAKRVKLD, PAAKRVKLDG, PGGKRVKLD, PGGKRVKLDG, PAGKRVKLDG, PGAKRVKLDG, PAAKRAKLDG, and PAAKRVKIDG.

27. The compound according to claim 25, or a pharmaceutically acceptable salt thereof, wherein the detection group is a dye.

28. The detection groups are Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, and HiLyte. ＴＭ Flour 488, Alexa Flour® 532, Alexa Flour® 546, Alexa Flour® 555, HiLyte ＴＭ Flour 555, Alexa Flour® 561, Alexa Flour® 568, Alexa Flour® 594, Alexa Flour® 647, HiLyte ＴＭ Fluor 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, or Alexa Fluor® 750, HiLyte ＴＭ Fluor 750, or a functional equivalent thereof, the compound according to claim 25, or a pharmaceutically acceptable salt thereof.

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

30. A compound according to claim 25, having one of the following structures, or a pharmaceutically acceptable salt thereof. 【Chemistry 2】 【change】 【change】

31. Use of a compound represented by formula V, according to any one of claims 25, 26, 29, or 30, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a pharmaceutical.

32. A compound represented by formula V, according to any one of claims 25, 26, 29, or 30, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a pharmaceutical for treating cancer.

33. array: P-x １ -x ２ -K-R-x ３ -K-x ４ -D-(x ５ ) ａ Sequence ID 10 A composition comprising a polypeptide containing, or a pharmaceutically acceptable salt thereof: Here: x １ , x ２ , x ３ , x ４ , and x ５ Each of these is independently either a natural or non-natural amino acid; and a is either 0 or 1.

34. Polypeptide, in sequence: P-x １ -x ２ -K-R-x ３ -K-x ４ -D Sequence ID 11 Includes, Here: x １ However, it is G or A; x ２ However, it is G or A; x ３ However, A or V; and x ４ The composition according to claim 33, or a pharmaceutically acceptable salt thereof, wherein L or I.

35. Polypeptide, in sequence: P-x １ -x ２ -K-R-x ３ -K-x ４ -D-x ５ Sequence ID 12 Includes, Here: x １ However, it is G or A; x ２ However, it is G or A; x ３ However, A or V; and x ４ However, it is L or I; and x ５ The composition according to claim 33, or a pharmaceutically acceptable salt thereof, wherein the composition is G or A.

36. Polypeptide, in sequence: P-x １ -x ２ -K-R-V-K-LD-(G) ａ Sequence ID 13 Includes, Here, x １ and x ２ are each independently A or G; and a is 0 or 1, the composition according to claim 33, or a pharmaceutically acceptable salt thereof.

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

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