Breast cancer treatment

Abstract

Provided include methods, compositions and kits for treating breast cancer in a subject. The method can comprise administrating to a subject a PLK1 inhibitor in combination with a chemotherapeutic agent or DNA damaging agent either in a standalone form or conjugated to an antibody targeting breast cancer cells to form an antibody drug conjugate in a manner sufficient to inhibit or reduce progression of the breast cancer. The breast cancer can be a HR+ breast cancer, or a breast cancer having negative, low or ultralow HER2 status. In some embodiments, the breast cancer is triple negative breast cancer with a negative, low or ultralow HER2 status.

Description

53NC-830004-WO PATENTBREAST CANCER TREATMENTRELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 63 / 724,371, filed November 24, 2024, and U.S. Provisional Patent Application Ser. No. 63 / 738,466, filed December 23, 2024, the contents of which are incorporated herein by reference in their entireties for all purposes.BACKGROUNDField

[0002] The present application generally relates to treatment for cancer, more specifically, combination therapies for treating breast cancer.Description of the Related Art

[0003] Breast cancer is the most common form of cancer and the second leading cause of cancer death in women worldwide. Hormone receptor-positive breast cancer is a type of breast cancer that grows in response to hormones (e.g., estrogen or progesterone). Approximately 67%- 80% of breast cancers in women are estrogen receptor positive, while approximately 90% of breast cancers in men are estrogen receptor positive. Standard treatments of hormone receptor-positive breast cancer currently include chemotherapy, hormone therapy, surgery, radiation therapy, and targeted therapy.

[0004] There is an urgent need to develop effective treatment for breast cancer patients, particularly breast cancer patients that have or developed resistance to first-line therapies.SUMMARY

[0005] Disclosed herein includes a method of treating breast cancer, including for example hormone receptor positive (HR+) breast cancer, HER2-negative, low, or ultralow (HER2- / low / ultralow) breast cancer, or triple negative breast cancer (TNBC). The method, in some embodiments, is a method of treating HR+ breast cancer, comprising: administering a Pololike kinase 1 (PLK1) inhibitor and deruxtecan (DXd) to a subject with the HR+ breast cancer, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject. In some embodiments, a method of treating hormone receptor positive (HR+) breast cancer comprises administering PLK1 inhibitor to a subject with HR+ breast cancer and being treated with DXd, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject. In some embodiments, a method of treating HR+ breast cancer comprises: administering an antibody drug conjugate (ADC) targeting breast cancer cells and a PLK1 inhibitor to a subject with the HR+ breast cancer, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject.In some embodiments, the method is a method of treating HR+ breast cancer, comprising: administering a PLK1 inhibitor to a subject with HR+ breast cancer and being treated with an ADC capable of targeting breast cancer cells, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject. The HR+ breast cancer can be, for example, estrogen receptor positive (ER+). In some embodiments, the HR+ breast cancer is progesterone receptor positive (PR+). In some embodiments, the HR+ breast cancer is progesterone receptor negative (PR-). In some embodiments, the HR+ breast cancer has a histological or cytological profile with ER > 1%, 10%, 20%, or higher.

[0006] The method, in some embodiments, is a method of treating triple negative breast cancer (TNBC), comprising: administering a PLK1 inhibitor and deruxtecan (DXd) to a subject with TNBC, thereby inhibiting or reducing progression of the TNBC in the subject. In some embodiments, a method of treating TNBC comprises administering a PLK1 inhibitor to a subject with the TNBC and being treated with DXd, thereby inhibiting or reducing progression of the TNBC in the subject. In some embodiments, a method of treating TNBC comprises administering an ADC capable of targeting breast cancer cells and a PLK1 inhibitor to a subject with TNBC, thereby inhibiting or reducing progression of the TNBC in the subject. In some embodiments, the method is a method of treating TNBC, comprising: administering the PLK1 inhibitor to a subj ect with TNBC and being treated with an ADC capable of targeting breast cancer cells, thereby inhibiting or reducing progression of the TNBC in the subject.

[0007] The method, in some embodiments, is a method of treating HER2-negative, low, or ultralow (HER2- / low / ultralow) breast cancer, comprising: administering a PLK1 inhibitor and DXd to a subject with the HER2- / low / ultralow breast cancer, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject. In some embodiments, a method of treating HER2- / low / ultralow breast cancer comprises administering a PLK1 inhibitor to a subject with the HER2- / low / ultralow breast cancer and being treated with DXd, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject. In some embodiments, a method of treating HER2- / low / ultralow breast cancer comprises administering an ADC capable of targeting breast cancer cells and a PLK1 inhibitor to a subject with the HER2- / low / ultralow breast cancer, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject. In some embodiments, the method is a method of treating HER2- / low / ultralow breast cancer, comprising: administering the PLK1 inhibitor to a subject with HER2- / low / ultralow breast cancer and being treated with an ADC capable of targeting breast cancer cells, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject.

[0008] In some embodiments, the HR+ breast cancer or TNBC is HER2 negative, lowor ultralow. In some embodiments, the HR+ breast cancer or TNBC patient has an HER2 immunohistochemistry (IHC) score of 0 or 1+. In some embodiments, the HR+ breast cancer or the TNBC patient has an HER2 IHC score of 2+ and a negative in situ hybridization (ISH) score. In some embodiments, the HR+ breast cancer or the TNBC is resistant to or does not respond effectively to first line treatments for HR+ breast cancer or the TNBC. In some embodiments, the HR+ breast cancer is HER2 negative, low or ultralow. In some embodiments, the subject with the HR+ breast cancer is resistant to or does not respond effectively to a hormone therapy.

[0009] In some embodiments, the hormone therapy comprises a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), an aromatase inhibitor, or a combination thereof. In some embodiments, the subject (e.g., the subject with the HR+ breast cancer) is resistant to a kinase inhibitor, optionally the kinase inhibitor is a CDK inhibitor, further optionally the CDK inhibitor is a CDK 4 / 6 inhibitor. In some embodiments, the subject with the HR+ breast cancer develops stable disease, progressive disease, or resistance to a CDK4 / 6 inhibitor and / or a SERD. In some embodiments, the subject is resistant to or develops stable or progressive disease following treatment with palbociclib, abemaciclib, fulvestrant, or a combination thereof. In some embodiments, the subject with the HR+ breast cancer is resistant to or does not respond effectively to alpelisib alone or in combination with a SERD, optionally the SERD is fulvestrant. In some embodiments, the subject with the TNBC is resistant to or does not respond effectively to chemotherapy. In some embodiments, the resistance is acquired resistance or intrinsic resistance. In some embodiments, the subject did not respond to the anti-HER2 ADC treatment. In some embodiments, the subject develops stable or progressive disease following the treatment with anti-HER2 ADC. In some embodiments, the subject is known to be resistant to the anti-HER2 ADC alone.

[0010] The ADC capable of targeting breast cancer cells and the PLK1 inhibitor can be co-administered simultaneously, separately, or sequentially. In some embodiments, the ADC, the PLK1 inhibitor, or both are administered to the subject in a cycle of 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, or 49 days. In some embodiments, the ADC is administered to the subject about once every two weeks or every three weeks, and the PLK1 inhibitor is administered to the subject about 5-7 days a week.

[0011] In some embodiments, each cycle of treatment is at least about 14 days. In some embodiments, each cycle of treatment is from about 14 days to about 28 days. In some embodiments, the PLK1 inhibitor is administered on at least five days, at least ten days, or at least fifteen days in a cycle. In some embodiments, the PLK1 inhibitor is not administered on at least one day, at least three days, or at least seven days in a cycle. In some embodiments, the ADC is administered once weekly, once every two weeks, once every three weeks, or once every fourweeks. In some embodiments, the ADC is administered once every three weeks for once, twice, three, or four times in a cycle. In some embodiments, the ADC is administered to the subject about once every three weeks and the PLK1 inhibitor is administered to the subject about 5 days a week. In some embodiments, the subject undergoes at least two cycles of the administration of the ADC and the PLK1 inhibitor.

[0012] The PLK1 inhibitor can be, e.g., onvansertib (NMS-P937), BI2536, volasertib (BI 6727), GSK461364, adavosertib (AZDI 775), CYC 140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, GTPL10072, Ro3280; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and any combinations thereof. In some embodiments, PLK1 inhibitor is onvansertib. In some embodiments, the ADC comprises an anti- HER2 antibody or an anti-TROP2 antibody and a chemotherapy drug. In some embodiments, the anti-HER2 ADC comprises an anti-HER2 antibody or an anti-TROP2 antibody and a DNA damaging agent. Non-limiting examples of the DNA damaging agent include radiomimetic neocarzinostatin, a platinating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an antimetabolite, an alkylating agent, an antibiotic, and a combination thereof. In some embodiments, the DNA damaging agent is a topoisomerase inhibitor. In some embodiments, the DNA damaging agent is selected from a group consisting of Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone, Teniposide, and Topotecan, Camptothecin, Rubitecan, Belotecan, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin, and a combination thereof. In some embodiments, the ADC capable of targeting breast cancer cells comprises deruxtecan (DXd) conjugated to an anti-HER2 antibody or an anti-TROP2 antibody. In some embodiments, the ADC described herein is trastuzumab deruxtecan (T-DXd). In some embodiments, the ADC described herein is datopotamab deruxtecan (Dato-DXd).

[0013] In some embodiments, the subject has received at least one prior cancer treatment. In some embodiments, the prior treatment does not comprise the use of an ADC, a PLK1 inhibitor, or both; and optionally the PLK1 inhibitor is onvansertib.

[0014] In some embodiments, the PLK1 inhibitor is administered orally; and optionally the PLK1 inhibitor is administered every day or five times a week. In some embodiments, the ADC is administered intravenously; and optionally wherein the ADC is administered every two weeks, three weeks, or four weeks. In some embodiments, the PLK1 inhibitor is administered at about 5 mg to about 80 mg; optionally at about 10 mg to about 30 mg; and further optionally at 10 mg, 15 mg, 20 mg, 25 mg, or 30 mg.

[0015] In some embodiments, the ADC capable of targeting breast cancer cells is administered at about 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kgor 9 mg / kg; optionally the ADC is administered at 5.4 mg / kg.

[0016] In some embodiments, the subject was in remission for cancer. In some embodiments, the subject in remission for cancer was in complete remission (CR) or in partial remission (PR). In some embodiments, the method further comprises determining cancer status of the subject. In some embodiments, the method further comprises determining responsiveness of the subject to the treatment of the ADC and / or the PLK1 inhibitor. In some embodiments, the method further comprises administering one or more additional cancer therapeutics or therapies for the cancer.

[0017] The subject can be human. In some embodiments, the subject achieves a complete response.

[0018] Also disclosed herein includes a kit, comprising: a PLK1 inhibitor; and a manual providing instructions for co-administering the PLK1 inhibitor in combination with an ADC capable of targeting breast cancer cells to a subject in need thereof for treating HR+ breast cancer, TNBC, or HER2- / low / ultralow breast cancer. In some embodiments, a kit comprises a PLK1 inhibitor and a manual providing instructions for co-administering the PLK1 inhibitor in combination with DXd to a subject in need thereof for treating HR+ breast cancer, TNBC, or HER2- / low / ultralow breast cancer. The PLK1 inhibitor can be, examples, onvansertib (NMS- P937), BI2536, volasertib (BI 6727), GSK461364, adavosertib (AZD1775), CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, GTPL10072, Ro3280; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and or a combination thereof. In some embodiments, the PLK1 inhibitor is onvansertib. In some embodiments, the instructions comprise instructions for co-administrating the ADC and the PLK1 inhibitor simultaneously, separately or sequentially.

[0019] In some embodiments, the instructions comprise instructions for administering to a subj ect that did not respond to treatment with the ADC treatment alone. In some embodiments, the instructions comprise instructions for administering to a subject resistant to a CDK 4 / 6 inhibitor, hormone therapy, alpelisib, or a combination thereof. The CDK 4 / 6 inhibitor can be, e.g., palbociclib or abemaciclib. In some embodiments, the hormone therapy comprise a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), an aromatase inhibitor, or a combination thereof, optionally the SERD is fulvestrant.

[0020] The kit can further comprise the ADC capable of targeting breast cancer cells. The ADC can comprise, e.g., an anti-HER2 antibody or an anti-TROP2 antibody and a DNA damaging agent selected from the group consisting of radiomimetic neocarzinostatin, a platinating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an antimetabolite, an alkylating agent, an antibiotic, or a combination thereof. The DNA damaging agent can be, e.g, atopoisomerase inhibitor, optionally, the DNA damaging agent is selected from a group consisting of Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone, Teniposide, and Topotecan, Camptothecin, Rubitecan, Belotecan, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin, or a combination thereof. In some embodiments, the ADC capable of targeting breast cancer cells comprises deruxtecan conjugated to an anti-HER2 antibody or an anti-TROP2 antibody. In some embodiments, the anti-HER2 ADC is T- trastuzumab deruxtecan (T-DXd). In some embodiments, the anti-TROP2 ADC is datopotamab deruxtecan (Dato-DXd).BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a table showing the characteristics of exemplary HER2-low breast cancer PDX models.

[0022] FIG. 2 depicts data showing an immunohistochemistry (IHC) image of HER2 staining (panel A), tumor volume overtime (panel B), tumor volume change at Day 29 (panel C), event-free survival rate (panel D) and tumor growth inhibition at Day 22 (panel E) in HBCx-246 PDX model treated with vehicle (Ctrl), onvansertib, trastuzumab deruxtecan (T-DXd), or both (O+T).

[0023] FIG. 3 depicts data showing an IHC image of HER2 staining (panel A), tumor volume overtime (panel B), tumor volume change at Day 39 (panel C), event-free survival rate (panel D), and tumor growth inhibition at Day 39 (panel E) in HBCx-139palbo+fulvR5 PDX model treated with vehicle (Ctrl), onvansertib, T-DXd, or both (O+T).

[0024] FIG. 4 depicts data showing an IHC image of HER2 staining (panel A), tumor volume overtime (panel B), tumor volume change at Day 42 (panel C), and tumor regression and complete response rates (panel D) in HBCx-3 PDX model treated with vehicle (Ctrl), onvansertib, T-DXd, or both (O+T).

[0025] FIG. 5 depicts data showing an IHC image of HER2 staining (panel A), tumor volume overtime (panel B), tumor volume change at Day 42 (panel C), and tumor regression and complete response rates (panel D) in HBCx-134palboR31 PDX model treated with vehicle (Ctrl), onvansertib, T-DXd, or both (O+T).

[0026] FIG. 6 are plots showing the percentage change in body weight (BW) of mice treated with vehicle (Ctrl), onvansertib (Onv), T-DXd, or both (O+T) as indicated in FIGs. 2-5.

[0027] FIG. 7 shows HER2 expression levels in selected breast cancer cell lines.

[0028] FIG. 8 shows dose matrix (9x9) evaluation of T-DXd and onvansertib combination in HER2-low TNBC cell lines. Top panel represents the Bliss synergy heatmaps and bottom panel represent the percent inhibition dose response matrices.

[0029] FIGs. 9A-9B show dose matrix (9x9) evaluation of T-DXd and onvansertibcombination in HER2-low HR+ breast cancer cell lines. Top panels represent the Bliss synergy heatmaps and bottom panels represent the percent inhibition dose response matrices.

[0030] FIGs. 10A-10B show dose matrix (9x9) evaluation of DXd payload and onvansertib combination in HER2-low TNBC and HR+ breast cancer cell lines. Top panels represent the Bliss synergy heatmaps and bottom panels represent the percent inhibition dose response matrices.

[0031] FIG. 11 are plots showing the percentage of TUNEL-positive cells in selected TNBC and HR+ HER2-low BC breast cancer cell lines DMSO (Ctrl), onvansertib, T-DXd, or both (O+T).

[0032] FIG. 12 is a flowchart showing the patient treatment history of the BC1732 TNBC PDX model.

[0033] FIG. 13 depicts data showing the tumor volume overtime (panel A), tumor volume change at Day 46 (panel B), and tumor regression and complete response rates (panel C) in BC7132 PDX model treated with vehicle (Ctrl), onvansertib, T-DXd, or both (O+T).

[0034] FIG. 14 depicts data showing tumor volume overtime (panel A), percentage of tumor volume change at Day 50 (panel B), and tumor regression and complete response rates (panel C) in vBC1220OvaRPalboR PDX model treated vehicle (Ctrl), onvansertib, T-DXd, or both (O+T).

[0035] FIG. 15 depicts data showing percent of yH2AX+ cells in HER2-low breast cancer cell lines treatment with DMSO (Ctrl), onvansertib, T-DXd, or both (O+T).

[0036] FIG. 16 are immunofluorescence images of MCF-7 cells treated with DMSO (Ctrl), onvansertib, T-DXd, or both (O+T) and stained with the DNA damage markers yH2AX (red) and 53BP1 (green), as well as the DNA marker DAPI (blue).DETAILED DESCRIPTION

[0037] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.

[0038] All patents, published patent applications, other publications, and sequencesfrom GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.Definitions

[0039] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0040] As used herein, a “patient” refers to a subject that is being treated by a medical professional, such as a Medical Doctor (i.e., Doctor of Allopathic medicine or Doctor of Osteopathic medicine) or a Doctor of Veterinary Medicine, to attempt to cure, or at least ameliorate the effects of, a particular disease or disorder or to prevent the disease or disorder from occurring in the first place. In some embodiments, the patient is a human or an animal. In some embodiments, the patient is a mammal. As used herein, the terms “individual,” “host,” “subject,” and “patient” are used interchangeably.

[0041] As used herein, “administration” or “administering” refers to a method of giving a dosage of a pharmaceutically active ingredient to a vertebrate.

[0042] As used herein, a “dosage” refers to the combined amount of the active ingredients (e.g., onvansertib).

[0043] As used herein, a “unit dosage” refers to an amount of therapeutic agent administered to a patient in a single dose.

[0044] As used herein, the term “daily dose” or “daily dosage” refers to a total amount of a pharmaceutical composition or a therapeutic agent that is to be taken within 24 hours.

[0045] As used herein, the term “delivery” refers to approaches, formulations, technologies, and systems for transporting a pharmaceutical composition or a therapeutic agent into the body of a patient as needed to safely achieve its desired therapeutic effect. In some embodiments, an effective amount of the composition or agent is formulated for delivery into the blood stream of a patient.

[0046] As used herein, the term “formulated” or “formulation” refers to the process in which different chemical substances, including one or more pharmaceutically active ingredients, are combined to produce a dosage form. In some embodiments, two or more pharmaceutically active ingredients can be co-formulated into a single dosage form or combined dosage unit, or formulated separately and subsequently combined into a combined dosage unit. A sustained release formulation is a formulation which is designed to slowly release a therapeutic agent in thebody over an extended period of time, whereas an immediate release formulation is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time.

[0047] As used herein, the term “pharmaceutically acceptable” indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile.

[0048] As used herein, the term “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body, or to deliver an agent to a diseased tissue or a tissue adjacent to the diseased tissue. Carriers or excipients can be used to produce compositions. The carriers or excipients can be chosen to facilitate administration of a drug or pro-drug. Examples of carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution, and dextrose.

[0049] As used herein, the term “pharmaceutically acceptable salt” refers to any acid or base addition salt whose counter-ions are non-toxic to the patient in pharmaceutical doses of the salts. A host of pharmaceutically acceptable salts are well known in the pharmaceutical field. If pharmaceutically acceptable salts of the compounds of this disclosure are utilized in these compositions, those salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, hydrohalides (e.g., hydrochlorides and hydrobromides), sulphates, phosphates, nitrates, sulphamates, malonates, salicylates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p- toluoyltartrates, ethanesulphonates, cyclohexylsulphamates, quinates, and the like. Pharmaceutically acceptable base addition salts include, without limitation, those derived fromalkali or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine, ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.

[0050] The pharmaceutically acceptable salts of the compounds can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, Md., 2000, p. 704; and “Handbook of Pharmaceutical Salts: Properties, Selection, and Use,” P. Heinrich Stahl and Camille G. Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

[0051] As used herein, the term “hydrate” refers to a complex formed by combination of water molecules with molecules or ions of the solute. As used herein, the term “solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Solvate is meant to include hydrate, hemi-hydrate, channel hydrate and the likes. Some examples of solvents include, but are not limited to, methanol, N, A-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.

[0052] As used herein, “therapeutically effective amount” or “pharmaceutically effective amount” refers to an amount of therapeutic agent, which has a therapeutic effect. The dosages of a pharmaceutically active ingredient which are useful in treatment when administered alone or in combination with one or more additional therapeutic agents are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount refers to an amount of therapeutic agent which produces the desired therapeutic effect as judged by clinical trial results and / or model animal studies. The therapeutically effective amount will vary depending on the compound, the disease, disorder or condition and its severity and the age, weight, etc., of the mammal to be treated. The dosage can be conveniently administered, e.g., in divided doses up to four times a day or in sustained-release form.

[0053] As used herein, the term “dosage regime” refers to drug administration regarding formulation, route of administration, drug dose, dosing interval and treatment duration.

[0054] As used herein, the term “treat,” “treatment,” or “treating,” refers to administering a therapeutic agent or pharmaceutical composition to a subject for prophylacticand / or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already suffering from a disease or condition. As used herein, a “therapeutic effect” relieves, to some extent, one or more of the symptoms of a disease or disorder. For example, a therapeutic effect may be observed by a reduction of the subjective discomfort that is communicated by a subject (e.g., reduced discomfort noted in self-administered patient questionnaire).

[0055] As used herein, each of the terms “partial response”, “partial remission” and “PR” refers to the amelioration of a cancerous state, as measured by, for example, tumor size and / or cancer marker levels, in response to a treatment. In some embodiments, a “partial response” means that a tumor or tumor-indicating blood marker has decreased in size or level by about 50% in response to a treatment. The treatment can be any treatment directed against cancer, including but not limited to, chemotherapy, radiation therapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The size of a tumor can be detected by clinical or by radiological means. Tumor-indicating markers can be detected by means well known to those of skill, e.g., ELISA or other antibody-based tests. A partial response of the target lesion can refer to at least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.

[0056] As used herein, each of the terms “complete response” or “complete remission” or “CR” means that a cancerous state, as measured by, for example, tumor size and / or cancer marker levels, has disappeared following a treatment, including but are not limited to, chemotherapy, radiation therapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The presence of a tumor can be detected by clinical or by radiological means. Tumor-indicating markers can be detected by means well known to those of skill, e.g., ELISA or other antibody-based tests. A “complete response” does not necessarily indicate that the cancer has been cured, however, a complete response may be followed by a relapse. A complete response of a target lesion includes disappearance of all target lesions and any pathological lymph nodes (whether target or non-target) having reduction in short axis to <10 mm. A complete response of a non-target lesion includes disappearance of all non-target lesions and normalization of tumor marker level (all lymph nodes must be non-pathological in size (<10 mm short axis)). If tumor markers are initially above the upper normal limit, they need to normalize for a patient to be considered in complete clinical response of a nontarget lesion. The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date thatprogressive disease is objectively documented, or death due to any cause. Participants without events reported are censored at the last disease evaluation.

[0057] As used herein, the term “stable disease” or “SD” means neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study. Duration of stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.

[0058] As used herein, the term “progressive disease” or “PD” when refers to a target lesion means at least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). When progressive disease or PD refers to a non-target lesion, it means the appearance of one or more new lesions and / or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase.

[0059] As used herein, the term “combination therapy” refers to treatment of a disease or symptom thereof, or a method for achieving a desired physiological change, including administering to an animal, such as a mammal, especially a human being, an effective amount of two or more chemical agents or components to treat the disease or symptom thereof, or to produce the physiological change, wherein the chemical agents or components are administered together, such as part of the same composition, or administered separately and independently at the same time or at different times (e.g., administration of each agent or component is separated by a finite period of time from each other). In the embodiments described herein, the combination therapy can refer to the combination of an anti-HER2 ADC and a PLK1 inhibitor. In some embodiments, the combination therapy can refer to the combination of trastuzumab deruxtecan and onvansertib.

[0060] As used herein, the term “characterizing” or “characterized” refers to assessing a patient, tissue sample, nucleic acid sample or cell for the presence or absence of a mutation.

[0061] As used herein, the term “breast cancer” refers to a condition characterized by anamalous rapid proliferation of abnormal cells in one or both breasts of a subject. The abnormal cells often are referred to as “neoplastic cells,” which refers to, in some embodiments, transformed cells that can form a solid tumor. The term “tumor”, in some embodiments, refers to an abnormal mass or population of cells (i.e. two or more cells) that result from excessive or abnormal cell division, whether malignant or benign, and pre-cancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cellularproliferation, they can invade surrounding tissues and can metastasize.

[0062] As used herein, the term “advanced breast cancer” refers to cancer that has spread to other places in the body and usually cannot be cured or controlled with current treatment.

[0063] As used herein, the term “metastasis” refers to a process in which cancer cells travel from one organ or tissue to another non-adjacent organ or tissue. Cancer cells in the breast(s) can spread to tissues and organs of a subject, and conversely, cancer cells from other organs or tissue can invade or metastasize to a breast. Cancerous cells from the breast(s) may invade or metastasize to any other organ or tissue of the body. Breast cancer cells often invade lymph node cells and / or metastasize to the liver, brain and / or bone and spread cancer in these tissues and organs. The term “invasion”, in some embodiments, refers to the spread of cancerous cells to adjacent surrounding tissues.

[0064] Disclosed herein includes methods for treating breast cancer, e.g., hormone receptor positive (HR+) breast cancer, a triple negative breast cancer (TNBC), or a breast cancer having a negative, low or ultralow HER2 status. In some embodiments, a method of treating breast cancer comprises administering an antibody drug conjugate capable of targeting breast cancer cells (e.g., anti-HER2 ADC) and a Polo-like kinase 1 (PLK1) inhibitor to a subject in need, thereby inhibiting or reducing progression of the breast cancer in the subject. In some embodiments, a method of treating breast cancer comprises administering a PLK 1 inhibitor to a subj ect with breast cancer and being treated with an ADC capable of targeting breast cancer cells, thereby inhibiting or reducing progression of the breast cancer in the subject. In some embodiments, the ADC capable of targeting breast cancer cells is an anti-HER2 ADC or an anti-TROP2 ADC. In some embodiments, a method of treating breast cancer comprises administering a PLK1 inhibitor and deruxtecan (DXd) to a subject in need, thereby inhibiting or reducing progression of the breast cancer in the subject. In some embodiments, a method of breast cancer comprises administering a PLK1 inhibitor to a subject with breast cancer and being treated with DXd, thereby inhibiting or reducing progression of the breast cancer in the subject. In some embodiments, the breast cancer is HER2 negative, low or ultralow HR+ breast cancer. In some embodiments, the breast cancer is a HER2 negative, low or ultralow TNBC.

[0065] Disclosed herein also includes compositions and kits for treating breast cancer (e.g., HR+ and / or HER2 negative, low or ultralow breast cancer, or TNBC). In some embodiments, a kit comprises a PLK1 inhibitor and a manual providing instructions for coadministering the PLK1 inhibitor in combination with an ADC capable of targeting breast cancer cells to a subject in need thereof for treating breast cancer. In some embodiments, the kit further comprises the ADC capable of targeting breast cancer cells. In some embodiments, the ADC capable of targeting breast cancer cells is an anti-HER2 ADC or an anti-TROP2 ADC. In someembodiments, a kit comprises a PLK1 inhibitor and a manual providing instructions for coadministering the PLK1 inhibitor in combination with deruxtecan to a subject in need thereof for treating breast cancer.Breast Cancer

[0066] Methods, compositions and kits disclosed herein can be used for treating cancer and / or tumor such as breast cancer.

[0067] In some embodiments, the cancer and / or tumor described herein can be a breast cancer and / or breast tumor. In some embodiments, the breast cancer cells only express a small amount or none of human epidermal growth factor receptor 2 (HER2) protein, such breast cancer cells are referred to as HER2 negative (HER2-) breast cancer, HER2 low breast cancer, or HER2 ultralow breast cancer, collectively referred to as HER2- / low / ultralow breast cancer. An immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and chromogenic in situ hybridization (CISH) are standard methods for the determination of HER2 status. IHC measures the level of HER2 receptor overexpression, while FISH quantifies the level of HER2 gene amplification.

[0068] In some embodiments, if the HER2 IHC result is 0 or 1+, the cancer is considered HER2 negative. In some embodiments, a HER2 IHC result showing no staining observed or incomplete membrane staining that is faint or barely perceptible and within < 10% of the invasive tumor cells indicates HER2 negative status. In some embodiments, a HER2 IHC result showing weak incomplete membrane staining in any proportion of tumor cells and / or weak, complete membrane staining in < 10% of tumor cells indicates HER2 negative status. In some embodiments, a HER2 IHC result showing incomplete membrane staining that is faint or barely perceptible and within > 10% of the invasive tumor cells indicates HER2 negative status. In some embodiments, if the IHC result is 1+ or 2+ along with a negative in situ hybridization (ISH) test, the cancer is considered as HER2 low or ultralow cancer. In some embodiments, a dual-probe assay result showing HER2 / CEP17 ratio of < 1.8 or HER2 / CEP17 ratio < 2.0 with average HER2 copy number <4.0 indicates HER2 negative status. In some embodiments, HER2 negative cancers include HER2 negative, low and ultralow cancers. In some embodiments, HER2 negative caners exclude HER2 low and ultralow cancers. Additional information about HER2 status determination in breast cancer can be found in published literatures and journal articles, such as J Pathol Transl Med. 2019 Nov 6;54(l):34-44. doi: 10.4132 / jptm.2019.11.03, the content of which is incorporated herein by reference in its entirety.

[0069] In some embodiments, the HER2 status is determined using HercepTest™ mAb. HercepTest™ mAb is a semiquantitative immunohistochemical assay with a scoring systemreflecting the cell membrane staining intensity and staining pattern. The assay is interpreted as negative for HER2 protein overexpression (score 0 and 1+ staining intensity), weakly positive (score 2+ staining intensity), and strongly positive (score 3+ staining intensity), respectively. In some embodiments, score 0 indicates that no staining is observed, or membrane staining is observed in less than 10% of the tumor cells. Score +1 indicates that a faint / barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane. Score 2+ indicates that a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells. Score 3+ indicates that a strong complete membrane staining is observed in more than 10% of the tumor cells. In some embodiments, a HER2-negative status indicates that no staining is observed from the HercepTest™ mAb test. A HER2-low status indicates that weak to moderate brown partial cell membrane staining is observed.

[0070] In some embodiments, the HER2 status is determined based on FDA guidelines. For example, a HER2 IHC score of 0 is considered as HER2 null or ultralow. A HER2 IHC score of 1+ or IHC2+ / FISH- is considered as HER2 low. In some embodiments, PATHWAY® anti-HER2 (4B5) Rabbit Monoclonal Primary Antibody assay is used as a companion diagnostic device to identify patients with HER2 -ultralow (IHC 0 with membrane staining) and HER2-low (IHC 1+ or IHC2+ / FISH-) breast cancer for treatment (e.g., treatment with T-DXd). As a person skilled in the art would understand, PATHWAY® anti-HER2 (4B5) Rabbit Monoclonal Primary Antibody is a rabbit monoclonal antibody intended for the semi- quantitative detection of HER2 antigen by IHC in sections of formalin-fixed, paraffin-embedded breast carcinoma and biliary tract cancer tissue. The HER2 status determined using the methods described above can be an indication for identifying patients who are eligible for receiving treatment with the therapies described herein.

[0071] In some embodiments, the breast cancer is hormone receptor positive (HR+) breast cancer. Hormone receptor positive breast cancers include breast cancers in which a portion of the cancer cells express hormone receptors including estrogen receptor, progesterone receptor, or both. Hormone receptor positive breast cancers are typically susceptible to hormone therapies. Hormone therapy can slow or stop the growth of hormone-sensitive tumors by blocking the body’s ability to produce hormones or by interfering with effects of hormones on breast cancer cells. Exemplary hormone therapies include, but are not limited to, selective estrogen receptor modulators or SERMs (e.g., tamoxifen, toremifene), aromatase inhibitors (e.g., anastrozole, letrozole), or selective estrogen receptor degraders or SERDs (e.g., fulvestrant). A person skilled in the art would understand that hormone therapies such as aromatase inhibitors block production of estrogens in the body, whereas SERMs and SERDs block the proliferative action of estrogenson the breast cancer cells.

[0072] In some embodiments, the HR+ breast cancer is estrogen receptor positive (ER+) breast cancer. The term “ER+ breast cancer” refers to breast cancer wherein at least a portion of the cancer cells express estrogen receptor (ER). In some embodiments, at least 1% of the cancer cells express ER. In some embodiments, at least 2%, 5%, 10%, 15%, 20% or more of the cancer cells express ER. In some embodiments, the ER+ breast cancer can comprise breast cancer cells also expressing progesterone receptor (PR). Accordingly, the ER+ breast cancer can be PR positive (PR+). In some embodiments, the ER+ breast cancer is PR negative (PR-). Immunohistochemistry (IHC) test can be performed to test if cancer cells have estrogen and / or progesterone receptors.

[0073] In some embodiments, the breast cancer described herein is a HR+ breast cancer having a negative, low or ultralow HER2 status (referred to as “HER2- / low / ultralow HR+ breast cancer” herein). The HR+ breast cancer described herein can include breast cancer, advanced breast cancer, metastatic breast cancer, ER+ / PR+ breast cancer, ER+ / PR- breast cancer, ER- / PR+ breast cancer, ER+ / PR+ / HER2- / low / ultralow breast cancer, ER+ / PR- / HER2- / low / ultralow breast cancer, ER positive breast cancer with or without expression of androgen receptor (AR), refractory breast cancer, breast cancers that have failed or are resistant to hormone therapies (e.g., estrogen receptor modulators, aromatase inhibitors, and / or selective estrogen receptor degraders), and / or breast cancers that have developed resistance to first line treatments such as first line treatments targeting HR+ / HER2- breast cancer types.

[0074] In some embodiments, the breast cancer described herein is triple negative breast cancer (TNBC) that tests negative for estrogen receptors and progesterone receptors, and negative, low or ultralow for the HER2 protein. In some embodiments, the TNBC described herein is a HER2 -negative, HER2-low or HER2 -ultralow TNBC. The TNBC can include advanced breast cancer, metastatic breast cancer, breast cancer with or without expression of androgen receptor (AR), refractory breast cancer, breast cancers that have failed or are resistant to hormone therapies (e.g., estrogen receptor modulators, aromatase inhibitors, and / or selective estrogen receptor degraders), and / or breast cancers that have developed resistance to first line treatments such as first line treatments targeting TNBC breast cancer types. In some embodiments, the subject with TNBC is resistant to or does not respond effectively to chemotherapy.

[0075] In some embodiments, the breast cancer (e.g., TNBC or HR+ or HER2- / low / ultralow) is refractory breast cancer that does not respond to treatment such as hormone drugs, kinase inhibitors, and / or drugs / therapies that target HER2. Refractory breast cancer can also be referred to as “resistant cancer”. The breast cancer may be resistant at the beginning of the treatment (intrinsic resistance) or it may become resistant during treatment (acquired or inducedresistance). In some embodiments, the HR+ breast cancer is resistant or does not respond to hormone therapies such as SERMs (e.g., tamoxifen, toremifene, raloxifene), aromatase inhibitors (e.g., anastrozole, letrozole, exemestane), or SERDs (e.g., fulvestrant). For example, the subject ER+ breast cancer can develop stable or progressive disease following hormone therapies. In some embodiments, the breast cancer have or developed resistance to fulvestrant, anastrozole, letrozole, exemestane, tamoxifen, toremifene, raloxifene, or a combination thereof. In some embodiments, the breast cancer (e.g., HR+ breast cancer) have or developed resistance to endocrine therapy (e.g., fulvestrant) alone or in combination with other drugs such as alpelisib and CDk4 / 6 inhibitors.

[0076] In some embodiments, the breast cancer (e.g., TNBC or HR+ or HER2- / low / ultralow breast cancer) is resistant or does not respond to kinase inhibitors. For example, the breast cancer can be resistant or does not respond effectively to phosphatidylinositol 3-kinase (PI3K) inhibitors that inhibit or block the activity of PI3K enzymes which are part of the PI3K / AKT / mTOR pathway that regulates cell growth and survival. Exemplary PI3K inhibitor include alpelisib and idelalisib. In some embodiments, the HR+ breast cancer is resistant or does not respond to cyclin-dependent kinase (CDK) inhibitors that inhibit a family of proline-binding serine / threonine protein kinases known as CDKs. In some embodiments, the CDK inhibitor comprises CDK4 and / or CDK6 inhibitor. The CDK4 / 6 complex acts as a checkpoint during the cell cycle transition from cell growth (Gl) to DNA synthesis (S) phase and its deregulation or overexpression induces abnormal cell proliferation and cancer development. Exemplary FDA- approved CDK inhibitors include, but are not limited to, Palbociclib, Ribociclib, and Abemaciclib. In some embodiments, the breast cancer (e.g., HR+ breast cancer) is resistant or does not respond to (e.g., develops stable or progressive disease following treatment) CDK4 / 6 inhibitors (e.g., Palbociclib) alone or in combination with hormone drugs such as SERDs (e.g., fulvestrant) or aromatase inhibitors. The resistance can be acquired resistance or intrinsic resistance. In some embodiments, the breast cancer is resistant to palbociclib. In some embodiments, the breast cancer is resistant to abemaciclib. In some embodiments, the breast cancer is resistant to palbociclib or abemaciclib alone in combination with fulvestrant. In some embodiments, the breast cancer develops stable disease, progressive disease, or acquired resistance to palbociclib and fulvestrant or to abemaciclib and fulvestrant.

[0077] The breast cancer and / or tumor (e.g., TNBC or HR+ or HER2- / low / ultralow breast cancer) can be a cancer and / or tumor having abnormal alterations to PLK1 gene or protein. It has been identified that polo-like kinase 1 (PLK1) is an important gene for growth and survival of breast cancer cells with unstable genome. For example, the abnormal alterations can include one or more PLK1 alterations and / or PLK1 aberrant activation such as copy number alteration (CNA), single-nucleotide variation (SNV), and gene rearrangement or fusions. Non-limitingexemplary cancer and / or tumor with PLK1 alterations include cancer with PLK1 gene or protein amplification, PLK1 gene or protein modification, PLK1 gene deletion, PLK1 gene or protein overexpression, elevated PLK1 gene or protein expression, and / or a combination thereof. In some embodiments, the cancer and / or tumor can be a PLK1 -amplified cancer in which PLK1 gene and / or protein is amplified, for example, as a result of gene duplication and / or aberrant gene transcriptional control. For example, the cancer with PLK1 amplification can be a cancer with higher PLK1 mRNA and / or protein levels as compared to healthy tissues. In heterogeneous cancer types, the breast cancer / or tumor can include a subtype that has an abnormal high expression of PLK1 gene and / or protein. In some embodiments, the breast cancer and / or tumor with amplified PLK1 can be node-positive tumors, aggressive tumors and / or invasive tumors. In some embodiments, the ER+ breast cancer and / or tumor with amplified PLK1 can have a shorter disease-free survival as compared to cancer and / or tumor with normal levels of PLK1. In some embodiments, the breast cancer and / or tumor exhibits a high relapse and / or resistance to traditional and / or mono-therapies, such as hormone therapy, chemotherapy and / or radiotherapy.

[0078] In some embodiments, the breast cancer described herein (e.g., TNBC or HR+ or HER2- / low / ultralow) is resistant to or does not respond effectively to (e.g., develops stable or progressive disease) HER2-directed antibody drug conjugate (e.g., trastuzumab deruxtecan “T- DXd”) alone. In some embodiments, the HER2-directed antibody drug conjugate (HER2-directed ADC) comprises an anti-HER2 antibody and a DNA breaking agent. Administering a PLK1 inhibitor drug (e.g., onvansertib) in combination with a HER2-directed antibody drug conjugate (e.g., T-DXd) to the subject having a breast cancer (e.g., HER2 negative, low or ultralow breast cancer) that is resistant to or does not respond effectively to the HER2-directed ADC (e.g., T- DXd) alone can unexpectedly enhance the therapeutic effect in treating the breast cancer. When combined, surprisingly, the combination can induce robust and durable anti-tumor activity and cancer survival rate / duration by the combination can be surprisingly synergistic (i.e., more than additive, superior to the cumulated anti-tumor efficacy caused by the single agents separately).PLK1 Inhibitors

[0079] Polo-like kinases (PLKs) are a family of five highly conserved serine / threonine protein kinases. PLK1 is a master regulator of mitosis and is involved in several steps of the cell cycle, including mitosis entry, centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. It is also critical for the entry and progression through mitosis, regulates progression of cells through the G2 phase of the cell cycle by phosphorylating forkhead box protein Ml (FOXM1), which then regulates the expression of cyclins and other genes necessary for cells to progress through the cell cycle. PLK1 has been shown to be overexpressedin solid tumors and hematologic malignancies, including breast cancers. Overall survival of breast cancer patients with high PLK1 expression is lower than breast cancer patients with low PLK1 expression. PLK1 expression level was higher in TNBC as compared to adeno-cavity Type A, adeno-cavity Type B, and HER2 overexpression type breast cancer. PLK1 inhibition induces G2- M-phase arrest with subsequent apoptosis in cancer cells, and has emerged as a promising targeted therapy. Several PLK inhibitors have been studied in clinical trials. In the early pre-clinical development of PLK1 targeted drugs, cancer cells with TP53 mutation (mutp53) were more responsive and had lower IC50 than cell lines with wild type (wtp53), which are consistent with the lack of checkpoint control and genomic instability associated with mutp53 and increases the importance of PLK1 function for progression through G2 and M phases of the cell cycle. The pyruvate dehydrogenases kinase 1 (PDK1), PLK1, and MYC have also been suggested to be important in driving the expression of a set of genes associated with cancer stem cell self-renewal. Thus, it is possible that blocking PLK1 function and affecting the ability of cancer cells with unstable genomes to progress through mitosis can increase the overall sensitivity of cells to taxanes, such as paclitaxel. PLK1 has been identified as a therapeutic target for TNBC through siRNA-mediated screen and inhibition of PLK1 by siRNA-mediated knockdown or a chemical inhibitor promoted cell cycle arrest and apoptosis in multiple TNBC lines. The lack of a druggable target is the problem for poor prognosis of TNBC, and the effectiveness and unique action profile of PLK1 inhibition, in addition to relatively specific expression to TNBC tissue, suggest that PLK1 is a promising molecular target for TNBC.

[0080] Several PLK inhibitors have been studied in clinical trials. In a randomized phase II study of patients with AML who were treatment naive yet unsuitable for induction therapy, the pan-PLK inhibitor, volasertib (BI6727), administered intravenously in combination with LDAC showed a significant increase in OS when compared with LDAC alone. A subsequent randomized phase III study identified no benefit of the combination and described an increased risk of severe infections. PLK1 facilitates HR during Double Strand DNA Break (DSB) Repair. PLK1 phosphorylates Rad51 and BRCA1, facilitating their recruitment to DSB sites and thereby HR-mediated DNA repair. The PLK1 inhibitor can be selective and / or specific for PLK1.

[0081] The PLK1 inhibitor can be a dihydropteridinone, a pyridopyrimidine, a aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5- f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or any combination thereof. The PLK1 inhibitor can be onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON- 01910), MLN0905, TKM-080301, TAK-960, or Ro3280.

[0082] Onvansertib (also known as PCM-075, NMS-1286937, NMS-937, “compoundof formula (I)” in U.S. Patent No. 8,927,530; IUPAC name l-(2-hydroxyethyl)-8-{[5-(4- methylpiperazin-l-yl)-2-(trifluoromethoxy) phenyl] amino}-4,5-dihydro-lH-pyrazolo[4,3-h] quinazoline-3 -carboxamide), or a pharmaceutically acceptable salt, is a selective ATP- competitive PLK1 inhibitor. Onvansertib can be formulated, for example, with an additive such as free base, lactose monohydrate, pregelatinized starch and glyceryl beneate. In some embodiments, the onvansertib is formulated for oral administration, such as in a hard gelatin capsule.

[0083] Biochemical assays demonstrated high specificity of onvansertib for PLK1 among a panel of 296 kinases, including other PLK members. Onvansertib has potent in vitro and in vivo antitumor activity in models of both solid and hematologic malignancies. Onvansertib is the first PLK1 specific ATP competitive inhibitor administered by oral route to enter clinical trials with proven antitumor activity in different preclinical models. Onvansertib has shown a promising safety profile in a phase 1 clinical trial as single agent. In addition, clinical investigations of onvansertib includes onvansertib in combination with abiraterone and prednisone in adult patients with metastatic castration-resistant prostate cancer, onvansertib in combination with FOLFIRI and bevacizumab in adult patients with KRAS-mutated metastatic colorectal cancer, and onvansertib in combination with nanoliposomal irinotecan and 5-FU in patients with metastatic pancreatic cancer. As described herein, onvansertib can synergize with anti-HER2 ADC (e.g., T-DXd) in HER2-low breast cancer patients (TNBC or HR+), and thereafter achieve robust anti-tumor activity compared to single agents, and without drug specific toxicity.

[0084] Onvansertib also inhibited cell proliferation at nanomolar concentrations in AML cell lines and tumor growth in xenograft models of AML. In addition, onvansertib significantly increased cytarabine antitumor activity in disseminated models of AML.

[0085] Onvansertib shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid as well as hematologic tumors.Onvansertib has a relative short half-life of 24 h and is highly potent against the PLK1 enzyme ([IC50] = 2 nM). In comparison, low or no activity was observed on a panel of 63 kinases (IC50 > 500 nM), including the PLK members PLK2 and PLK3 (IC50 > 10 pM). Onvansertib potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with a clear PLKl-related mechanism of action at well tolerated doses in mice after oral administration. In addition, onvansertib shows activity in combination therapy with approved cytotoxic drugs, such as irinotecan, in which there is enhanced tumor regression in HT29 human colon adenocarcinoma xenografts compared to each agent alone, and shows prolonged survival of animals in a disseminated model of AML in combination therapy with cytarabine. Onvansertib has favorable pharmacologic parameters and good oral bioavailability in rodent and nonrodent species, as well as proven antitumor activity in different nonclinical models using a variety of dosing regimens, which may potentially provide a high degree of flexibility in dosing schedules, warranting investigation in clinical settings. Onvansertib has several advantages over volasertib (BI6727, another PLK1 inhibitor), including a higher degree of potency and specificity for the PLK1 isozyme, and oral bioavailability. In addition, onvansertib has proven antitumor activity in different nonclinical models using a variety of dosing regimens, which can provide flexibility in dosing schedules, and therefore, warrants investigation in clinical settings.

[0086] A phase I, first-in-human, dose-escalation study of onvansertib in patients with advanced / metastatic solid tumors identified neutropenia and thrombocytopenia as the primary dose-limiting toxicities. These hematologic toxicities were anticipated on the basis of the mechanism of action of the drug and were reversible, with recovery occurring within 3 weeks. The half-life of onvansertib was established between 20 and 30 hours. The oral bioavailability of onvansertib plus its short half-life provide the opportunity for convenient, controlled, and flexible dosing schedules with the potential to minimize toxicities and improve the therapeutic window. Pharmacodynamics and biomarker studies, including baseline genomic profiling, serial monitoring of mutant allele fractions in plasma, and the extent of PLK 1 inhibition in circulating blasts, have been performed to identify biomarkers associated with clinical response and are described in PCT Application No. PCT / US2021 / 013287, the content of which is incorporated herein by reference in its entirety.

[0087] The major metabolic pathways found in the different animal species were N- oxidation of the N methyl-piperazine ring to give N-oxide M2 and hydroxylation on an aliphatic carbon atom of the methylene bridge of the pyrazoloquinazoline moiety to give metabolite ML Qualitatively, no marked differences in the metabolism of onvansertib were observed between species and, quantitatively, some differences were observed cross-species.

[0088] The potential inhibitory capacity of onvansertib towards the major humancytochrome P450 (CYP) isoforms that are responsible for hepatic drug metabolism in man (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) was investigated using human liver microsomes. Onvansertib was able to inhibit the metabolic activities of CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoforms to different extents, with 50% inhibitory concentration (IC50) values ranging from 20 pM to 66 pM. No significant inhibitory effects against CYP1 A2 were detected. Considering that the concentrations relevant to achieve significant anti-tumoral activity of the compound in mice were in the order of 1 pM, the likelihood that onvansertib would show clinically relevant metabolic drug-drug interactions is considered low.

[0089] A Phase 1 safety study with onvansertib has been completed in adult patients with advanced / metastatic solid tumors at a single study site in the US. First cycle dose-limiting toxi cities (DLTs) and the maximum tolerated dose (MTD) of onvansertib administered orally for 5 consecutive days every 3 weeks (i.e., a 21 day treatment cycle) was conducted. Safety profile of onvansertib, to determine the pharmacokinetics (PK) of onvansertib in plasma (at the MTD), and to document any antitumor activity has been determined. In one study, a total of 21 patients were enrolled, and 19 patients were treated. No DLTs occurred at the first 3 dose levels (doses of 6, 12, and 24 mg / m2 / day). At the subsequent dose level (dose of 48 mg / m2 / day), 2 of 3 patients developed DLTs. An intermediate dose level of 36 mg / m2 / day was investigated. At the intermediate dose level, 4 patients were treated and 2 DLTs were observed. After further cohort expansion, the MTD was determined to be 24 mg / m2 / day. The best observed treatment response was stable disease (SD); SD occurred in 5 of the 16 evaluable patients. The study identified thrombocytopenia and neutropenia as the primary toxicities; this is consistent with the expected mechanism of action of onvansertib and with results from the preclinical studies. These hematologic toxicities were reversible, with recovery usually occurring within 3 weeks. No other clinically relevant safety findings emerged with treatment with onvansertib as a single agent. Other mechanism-related, possibly expected events such as gastrointestinal disorders, mucositis, and alopecia were not observed, confirming that with this schedule, the bone marrow is the most sensitive target of onvansertib in humans.DNA Damaging Agent / Chemotherapeutic Agent

[0090] A PLK1 inhibitor described herein can be used in combination with a DNA damaging agent or a chemotherapeutic agent to treat breast cancer, including HER2-negative, low, or ultralow breast cancer (HER2- / low / ultralow breast cancer), HR+ breast cancer, and triple negative breast cancer (TNBC). In some embodiments, the breast cancer is HR+ and HER2- / low / ultralow or TNBC and HER2- / low / ultralow.

[0091] A DNA damaging agent can be any substance capable of causing damage tothe DNA structure or interfering with DNA replication in a cell, leading to mutations, alterations or breaks in the DNA. In some embodiments, a DNA damaging agent can be selected from the group consisting of radiomimetic neocarzinostatin, a platinating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an antimetabolite, an alkylating agent (e.g., alkyl sulphonate), an antibiotic, and a combination thereof. In some embodiments, the DNA damaging agent is a platinating agent selected from the group consisting of Cisplatin, Oxaliplatin, Carboplatin, Nedaplatin, Lobaplatin, Triplatin Tetranitrate, Picoplatin, Satraplatin, ProLindac, Aroplatin, and a combination thereof. In some embodiments, the DNA damaging agent is a topoisomerase I inhibitor selected from the group consisting of Camptothecin, Topotecan, Irinotecan / SN38, Rubitecan, and Belotecan. In some embodiments, the DNA damaging agent is a topoisomerase II inhibitor selected from the group consisting of Etoposide, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin, Teniposide, and a combination thereof. In some embodiments, the DNA damaging agent is an antimetabolite selected from the group consisting of Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Pentostatin, Cladribine, Clofarabine, Fludarabine, Thioguanine, Mercaptopurine, Fluorouracil, Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine, Gemcitabine, 6- Mercaptopurine, 5-Fluorouracil, Azacitidine, Hydroxyurea, and a combination thereof. In some embodiments, the DNA damaging agent is an alkylating agent selected from the group consisting of Mechlorethamine, Cyclophosphamide, Ifosfamide, Trofosfamide, Chlorambucil, Melphalan, Prednimustine, Bendamustine, Uramustine, Estramustine, Carmustine, Lomustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin, Busulfan, Mannosulfan, Treosulfan, Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine, Procarbazine, Dacarbazine, Temozolomide, Altretamine, Mitobronitol, Actinomycin, Bleomycin, Mitomycin, nitrogen mustards, nitrosoureas, triazenes, alkyl sulfonates, Procarbazine, aziridines, Plicamycin, and a combination thereof. In some embodiments, the DNA damaging agent is an antibiotic selected from the group consisting of Hydroxyurea, Anthracyclines, Anthracenediones, and antibiotics from the Streptomyces family.

[0092] In some embodiments, the DNA damaging agent is selected from the group consisting of Cisplatin, Oxaliplatin, Carboplatin, Nedaplatin, Lobaplatin, Triplatin Tetranitrate, Picoplatin, Satraplatin, ProLindac, Aroplatin, Camptothecin, Topotecan, Irinotecan / SN38, Rubitecan, Belotecan, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin, Teniposide, Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Pentostatin, Cladribine, Clofarabine, Fludarabine, Thioguanine, Mercaptopurine, Fluorouracil, Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine, Gemcitabine, Azacitidine, Hydroxyurea, Mechlorethamine, Cyclophosphamide, Ifosfamide, Trofosfamide, Chlorambucil, Melphalan, Prednimustine,Bendamustine, Uramustine, Estramustine, Carmustine, Lomustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin, Busulfan, Mannosulfan, Treosulfan, Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine, Procarbazine, Dacarbazine, etopside, Tern ozolom ide, Altretamine, Mitobronitol, Actinomycin, Bleomycin, Mitomycin, Plicamycin, and a combination thereof.

[0093] In some embodiments, the PLK1 inhibitor is used in combination with a chemotherapy drug. Chemotherapy drugs in general refers to any drug capable of killing cancer cells. Chemotherapy drugs include alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antitumor antibiotics, and other chemotherapy drugs identifiable to a person skilled in the art. Exemplary chemotherapy drugs include, but are not limited to, Altretamine, Bendamustine, Busulfan, Carboplatin, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine, Ifosfamide, Mechlorethamine, Melphalan, Oxaliplatin, Procarbazine, Temozolomide, Thiotepa, Trabectedin, Carmustine, Lomustine, Streptozocin, 5-fluorouracil, 6-mercaptopurine, Azacitidine, Capecitabine, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Gemcitabine, Hydroxyurea, Methotrexate, Nelarabine, Pemetrexed, Pentostatin, Pralatrexate, Thioguanine, Trifluridine / tipiracil combination, Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone, Teniposide, Topotecan, Cabazitaxel, Docetaxel, Nab-paclitaxel, Paclitaxel, Vinblastine, Vincristine, Vincristine liposomal, Vinorelbine, Daunorubicin, Doxorubicin, Doxorubicin liposomal, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin, Bleomycin, Dactinomycin, Mitomycin-C, All-trans-retinoic acid, Arsenic trioxide, Eribulin, Asparaginase, Ixabepilone, Mitotane, Omacetaxine, Pegaspargase, Procarbazine, Romidepsin, and Vorinostat.

[0094] In some embodiments, the DNA damaging agent and / or chemotherapeutic agent described can enable a bystander antitumor effect, resulting in the elimination of both target cells and surrounding tumor cells, especially when conjugated with a cancer cell targeting antibody.

[0095] In some embodiments, the PLK1 inhibitor is used in combination with a topoisomerase inhibitor. A topoisomerase inhibitor prevents topoisomerase from performing DNA strand breaks, or associate with topoisomerase-DNA complex and prevent the re-ligation step of the topoisomerase mechanism. In some embodiments, the topoisomerase inhibitor is a topoisomerase I inhibitor or a topoisomerase II inhibitor. Exemplary topoisomerase inhibitors include, but are not limited to, Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone, Teniposide, and Topotecan, Camptothecin, Rubitecan, Belotecan, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, and Zorubicin. In some embodiments, the topoisomerase inhibitor is an exatecan derivative.

[0096] In some embodiments, the PLK1 inhibitor is used in combination with deruxtecan (DXd). DXd is a highly potent topoisomerase I inhibitor that can interfere with DNA replication by stabilizing the topoisomerase I-DNA cleavage complex, leading to DNA strand breaks and apoptosis in rapidly proliferating cancer cells. In some embodiments, DXd can be used as a standalone molecule with a PLK1 inhibitor (e.g., onvansertib). In some embodiments, DXd can be used as a payload incorporated in an antibody drug conjugate (ADC). DXd exhibits markedly enhanced membrane permeability compared to other ADC payloads, thereby amplifying the bystander effect. This unique feature enables DXd to exert cytotoxic activity not only in antigen-expressing tumor cells but also in adjacent tumor cells, effectively addressing tumor heterogeneity, a major challenge in cancer treatment.Antibody Drug Conjugate (ADC)

[0097] In some embodiments, the DNA damaging agent and / or chemotherapeutic agent described herein can be conjugated with an antibody to form an antibody drug conjugate, and then administrated in combination with a PLK1 inhibitor to a subject in need to treat breast cancer, including HER2-negative, low, or ultralow (HER2- / low / ultralow) breast cancer, HR+ breast cancer, and triple negative breast cancer (TNBC). In some embodiments, the breast cancer is HR+ and HER2- / low / ultralow. In some embodiments, the breast cancer is TNBC and HER2- / low / ultralow.

[0098] An antibody drug conjugate (ADC) is a targeted cancer drug that combines a monoclonal antibody with a cytotoxic drug to deliver chemotherapy to cancer cells. The ADC can bind to a receptor on a cancer cell and internalizes and releases the drug. The ADC comprises a monoclonal antibody capable of binding to specific proteins or receptors found on certain types of cells (e.g., cancer cells). The monoclonal antibody is linked to a payload, such as a cytotoxic drug, either directly or indirectly via a linker. The linker can stabilize the ADC in the blood to prevent the drug from being released in healthy tissues / cells, but can also release the drug when the ADC is internalized by the cancer cell. The linker can be cleavable or non-cleavable. The drug to antibody ratio can vary in different embodiments. In some embodiments, the drug to antibody ratio can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0099] In some embodiments, the ADC is an ADC capable of targeting breast cancer cells, e.g., an ADC comprising a monoclonal antibody capable of binding to a receptor protein or marker expressed or overexpressed on breast cancer cells. Non-limiting exemplary targets expressed or overexpressed in breast cancer cells include, but are not limited to, HER2 (human epidermal growth factor receptor 2), TROP2 (trophoblast surface antigen 2), HER3 (ERBB3), LIV-1, PTK7, and others identifiable to a person of skill in the art. The ADC can comprise one ormore drugs (e.g., payload), for example a chemotherapy agent, or a

[0100] In some embodiments, the ADC comprises an anti-HER2 antibody and is a HER2-directed ADC comprising a monoclonal antibody capable of binding to HER2 protein that is expressed or overexpressed in certain cancer cells such as breast cancer. HER2 is a membrane tyrosine kinase and oncogene that is overexpressed and gene amplified in about 20% of breast cancers. When activated it provides the cell with potent proliferative and anti -apoptosis signals and it is the major driver of tumor development and progression for this subset of breast cancer. Exemplary anti-HER2 antibody include, but are not limited to, trastuzumab, pertuzumab, margetuximab, zanidatamab, disitamab, and others identifiable to a person skilled in the art.

[0101] In some embodiments, the ADC is a HER2-directed ADC or an anti-HER2 ADC comprising an anti-HER2 antibody covalently linked to a DNA damaging agent described above either directly or indirectly via a linker. In some embodiments, the ADC is an anti-HER2 ADC comprising an anti-HER2 antibody covalently linked to a chemotherapy drug described herein either directly or indirectly via a linker.

[0102] In some embodiments, the anti-HER2 ADC comprises an anti-HER2 antibody covalently linked to a topoisomerase inhibitor directly or indirectly via a linker. In some embodiments, the anti-HER2 ADC comprises an anti-HER2 antibody covalently linked to deruxtecan (DXd) via a linker. DXd has exhibited markedly enhanced membrane permeability compared to that of other ADC payloads, thereby amplifying the bystander effect. In some embodiments, DXd-based ADCs feature a high drug-to-antibody ratio (DAR) and a stable, cleavable linker that enables efficient drug release upon internalization. Upon binding to its target antigen, the ADC-receptor complex is internalized into the cell via receptor-mediated endocytosis. Once internalized, the ADC undergoes progressive transport through early and late endosomes before reaching lysosomes. Within the lysosomal compartment, the acidic microenvironment and proteolytic enzymes facilitate linker degradation, resulting in the controlled release of a given cytotoxic payload such as DXd into the cytoplasm. The released DXd then diffuses throughout the cytoplasm and enters the nucleus, where it inhibits topoisomerase I, thereby inducing DNA damage and promoting tumor cell death. This mechanism ensures selective drug activation, thereby minimizing systemic toxicity while maximizing therapeutic efficacy.

[0103] In some embodiments, the anti-HER2 ADC is Trastuzumab deruxtecan (T- DXd). T-DXd is a HER2-directed antibody-drug conjugate consisting of the humanized monoclonal antibody trastuzumab covalently linked to deruxtecan (DXd), an exatecan derivative and a topoisomerase I inhibitor, via a linker (e.g., a cleavable GGFG tetrapeptide linker). Trastuzumab can bind to HER2 expressed on the surface of certain tumor cells. After binding, the T-DXd complex then undergoes internalization and intracellular linker cleavage by lysosomalenzymes that are upregulated in cancer cells. Upon release, the membrane permeable DXd can prevent re-ligation of the DNA strand, resulting in DNA damage, apoptosis, and cell death. T- DXd has a high drug to antibody ratio (i.e., approximately 8 molecules of DXd for each antibody molecule) which enables effective delivery of DXd into tumor cells and activity against low or ultralow HER2-expressing tumors. Furthermore, DXd can also penetrate into the microenvironment and neighboring tumor cells resulting in a potent bystander effect upon release within the target cells. DXd is also cell cycle phase-specific and can stall cell cycle progression at S phase.

[0104] T-DXd is approved in the United States, Japan, and Europe for the treatment of unresectable or metastatic HER2 -positive breast cancer in patients who have received 2 or more prior anti-HER2 based regimens in the metastatic setting, and in the United States and Japan for the treatment of HER2-positive unresectable advanced or recurrent gastric cancer that has progressed after chemotherapy. Recent phase I and II clinical studies have shown the antitumor activity of T-DXd in the treatment of HER2 -low-expressing breast cancer, HER2-expressing gastric or gastroesophageal junction adenocarcinoma, Zffi7?2-mutant non-small cell lung cancer, and HER2-positive colorectal cancer.

[0105] The anti-tumor efficacy of T-DXd has been evaluated in various clinical trials. T-DXd has been used for treating HER2 -positive breast cancer in patients who have received one or more prior anti-HER2 based regimens. In a phase I study (DS8201-A-J101; NCT02564900), T-DXd demonstrated a manageable safety profile and preliminary antitumor activity across many different HER2-expressing or Zffi7?2-mutated advanced solid-tumor types. The efficacy and safety of T-DXd were also studied in several open-label, randomized phase 2 and 3 clinical trials that enrolled patients with HER2-positive, unresectable or metastatic breast cancer. The trials enrolled patients who have received prior trastuzumab and taxane therapy for metastatic disease or developed disease recurrence during or within 6 months of completing adjuvant therapy, patients who were resistant or refractory to prior T-DM1 therapy, or patients who have received two or more prior anti-HER2-based regimens including trastuzumab emtansine, trastuzumab and pertuzumab. Consistent anti-tumour activity was observed across prespecified subgroups based on prior therapy and hormone receptor status.

[0106] T-DXd has also been evaluated in clinical trials for the treatment of adult patients with unresectable or metastatic HER2-low (e.g., immunohistochemistry (IHC) Score 1+ or 2+ / in situ hybridization-negative) breast cancer, including patients who have received prior chemotherapy in the metastatic setting or developed disease recurrence during or within 6 months of completing adjuvant chemotherapy. The efficacy and safety of T-DXd were studied in a randomized, multicenter, open-label study (NCT03734029) that enrolled 557 adult patients withunresectable or metastatic HER2-low breast cancer. Consistent overall survival (OS) and progression-free survival (PFS) benefit were observed across prespecified subgroups including HR status (HR+ and HR-), prior CDK4 / 6 treatment, number of prior chemotherapies and IHC 1+ and IHC 2+ / ISH- status. T-DXd can be administered intravenously.

[0107] In some embodiments, the ADC is a TROP2-directed ADC or an anti-TROP2 ADC comprising an anti-TROP2 antibody and a DNA damaging agent / chemotherapeutic agent described above. Trophoblast surface antigen 2 (TROP-2) is a type I transmembrane glycoprotein involved in Ca2+signaling in tumor cells. It is highly expressed in various tumor tissues than in normal tissues and represents a novel and promising molecular target for caner targeted therapy. TROP-2 plays an important role in cell proliferation, apoptosis, cell adhesion, epithelial- mesenchymal transition, as well as tumorigenesis and tumor progression. Exemplary anti-TROP2 antibody include, but are not limited to, datopotamab, sacituzumab, and others identifiable to a person skilled in the art.

[0108] In some embodiment, the anti-TROP2 ADC is an anti-TROP2 antibody conjugated with DXd payload. In some embodiments, the anti-TROP2 ADC is datopotamab deruxtecan (Dato-DXd). Early-phase clinical trials have demonstrated promising antitumor activity and a manageable safety profile for Dato-DXd in heavily pretreated patients. Unlike conventional chemotherapy, Dato-DXd facilitates targeted delivery of a topoisomerase I inhibitor payload, potentially enhancing the therapeutic index while reducing systemic toxicity. Dato-DXd also employs a cleavable GGFG tetrapeptide linker, similar to that used in T-DXd, which enables selective cleavage by lysosomal proteases within tumor cells. It has a lower DAR of approximately 4, a design feature intended to balance therapeutic efficacy with reduced systemic toxicity due to TROP2 expression in some normal tissues. Data-DXd has been approved by FDA for the treatment of adult patients with unresectable or metastatic HR-positive, HER2 -negative breast cancer who had received prior endocrine-based therapy and chemotherapy.

[0109] In some embodiments, the ADC is a HER3-directed ADC or an anti-HER3 ADC comprising an anti-HER3 antibody and a DNA damaging agent / chemotherapeutic agent described above. HER3 (ErbB3) is a member of the human epidermal growth factor receptor family, and is frequently overexpressed in many solid tumors, including breast, head and neck, lung, colorectal, prostate and ovarian cancers. High HER3 expression is also linked to disease progression and poor prognosis in many cancer types. Due to its common expression in numerous tumors and efficient cancer cell internalization upon antibody binding, HER3 has become a feasible target especially for ADC development.

[0110] In some embodiment, the anti-HER3 ADC is an anti-HER3 antibody conjugated with DXd payload. In some embodiments, the anti-HER3 ADC is patribumabderuxtecan (HER3-DXd). Patribumab deruxtecan is a HER3 -targeting ADC in clinical development and has shown clinical benefit in breast cancer and other cancer patient with or without EGFR mutations.[OHl] In some embodiments described herein, the ADCs targeting breast cancer cells can induce a bystander effect, also known as bystander killing. The bystander effects refers to the phenomenon where cytotoxic drugs released from ADCs can diffuse from targeted cancer cells to neighboring cancer cells that may not express the specific antigen recognized by the ADC. By leveraging the bystander effect, ADCs used herein can target and eliminate both antigen-positive and antigen-negative cancer cells within the tumor, thereby improving treatment outcomes. Accordingly, as demonstrated in some embodiments herein, the anti-HER2 ADCs can effectively target not only HER2-positive cancer cells, but also HER2 -negative, low, or ultralow cancer cells.Combination Therapy for Treating Breast Cancer

[0112] Provided herein includes a combinational therapy using a PLK1 inhibitor in combination with a chemotherapeutic agent / DNA damaging agent for treating cancers, such as breast cancer, and particularly HER2 negative, low or ultralow (HER2- / low / ultralow) breast cancer, hormone receptor positive (HR+) breast cancer or triple negative breast cancer (TNBC). The chemotherapeutic agent / DNA damaging agent can be either in standalone form or conjugated with an antibody to form an antibody drug conjugate (e.g., T-DXd). The breast cancer can be HR+ and HER2- / low / ultralow. The breast cancer can be a TNBC, with a negative, low or ultralow HER2 protein. In some embodiments, the method of treating HR+ breast cancer comprises administering a PLK1 inhibitor in combination with an antibody drug conjugate capable of targeting breast cancer cells (e.g., anti-HER2 ADC) to a subject with the HR+ breast cancer, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject. In some embodiments, a method of treating TNBC comprises administering an antibody drug conjugate capable of targeting breast cancer cells (e.g., anti-HER2 ADC) and a PLK1 inhibitor to a subject with the TNBC, thereby inhibiting or reducing progression of the TNBC in the subject. In some embodiments, a method of treating HER2- / low / ultralow breast cancer comprises administering an antibody drug conjugate capable of targeting breast cancer cells (e.g., anti-HER2 ADC) and a PLK1 inhibitor to a subject with the HER2- / low / ultralow breast cancer, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject. The ADC (e.g., anti- HER2 ADC) and the PLK1 inhibitor can be administered to the subject with breast cancer (e.g., TNBC or HR+ or HER2- / low / ultralow breast cancer) in a manner sufficient to inhibit or reduce progression of the cancer. In some embodiments, the ADC is anti-HER2 ADC. In some embodiments, the ADC is anti-TROP2. In some embodiments, the ADC is anti-HER3 ADC. TheADC (e.g., anti-HER2 ADC) and the PLK1 inhibitor can be administrated to a subject with cancer simultaneously, separately, or sequentially. The ADC and the PLK1 inhibitor can be administered in any suitable order. For example, the ADC can be administered followed by the PLK1 inhibitor. Alternatively or in combination, the PLK1 inhibitor can be administered followed by the ADC.

[0113] In some embodiments, the breast cancer (e.g., HR+ breast cancer) is resistant or has developed resistance to endocrine therapy and CDK4 / 6 and the patient is under the ADC treatment. The combination of a PLK1 inhibitor with the ADC treatment can enhance the antitumor efficacy of the ADC and induce tumor regression in the patient. In some embodiments, the method of treating breast cancer comprises administering a PLK1 inhibitor to a subject with the breast cancer (e.g., TNBC or HR+ or HER2- / low / ultralow breast cancer) and being treated with the ADC (e.g., T-DXd).

[0114] It is expected that the combination treatment using the ADC and the PLK1 inhibitor can result in significantly enhanced efficacy against breast cancer, and particularly breast cancer with a negative, low or ultralow HER2 status, causing tumor regression and cancer survival. The resulted tumor regression and cancer survival rate / duration by the combination can be surprisingly synergistic (i.e., more than additive, superior to the cumulated anti-tumor efficacy caused by the ADC and the PLK1 inhibitor separately). In the embodiments described herein, the combination therapeutic strategies combining onvansertib with the ADC can extend the clinical benefit of the ADC in treating HR+ metastatic breast cancer and induce robust and durable antitumor activity in HR+ breast cancer models resistant to hormone therapy treatments and / or CDK4 / 6 inhibitor treatments as well as other first line therapies for HR+ breast cancer. In some embodiments described herein, combining a PLK1 inhibitor such as onvansertib with an ADC such as T-DXd can overcome T-DXd resistance and delay tumor progression due to intrinsic or acquired resistance to various first line treatments for breast cancer including hormone therapy and CDK4 / 6 inhibitor treatments.

[0115] In some embodiments, the inhibition or reduction of cancer progression is not merely additive, but is enhanced or synergistic (that is, the inhibition is greater than the combined inhibition of progression caused by the ADC and the PLK1 inhibitor alone). The enhanced or synergistic efficacy or inhibition of any combination of the ADC and a PLK1 inhibitor of the present disclosure can be different in different embodiments. In some embodiments, the enhanced or synergistic efficacy or inhibition of the combination treatment of the present disclosure is, is about, is at least, is at least about, is at most, or is at most about, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, or a number or a range between any two of these values, higher thanthe combined inhibition of progression caused by the ADC and the PLK1 inhibitor alone. In some embodiments, the combination treatment of the present disclosure can extend the clinical benefit of the ADC (e.g., T-DXd) in treating breast cancer. For example, the enhanced efficacy or inhibition of cancer progression caused by the combination of an ADC (e.g., T-DXd) and a PLK1 inhibitor (e.g., onvansertib) can be, for example, 50%, 60%, 70%, 80%, 90%, 100%, or more higher than the combined inhibition of progression caused by the ADC (T-DXd) alone plus the PLK1 inhibitor (onvansertib) alone.

[0116] As described herein, treatment with one or more dose cycles of the PLK1 inhibitor in combination with the ADC capable of targeting breast cancer cells can lead to remarkable therapeutic effect. In some embodiments, patients treated with the combinational therapy can achieve complete response or partial response. In some embodiments, the patient who did not respond to or developed stable or progressive disease following treatment with the ADC alone can achieve partial or complete response after the combinational treatment with the PLK1 inhibitor and the ADC. The combinational therapy described herein can lead to a complete response or complete remission of the cancer, a progression-free survival, an overall survival rate exceeding values obtained from single agent treatments. In some embodiments, the combinational therapy exhibits tolerable or undetectable adverse effect.

[0117] The ADC and the PLK1 inhibitor can be administered to the patient in any manner deemed effective to treat the cancer. The ADC can be administered together with, or separately from, the PLK1 inhibitor. When administered separately, the ADC can be administered before or after the PLK1 inhibitor, or in different administration cycles. In some embodiments, the patient are under the treatment with the ADC, but did not respond to or developed stable or progressive disease following the ADC treatment. The addition of a PLK inhibitor to the treatment regimen can result in a complete or partial response or robust tumor regression in the patients.

[0118] The ADC and the PLK1 inhibitor can be administered to the patient at any appropriate dosage in different embodiments. In some embodiments, the combination treatment with the ADC and the PLK1 inhibitor can be administered at the same dose as single treatment with the ADC and the PLK1 inhibitor.

[0119] In some embodiments, the PLK1 inhibitor can be administered to the patient at a dosage of about, at least or at most 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 105 mg / kg, 110 mg / kg, 115 mg / kg, 125 mg / kg, 130 mg / kg, 135 mg / kg, 140 mg / kg, 145 mg / kg, 150 mg / kg, 155 mg / kg, 160 mg / kg, 165 mg / kg, 170 mg / kg, 175 mg / kg, 180 mg / kg, 185 mg / kg, 190 mg / kg, 195 mg / kg, 200 mg / kg or a number between any two of these values.

[0120] In some embodiments, the PLK1 inhibitor is onvansertib. The onvansertib can be administered to the patient at any appropriate dosage, e.g., a dosage of less than 12 mg / m2, less than or equal to 24 mg / m2, or greater than 24 mg / m2. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is be administered at a dosage from about 10 mg / kg of body weight to about 80 mg / kg of body weight, optionally at a dose from about 20 mg / kg of body weight to about 60 mg / kg of body weight, optionally at a dose from about 30 mg / kg of body weight to about 50 mg / kg of body weight. In some embodiments, onvansertib is administered to the patient daily. In some embodiments, onvansertib is administered in a cycle of 5-14 days of daily onvansertib administration with 2-16 days with no onvansertib administration. The PLK1 inhibitor (e.g., onvansertib) can, e.g., be administered at a dosage of 5-50 mg, e.g., 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, or a range between any two of these values. For example, in some embodiments, onvansertib is administered daily for 21 consecutive days followed by no onvansertib administration for 7 days in a cycle. In some embodiments, onvansertib is administered for 5 consecutive days a week followed by no onvansertib administration for 2 days each week or on selected weeks of an administration cycle.

[0121] The ADC can be administered to the patient at any appropriate dosage in different embodiments. The ADC can be administered to the patient at a dosage of about, at least or at most 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 105 mg / kg, 110 mg / kg, 115 mg / kg, 125 mg / kg, 130 mg / kg, 135 mg / kg, 140 mg / kg, 145 mg / kg, 150 mg / kg, 155 mg / kg, 160 mg / kg, 165 mg / kg, 170 mg / kg, 175 mg / kg, 180 mg / kg, 185 mg / kg, 190 mg / kg, 195 mg / kg, 200 mg / kg or a number between any two of these values.

[0122] In some embodiments, the ADC is T-DXd. T-DXd can be administered to the patient at a dosage of about, at least or at most 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg or a number between any two of these values. In some embodiments, T-DXd is administered at about 2 mg / kg to about 15 mg / kg of body weight, optionally at about 4 mg / kg to about 10 mg / kg of body weight. In some embodiments, T-DXd is administered to the patient once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, T-DXd is administered once every 21 days in a treatment cycle of at least 21 days. For example, in some embodiments, T-DXd is administered on day 1 of a treatment cycle of 21 days. In some embodiments, T-DXd is administered on day 1 and day 22 of a treatment cycle of at least 42 days. In some embodiments, T-DXd is administered on day 1, day 22, and day 43 of a treatment cycle of at least 63 days. In some embodiments, T-DXd is administered on day 1, day22, day 43, and day 64 of a treatment cycle of at least 84 days.

[0123] As can be appreciated by one of skill in the art, the amount of co-administration of the ADC and the PLK1 inhibitor, and the timing of co-administration, can depend on the type (species, gender, age, weight, etc.) and condition of the subject being treated and the severity of the disease or condition being treated. The ADC and the PLK1 inhibitor can be formulated into a single pharmaceutical composition, or two separate pharmaceutical compositions. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interracial polymerization, for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.

[0124] The ADC and the PLK1 inhibitor can be administered by any suitable routes, including but not limited to oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal, epidural, and intranasal administration. Parenteral administration (e.g., injection) can include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the ADC (e.g., T-DXd) can be, for example, administered by intravenous infusion (e.g., over about 30 minutes) and the PLK1 inhibitor (e.g., onvansertib) can be, for example, administered orally.

[0125] Methods, compositions, kits and systems disclosed herein can be applied to different types of subjects. For example, the subject can be a subject receiving a cancer treatment, a subject at cancer remission, a subject has received one or more cancer treatment, or a subject suspected of having cancer. The subject can have a stage I cancer, a stage II cancer, a stage III cancer, and / or a stage IV cancer. The methods can further comprise administering an additional therapeutic intervention to the subject. The additional therapeutic intervention can comprise a therapeutic intervention such as an antibody, an adoptive T cell therapy, a chimeric antigen receptor (CAR) T cell therapy, an antibody-drug conjugate, a cytokine therapy, a cancer vaccine, a checkpoint inhibitor, a radiation therapy, surgery, a chemotherapeutic agent, or any combination thereof. The therapeutic intervention can be administered at any time of the treatment, for example at a time when the subject has an early-stage cancer, and wherein the therapeutic intervention is more effective that if the therapeutic intervention were to be administered to the subject at a later time.Dosing and Pharmacokinetics

[0126] The treatment described in the present disclosure can comprise administrationof a PLK1 inhibitor (e.g., onvansertib) for a desired duration in one or more cycles of treatment together with an ADC (e.g., T-DXd).

[0127] In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered for 1 to 10 cycles, for example, 1 to 9 cycles, 1 to 8 cycles, 1 to 7 cycles, 1 to 6 cycles, 1 to 5 cycles, 1 to 4 cycles, 1 to 3 cycles, 1 to 2 cycles, or 1 cycle. Each cycle of treatment can have various lengths, for example, at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more.

[0128] In some embodiments, the administration of the PLK1 inhibitor can be daily or with break(s) between days of administrations. The break can be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more. The administration can be once, twice, three times, four times, or more on a day when PLK1 inhibitor (e.g., onvansertib) is administered to the patient. The administration can be, for example, once every two days, every three days, every four days, every five days, every six days, or every seven days. The length of the desired duration can vary, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or more days. Each cycle of treatment can have various lengths, for example, at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more. For example, a single cycle of the treatment can comprise administration of the PLK1 inhibitor for four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, twenty days, twenty-one days, twenty-two days, twenty-three days, twenty-four days, twenty-five days, twenty-six days, twenty-seven days, twenty-eight days, or more in a cycle (e.g., in a cycle of at least 21 days (e.g., 21 to 28 days)). In some embodiments, the treatment can comprise administration of the PLK1 inhibitor (e.g., onvansertib) and / or one or more chemotherapeutic agents for, or for at least, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, twenty days, or a range between any two of these values, in a cycle (e.g., a cycle of at least 21 days (e.g., 21 to 28 days)). The administration of the PLK1 inhibitor in a single cycle of the treatment can be continuous or with one or more intervals (e.g., one day or two days of break). In some embodiments, the treatment comprises administration of the PLK1 inhibitor (e.g., onvansertib) for five days in a cycle of 14 to 28 days. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered daily for about 14 days, followed by a 7-day off. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered orally. In some embodiments the PLK1 inhibitor (e.g., onvansertib) is administered without any catch-up doses.

[0129] The PLK1 inhibitor (e.g., onvansertib) can be administered to the subject in need thereof on twenty days (e.g., Days 1-10 and 15-24) during a 28-day cycle. The twenty days can be, for example, a continuous daily administration for ten days (e.g., Days 1-10) and another continuous daily administration (e.g., Days 15-24) for ten days, or a continuous daily administration for four sets of five days (e.g., Days 1-5, 8-12, 15-19, and 22-26). In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered to the subject in need thereof on twenty-one days (e.g., Days 1-21) during a 28-day cycle. In some embodiments, for example when the patient is identified to have low tolerance to the PLK1 inhibitor (e.g., onvansertib), the PLK1 inhibitor is administered to the subject in need thereof on ten days (e.g., Days 1-5 and 15- 19) during a 28-day cycle. The ten days can be, for example, a continuous daily administration for ten days (e.g., Days 1-10) or two continuous daily admiration for five days each (e.g., Days 1-5 and Days 15-19). In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered to the subject in need thereof daily throughout the whole cycle (e.g., daily for 28 days in a cycle of 28 days). Depending on the needs of inhibition / reversion of cancer progression in the subject, the subject can receive one, two, three, four, five, six, or more cycles of treatment.

[0130] For combination treatment, the administration cycles, dosing schedules, and / or dosage amounts of the ADC and the PLK1 inhibitor can be the same or different. For combination treatment, the administration cycle, dosing schedule, and / or dosage amount of the ADC can be adjusted according to the administration cycle, dosing schedule, and / or dosage amount of the PLK1 inhibitor. For example, the ADC (e.g., T-DXd) can be administered two times in a 28-day cycles (e.g., on Days 1 and 22), which corresponds to a 28-day cycle for administration of the PLK1 inhibitor (e.g., onvansertib).

[0131] The treatment can comprise administration of the PLK1 inhibitor (e.g., onvansertib) at, or at about, 6 mg / m2- 90 mg / m2drug / body surface area, for example, as a daily dose. For example, the treatment can comprise daily administration of the PLK1 inhibitor (e.g., onvansertib) at, or at about, 6 mg / m2, 8 mg / m2, 10 mg / m2, 12 mg / m2, 14 mg / m2, 16 mg / m2, 18 mg / m2, 20 mg / m2, 23 mg / m2, 27 mg / m2, 30 mg / m2, 35 mg / m2, 40 mg / m2, 45 mg / m2, 50 mg / m2, 55 mg / m2, 60 mg / m2, 65 mg / m2, 70 mg / m2, 80 mg / m2, 85 mg / m2, 90 mg / m2, a number or a range between any two of these values, or any value between 8 mg / m2- 90 mg / m2. In some embodiments, the daily dose of the PLK1 inhibitor (e.g., onvansertib) can be adjusted (e.g., increased or decreased with the range) during the treatment, or during a single cycle (e.g., the first cycle, the second cycle, the third cycle, and a subsequent cycle) of the treatment, for the subject. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 12 mg / m2on twenty days (e.g., Days 1-10 and 15-24) during a 28-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 15 mg / m2on ten days (e.g., Days 1-5 and 15-19) during a28-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 8 mg / m2or 10 mg / m2everyday (e.g., Days 11-28) during a 28-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg / kg 5 days a week during a 18-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg / kg 5 days a week during a 32-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg / kg 5 days a week during a 39-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg / kg 5 days a week during a 45-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg / kg 5 days a week during a cycle (e.g., 30-day, 31-day, 32-day, 33-day, 34-day, 35-day, 36-day, 37-day, 38- day, 39-day or 40-day cycle) with no administration of the PLK inhibitor (e.g., onvansertib) for one week.

[0132] In some embodiments, the daily dose of the PLK1 inhibitor can be adjusted (e.g., increased or decreased with the range) during the treatment, or during a single cycle (e.g., the first cycle, the second cycle, the third cycle, and a subsequent cycle) of the treatment, for the subject.

[0133] A maximum concentration (Cmax) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject (during the treatment or after the treatment) when the PLK1 inhibitor is administered alone or in combination with the ADC (e.g., T-DXd) can be from about 100 nmol / L to about 1500 nmol / L. For example, the Cmax of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the MTA can be, or be about, 100 nmol / L, 200 nmol / L, 300 nmol / L, 400 nmol / L, 500 nmol / L, 600 nmol / L, 700 nmol / L, 800 nmol / L, 900 nmol / L, 1000 nmol / L, 1100 nmol / L, 1200 nmol / L, 1300 nmol / L, 1400 nmol / L, 1500 nmol / L, a range between any two of these values, or any value between 200 nmol / L to 1500 nmol / L.

[0134] An area under curve (AUC) of a plot of a concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject over time (e.g., AUC0-24 for the first 24 hours after administration) when the PLK1 inhibitor is administered alone or in combination with the ADC (e.g., T-DXd) can be from about 1000 nmol / L. hour to about 400000 nmol / L. hour. For example, the AUC of a plot of a concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject over time (e.g., AUC0-24 for the first 24 hours after administration) when the PLK1 inhibitor is administered alone or in combination with the ADC (e.g., T-DXd) can be, or be about, 1000 nmol / L. hour, 5000 nmol / L. hour, 10000 nmol / L. hour, 15000 nmol / L. hour, 20000 nmol / L. hour, 25000 nmol / L. hour, 30000 nmol / L. hour, 35000 nmol / L. hour, 40000 nmol / L. hour, a range between any two of these values, or any value between 1000 nmol / L. hour and 400000 nmol / L. hour.

[0135] A time (T max ) to reach a maximum concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the ADC (e.g., T-DXd) can be from about 1 hour to about 5 hours. For example, the time (Tmax) to reach a maximum concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the ADC (e.g., T-DXd) can be, or be about, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, a range between any two of these values, or any value between 1 hour and 5 hours.

[0136] An elimination half-life (T1 / 2) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the ADC (e.g., T-DXd) can be from about 10 hours to about 60 hours. For example, the elimination half-life (T1 / 2) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the ADC (e.g., T-DXd) can be, or be about, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, a range between any two of these values, or any value between 10 hours and 60 hours.

[0137] The PLK1 inhibitor can be administered to a breast cancer patient being treated with an ADC capable of targeting breast cancer (e.g., anti-HER2 ADC). Alternatively or in combination, the PLK1 inhibitor can be administered in combination with the ADC to a breast cancer patient.

[0138] In some embodiments, the ADC (e.g., T-DXd) is administered for 1 to 10 cycles, for example, 1 to 9 cycles, 1 to 8 cycles, 1 to 7 cycles, 1 to 6 cycles, 1 to 5 cycles, 1 to 4 cycles, 1 to 3 cycles, 1 to 2 cycles, or 1 cycle. Each cycle of treatment can have various lengths, for example, at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more. In some embodiments, the administration of the ADC can be daily or with break(s) between days of administrations. The break can be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, or more. The administration can be, for example, once every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every week, every 2 weeks, every three weeks, or more weeks. In some embodiments, the ADC (e.g., T-DXd) is administered intravenously.

[0139] In some embodiments, the ADC is T-DXd. T-DXd can be administered for 1 to 5 cycles, for example, 1 to 5 cycles, 1 to 4 cycles, 1 to 3 cycles, 1 to 2 cycles, or 1 cycle. Each cycle of treatment can have various lengths, for example, at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more.

[0140] In some embodiments, the administration of T-DXd can be once every week, once every two weeks, once every three weeks, or once every four weeks. The break between two consecutive administrations can be, for example, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or more. In some embodiments, T-DXd is administered once followed by a 20-day break without administration. For example, T-DXd can be administered on day 1 of a treatment cycle of 21 days. T-DXd can be administered on day 1 and day 22 of a treatment cycle of at least 42 days. T-DXd can be administered on day 1, day 22, and day 43 of a treatment cycle of at least 63 days. T-DXd can be administered on day 1, day 22, day 43, and day 64 of a treatment cycle of at least 84 days.

[0141] The ADC can be administered to the patient at any appropriate dosage in different embodiments. The ADC can be administered to the patient at a dosage of about, at least or at most 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 105 mg / kg, 110 mg / kg, 115 mg / kg, 125 mg / kg, 130 mg / kg, 135 mg / kg, 140 mg / kg, 145 mg / kg, 150 mg / kg, 155 mg / kg, 160 mg / kg, 165 mg / kg, 170 mg / kg, 175 mg / kg, 180 mg / kg, 185 mg / kg, 190 mg / kg, 195 mg / kg, 200 mg / kg or a number between any two of these values. In some embodiments, the ADC is administered to the patient at a dosage of 5-6 mg / kg, e.g., 5.4 mg / kg.

[0142] In some embodiments, the anti-HER2 ADC is T-DXd. T-DXd can be administered to the patient at a dosage of about, at least or at most 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg or a number between any two of these values. In some embodiments, T-DXd is administered at about 2 mg / kg to about 15 mg / kg of body weight, optionally at about 4 mg / kg to about 10 mg / kg (e.g., 5.4 mg / kg) of body weight. The daily dose of T-DXd can be adjusted (e.g., increased or decreased with the range) during the treatment, or during a single cycle (e.g., the first cycle, the second cycle, the third cycle, and a subsequent cycle) of the treatment, for the subject. In some embodiments, T-DXd is administered at about 4 mg / kg or 5.4 mg / kg once every three weeks during a 21-day cycle. In some embodiments, T-DXd is administered at about 4 mg / kg or 5.4 mg / kg once every three weeks during a 42-day cycle. In some embodiments, T-DXd is administered at about 4 mg / kg once every three weeks during a 63- day cycle. In some embodiments, T-DXd is administered at about 4 mg / kg once every three weeks during an 84-day cycle. The anti-HER.2 ADC (e.g., T-DXd) can be administered to the patient via, e.g., IV.

[0143] The pharmacokinetics of T-DXd can depend on patient-related factors. In some embodiments, a maximum concentration (Cmax) of T-DXd in a blood of the subject (during thetreatment or after the treatment) when T-DXd is administered alone or in combination with a PLK1 inhibitor can be from about 100 pg / mL to about 200 pg / mL. The Cmax of DXd in a blood of the subject (during the treatment or after the treatment) when T-DXd is administered alone or in combination with a PLK1 inhibitor can be from about 5 ng / mL to 15 ng / mL. An area under curve (AUC) of a plot of a concentration of T-DXd in a blood of the subject over time (e.g., AUCO-24 for the first 24 hours after administration) when T-DXd is administered alone or in combination with the PLK1 inhibitor can be from about 500 ug.day / mL to about 1000 ug.day / mL. For example, the AUC of a plot of a concentration of T-DXd in a blood of the subject over time when T-DXd is administered alone or in combination with the PLK1 inhibitor can be about 700- 800 ug.day / mL. The AUC of a plot of a concentration of DXd in a blood of the subject over time when T-DXd is administered alone or in combination with the PLK1 inhibitor can be from about 20 ng.day / mL to about 50 ng.day / mL. An elimination half-life (T1 / 2) of T-DXd in a blood of the subject when T-DXd is administered alone or in combination with the PLK1 inhibitor can be from about 5 days to 10 days. A time (Tmax) to reach a maximum concentration of T-DXd in a blood of the subject when T-DXd is administered alone or in combination with the PLK1 inhibitor can be from about 1 hours to 10 hours. For example, the time (Tmax) to reach a maximum concentration of T-DXd in a blood of the subject when T-DXd is administered alone or in combination with the PLK1 inhibitor can be, or be about, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, a range between any two of these values. The Tmax of DXd in a blood of the subject when T-DXd is administered alone or in combination with the PLK1 inhibitor can be about 6-15 hours, for example, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours or 15 hours.Additional Cancer Therapeutics or Therapy

[0144] Methods, compositions and kits disclosed herein can be used for treating cancer, for example breast cancer (e.g., HR+ breast cancer, HER2-negative, -low or -ultralow breast cancer, or TNBC). In some embodiments, a method for treating breast cancer comprises administrating a PLK1 inhibitor (e.g., onvansertib) in combination with an ADC capable of targeting breast cancer cells (e.g., anti-HER2 ADC such as T-DXd) to a subject (e.g., a patient) in need thereof. The method can comprise administering a therapeutically effective amount of the ADC capable of targeting breast cancer cells (e.g., T-DXd) and a therapeutically effective amount of a PLK1 inhibitor. The treatment can comprise administration of at least one additional cancer therapeutics or cancer therapy. The treatment can comprise administration a therapeutically effective amount of at least one additional cancer therapeutics or cancer therapy. The ADC (e.g., T-DXd) and the additional cancer therapeutics or cancer therapy can, for example, co-administered simultaneously or sequentially. The PLK1 inhibitor and the additional cancer therapeutics or cancer therapy can, for example, co-administered simultaneously or sequentially. Additional cancer therapeutics or therapies in treating breast cancer (e.g., HR+ breast cancer, HER2 negative, low or ultralow breast cancer, TNBC) are identifiable to a person skilled in the art. Exemplary additional cancer therapeutics or therapies in treating breast cancer include, but are not limited to, surgery, chemotherapy, targeted therapy, immunotherapy, radiation therapy, hormone therapy, and neoadjuvant systemic therapy.Methods for Predicting / Determining Treatment Efficacy and Status for Cancer

[0145] Also disclosed herein include methods, compositions, kits, and systems for predicting / determining clinical outcome for a combination treatment of cancer of the present disclosure, monitoring of the combination treatment, predicting / determining responsiveness of a subject to the combination treatment, determining the status of the cancer in a subject, and improving combination treatment outcome. The methods, compositions, kits and systems can be used to guide the combination treatment, provide combination treatment recommendations, reduce or avoid unnecessary ineffective combination treatment for patients. ctDNA can be analyzed to predict / determine clinical outcome for cancer treatment using a combination of ADC (e.g., T- DXd) and a PLK1 inhibitor of the present disclosure, monitor the combination treatment, predict / determine responsiveness of a subject to the combination treatment, determine cancer status in a subject, improve combination treatment outcome, guide combination treatment, provide combination treatment recommendations, and / or to reduce or avoid ineffective combination treatment. ctDNA can be analyzed to predict / determine clinical outcome for cancer treatment, monitor cancer treatment, predict / determine responsiveness of a subject to a cancer treatment, determine cancer status in a subject, improve cancer treatment outcome, guide cancer treatment, provide treatment recommendations, and / or to reduce or avoid ineffective cancer treatment. Such analysis of ctDNA has been described in WO2021146322, the content of which is incorporated herein by reference in its entirety.

[0146] A method of determining responsiveness of a subject to a combination treatment comprising an ADC capable of targeting breast cancer (e.g., T-DXd) and a PLK1 inhibitor of the disclosure can comprise, for example, analyzing circulating tumor DNA (ctDNA) of a subject with cancer, the subject is undergoing a treatment and / or has received the combination treatment, thereby determining the responsiveness of the subject to the combination treatment. In some embodiments, determining the responsiveness of the subject comprises determining if the subject is a responder of the treatment, if the subject is or is going to be in CR, or if the subject is or is going to be in partial remission (PR). For example, analyzing ctDNA can comprise detectingvariant allele frequency in the ctDNA in a first sample obtained from the subject at a first time point, detecting variant allele frequency in the ctDNA obtained from the subject at one or more additional time points in one or more additional samples, and determining the difference of the variant allele frequency in ctDNA between the first and at least one of the one or more additional samples, a decrease in the variant allele frequency in at least one of the additional samples relative to the first sample indicates the subject as responsive to the cancer treatment.

[0147] In some embodiments, the first time point is prior to or immediately prior to the combination treatment, and at least one of the one or more additional time points are at the end of or after at least a cycle of the combination treatment. In some embodiments, the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment.

[0148] In some embodiments, the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment. In some embodiments, the method comprises continuing the combination treatment to the subject if the subject is indicated as responsive to the combination treatment. In some embodiments, the method comprises discontinuing the combination treatment to the subject and / or starting a different combination treatment to the subject if the subject is not indicated as responsive to the combination treatment.

[0149] Disclosed herein include methods of determining cancer status of a subject, comprising analyzing ctDNA of a subject, thereby determining cancer status of the subject. The subject can be a subject undergoing a current combination treatment of the present disclosure, a subject that has received a prior combination treatment of the present disclosure, and / or a subject that is in remission for the cancer. The subj ect in remission for cancer can be in complete remission (CR), or in partial remission (PR).

[0150] In some embodiments, analyzing the ctDNA comprises detecting variant allele frequency in the ctDNA. In some embodiments, analyzing the ctDNA comprises detecting variant allele frequency in the ctDNA obtained from the subject at a first time point in a first sample, detecting variant allele frequency in the ctDNA obtained from the subject at one or more additional time points in one or more additional samples, and determining the difference of the variant allele frequency in ctDNA between the first and at least one of the one or more additional samples, an increase in the variant allele frequency at the additional sample(s) relative to the first sample indicates that the subject is at risk of cancer relapse or is in cancer relapse.

[0151] In some embodiments, the first time point is prior or immediately prior to the combination treatment, and the one or more additional time points are at the end of or after at leasta cycle of the combination treatment, optionally the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment, optionally the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment.

[0152] In some embodiments, the method comprises starting an additional treatment to the subject if the subject is indicated as in cancer relapse. The additional treatment can be the same or different from the current or prior combination treatment.

[0153] The variant allele frequency in ctDNA can be determined, for example, by total mutation count in the ctDNA in each of the first sample and one or more additional samples, or by the mean variant allele frequency in each of the first sample and one or more additional samples. In some embodiments, the variant allele frequency is mutant allelic frequency (MAF) for a driver mutation of the cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, the variant allele frequency is MAF for one or more driver mutations of the cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, Log2(Ci / Co) < a MAF threshold indicates a decrease in ctDNA MAF Co is ctDNA MAF in the first sample and Ci is ctDNA MAF in one of the additional samples. In some embodiments, the MAF threshold is, or is about, 0.01 to -0.10. In some embodiments, the MAF threshold is, or is about, 0.06. In some embodiments, the MAF threshold is, or is about, 0.05.

[0154] In some embodiments, the first sample comprises ctDNA from the subject before treatment, and the one of additional samples comprises ctDNA from the subject after treatment. In some embodiments, the driver mutation is a mutation in one of the below 75 genes ABL1, ANKRD26, ASXL1, ATRX, BCOR, BCORL1, BRAF, BTK, CALR, CBL, CBLB, CBLC, CCND2, CDC25C, CDKN2A, CEBPA, CSF3R, CUX1, CXCR4, DCK, DDX41, DHX15, DNMT3A, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC7L2, MAP2K1, MPL, MYC, MYD88, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PPM1D, PTEN, PTPN11, RAD21, RBBP6, RPS14, RUNX1, SETBP1, SF3B1, SH2B3, SLC29A1, SMC1A, SMC3, SRSF2, STAG2, STAT3, TET2, TP53, U2AF1, U2AF2, WT1, XPO1, and ZRSR2. In some embodiments, at least one of the one or more the driver mutations is a mutation in in the 75 genes. In some embodiments, one or more the driver mutations are mutations in the 75 genes.

[0155] The driver mutation or at least one of the one or more driver mutations can be in a gene selected from the group consisting of TP53, ASXL1, DNMT3A, NRAS, SRSF2, TET2,SF3B1, FLT3, FLT3 ITD, IDH2, NPM1, RUNX1, CDKN2A, KRAS, STAG2, CALR, CBL, CSF3R, DDX41, GATA2, JAK2, PHF6, and SETBP1. In some embodiments, the driver mutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of DNMT3A, TET2, NPM1, SRSF2, NRAS, CDKN2A, SF3B1, FLT3, ASXL1, SRSF2, IDH2, NRAS, and SF3B1. In some embodiments, the method further comprises determining variant allele frequency in one or more of the ctDNA, PBMCs and BMMCs of the subject.

[0156] The ctDNA can be analyzed using, for example, polymerase chain reaction (PCR), next generation sequencing (NGS), and / or droplet digital PCR (ddPCR). The sample disclosed herein can be derived from, for example, whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof. In some embodiments, the ctDNA is from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof.

[0157] In some embodiments, the method comprises analyzing ctDNA of the subject before the treatment. In some embodiments, the treatment comprises one or more cycles, and the ctDNA is analyzed before, during and after each cycle of the treatment. Each cycle of treatment can be at least 21 days. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, the subject is human.

[0158] Disclosed herein include methods of improving treatment outcome for the cancer. The method can comprise: detecting variant allele frequency in circulating tumor DNA (ctDNA) obtained from a subject at a first time point in a first sample before the subject undergoes a combination treatment of the present disclosure; detecting variant allele frequency in ctDNA obtained from the subject at one or more additional time points in one or more additional samples after the subject undergoes the combination treatment; determining the difference of the variant allele frequency in ctDNA between the first and at least one of the one or more additional samples, a decrease in the variant allele frequency in at least one of the additional samples relative to the first sample indicates the subject as responsive to the combination treatment; and continuing the combination treatment to the subject if the subject is indicated as responsive to the combination treatment, or discontinuing the combination treatment to the subject and / or starting a different cancer treatment to the subject if the subject is not indicated as responsive to the combination treatment.

[0159] Also disclosed herein include methods of treating breast cancer (e.g., HR+ and / or HER2- / low / ultralow, or TNBC). The method can comprise: administering a combination treatment comprising ADC (e.g., T-DXd) and a PLK1 inhibitor to a subject in need thereof; determining a decrease, relative to a variant allele frequency in a first sample of the subject obtained at a first time point before the subject receives the combination treatment, in a variant allele frequency in a second sample of the subject obtained at a second time point after the subjectreceives the combination treatment; and continuing with the combination treatment. In some embodiments, the subject is a subject newly diagnosed with cancer, for example a subject that has not received any prior cancer treatment before the combination treatment. In some embodiments, the subject has received prior cancer treatment and was in remission for the cancer, for example a subject in complete remission (CR), or in partial remission (PR) after receiving the prior combination treatment.

[0160] The first time point can be, for example, prior or immediately prior to the combination treatment. The at least one of the one or more additional time points can be, for example, at the end of or after at least a cycle of the combination treatment. In some embodiments, the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment. In some embodiments, the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment.

[0161] The variant allele frequency in ctDNA can be determined, for example, by total mutation count in the ctDNA in each of the first sample and one or more additional samples, and / or by the mean variant allele frequency in each of the first sample and one or more additional samples. In some embodiments, the variant allele frequency is mutant allelic frequency (MAF) for a driver mutation of the cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, the variant allele frequency is mutant allelic frequency (MAF) for one or more driver mutations of the cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, Log2(Ci / Co) < a MAF threshold indicates a decrease in ctDNA MAF Co is ctDNA MAF in the first sample and Ci is ctDNA MAF in one of the additional samples. In some embodiments, the MAF threshold is -0.05.

[0162] The driver mutation can be, for example, a mutation in one of the 75 genes set forth in Table 3, at least one of the one or more the driver mutations is a mutation in one of the below 75 genes ABL1, ANKRD26, ASXL1, ATRX, BCOR, BCORL1, BRAF, BTK, CALR, CBL, CBLB, CBLC, CCND2, CDC25C, CDKN2A, CEBPA, CSF3R, CUX1, CXCR4, DCK, DDX41, DHX15, DNMT3A, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC7L2, MAP2K1, MPL, MYC, MYD88, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PPM1D, PTEN, PTPN11, RAD21, RBBP6, RPS14, RUNX1, SETBP1, SF3B1, SH2B3, SLC29A1, SMC1A, SMC3, SRSF2, STAG2, STAT3, TET2, TP53, U2AF1, U2AF2, WT1, XPO1, and ZRSR2, and / or one or more the driver mutations are mutations in the 75 genes. In some embodiments, the drivermutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of TP53, ASXL1, DNMT3A, NRAS, SRSF2, TET2, SF3B1, FLT3, FLT3 ITD, IDH2, NPM1, RUNX1, CDKN2A, KRAS, STAG2, CALR, CBL, CSF3R, DDX41, GATA2, JAK2, PHF6, and SETBP1. In some embodiments, the driver mutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of DNMT3 A, TET2, NPM1, SRSF2, NRAS, CDKN2A, SF3B1, FLT3, ASXL1, SRSF2, IDH2, NRAS, and SF3B1.

[0163] In some embodiments, the method further comprises determining variant allele frequency in one or more of the ctDNA, PBMCs and BMMCs of the subject. The variant allele frequency in ctDNA can be detected, for example, using polymerase chain reaction (PCR) or next generation sequencing (NGS). In some embodiments, the variant allele frequency in ctDNA is detected using droplet digital PCR (ddPCR).

[0164] At least one of the first sample, the one or more additional samples, and the second sample can be derived from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof. In some embodiments, the ctDNA is from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof.

[0165] In some embodiments, the subject whose ctDNA is analyzed is undergoing or will be undergoing treatment for the cancer. The method can comprise analyzing ctDNA of the subject before the treatment. The treatment can comprise one or more cycles, and the ctDNA is analyzed before, during and after one or more cycles of the treatment. For example, the ctDNA can be analyzed before, during and after two or more cycle of the treatment, three or more cycle of the treatment, or each cycle of the treatment. Each cycle of treatment can be at least 21 days, for example, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more, or a range between any two of these values. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, each cycle of treatment is from 21 days to 28 days. In some embodiments, the subject is human.Compositions and Kits

[0166] Disclosed herein include compositions and kits for treating breast cancer (e.g., HR+ and / or HER2 -negative, -low or -ultralow breast cancer, or TNBC). In some embodiments, a kit comprises: a PLK1 inhibitor, and a manual providing instructions for co-administering the PLK1 inhibitor in combination with an antibody-drug conjugate (ADC) capable of targeting breast cancer cells (e.g., T-DXd) to a subject in need thereof for treating breast cancer (e.g., HR+ and / or HER2-negative, -low or -ultralow breast cancer, or TNBC). In some embodiments, the PLK1 inhibitor is onvansertib. In some embodiments, the kit further comprises the ADC described herein. In some embodiments, the ADC comprises an anti-HER2 antibody linked to atopoisomerase inhibitor (e.g., T-DXd). In some embodiments, the ADC comprises an anti-TROP2 antibody linked to a topoisomerase inhibitor (e.g., Dato-DXd).

[0167] In some embodiments, the instructions comprise instructions for coadministrating the ADC and the PLK1 inhibitor simultaneously. In some embodiments, the instructions comprise instructions for co-administrating the ADC and the PLK1 inhibitor sequentially. In some embodiments, the instructions comprise instructions for administering the ADC intravenously. In some embodiments, the instructions comprise instructions for administrating the PLK1 inhibitor orally. In some embodiments, the instructions comprise instructions for administering the ADC intravenously and the PLK1 inhibitor orally.

[0168] In some embodiments, the instructions comprise instructions for subjects who have received a prior treatment with the ADC (e.g., T-DXd). In some embodiments, the instructions comprise instructions for subjects who did not respond to treatment with the ADC alone. In some embodiments, the instructions comprise instructions for subjects who are known to be resistant to the ADC treatment.

[0169] In some embodiments, the instructions comprise instructions for subjects who have received a hormone therapy treatment. In some embodiments, the instructions comprise instructions for subjects who did not respond to treatment with the hormone therapy. In some embodiments, the instructions comprise instructions for subjects who are known to be resistant to a hormone therapy. In some embodiments, the hormone therapy comprises using one or more of selective estrogen receptor modulators or SERMs (e.g., tamoxifen, toremifene), aromatase inhibitors (e.g., anastrozole), or selective estrogen receptor degraders or SERDs (e.g., fulvestrant).

[0170] In some embodiments, the instructions comprise instruction for subjects who have received a kinase inhibitor treatment such as PI3K inhibitor and / or CDK4 / 6 inhibitor treatment. In some embodiments, the instructions comprise instructions for treating subjects who did not respond to the PI3K inhibitor and / or CDK4 / 6 inhibitor treatment. In some embodiments, the instructions comprise instructions for subjects who are known to be resistant to a PI3K inhibitor and / or CDK4 / 6 inhibitor. In some embodiments, the CDK4 / 6 inhibitor comprises palbociclib and abemaciclib, and PI3K inhibitor comprises alpelisib and idelalisib. In some embodiments, the instructions comprise instructions for subjects who are known to be resistant to both kinse inhibitor and hormone therapy treatments.

[0171] In some embodiments, the instructions comprise instructions for subjects who have received at least one prior treatment for the cancer. In some embodiments, the prior treatment does not comprise the use of an ADC, a PLK inhibitor, or both. In some embodiments, the instructions comprise instructions the subject was in remission for the cancer. In some embodiments, the subject in remission for cancer was in complete remission (CR), or in partialremission (PR).

[0172] The instructions can comprise instructions for administering each of the ADC and the PLK1 inhibitor to the subject in a treatment cycle. In some embodiments, the instructions comprise instructions for administering the ADC to the subject in a cycle of at least once every three weeks. In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor to the subject in a cycle of at least five times within a week. In some embodiments, the instructions comprise administering the ADC and the PLK1 inhibitor, or both in a cycle of at least 7, 14, 21, 28, 35, 42, or 49 days. In some embodiments, each cycle of treatment is at least about 14 days to about 21 days. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, each cycle of treatment is from about 28 days to about 35 days. In some embodiments, each cycle of treatment is from about 35 days to about 42 days. In some embodiments, each cycle of treatment is from about 42 days to about 49 days. In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor on at least four days in the cycle. In some embodiments, the instructions comprise instructions for not administering the PLK1 inhibitor on at least one day in the cycle. In some embodiments, the instructions comprise instructions for administrating the ADC once a week, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the instructions comprise instructions for administrating the ADC and the PLK1 inhibitor for at least two cycles.

[0173] In some embodiments, the PLK1 inhibitor can be selective and / or specific for PLK1. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, a aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5- f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a stilbene derivative, or any combination thereof. In some embodiments, the PLK1 inhibitor is onvansertib, BI2536, Volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960 or Ro3280. In some embodiments, the PLK1 inhibitor is onvansertib.

[0174] In some embodiments, the ADC comprises an anti-HER2 antibody or an anti- TROP2 antibody linked to a DNA damaging agent. The DNA damaging agent can be selected from the group consisting of radiomimetic neocarzinostatin, a platinating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an antimetabolite, an alkylating agent, an antibiotic, and a combination thereof. In some embodiments, the ADC comprises an anti-HER2 antibody or an anti-TROP2 antibody linked to a chemotherapy drug. In some embodiments, the ADC comprises an anti-HER2 antibody or an anti-TROP2 antibody linked to a topoisomerase inhibitor (e.g., topo I inhibitor or topo II inhibitor). In some embodiments, the topoisomerase inhibitor is selected from a group consisting of Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone, Teniposide, andTopotecan, Camptothecin, Rubitecan, Belotecan, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin, and a combination thereof. In some embodiments, the ADC comprises deruxtecan conjugated to an anti-HER2 antibody or an anti-TROP2 antibody. In some embodiments, the ADC is T-DXd or Dato-DXd.

[0175] In some embodiments, the instructions comprises dosing guidelines for administering the ADC (e.g., T-DXd) and the PLK1 inhibitor (e.g., onvansertib). In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor at 8 mg / m2- 90 mg / m2. In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor (e.g., onvansertib) at a dose from about 10 mg / kg of body weight to about 80 mg / kg of body weight, optionally at a dose from about 20 mg / kg of body weight to about 60 mg / kg of body weight, optionally at a dose from about 30 mg / kg of body weight to about 50 mg / kg of body weight. In some embodiments, the instructions comprise instructions for administering the ADC at a dose from about 1 mg / kg of body weight to about 20 mg / kg of body weight, optionally at a dose from about 2 mg / kg of body weight to about 15 mg / kg of body weight, optionally at a dose from about 4 mg / kg of body weight to about 10 mg / kg of body weight.EXAMPLES

[0176] Some aspects of the embodiments discussed above are disclosed in further detail in the following example, which are not in any way intended to limit the scope of the present disclosure.Example 1 Onvansertib and T-DXd combination induces robust anti -turn or activity in various breast cancer PDX models

[0177] The breast cancer PDX models used in the example are HER2-low breast cancer PDX models with HER2 status being negative or low. FIG. 1 provides the characteristics of exemplary HER2-low breast cancer PDX models. AC denotes Adriamycin + Cyclophosphamide, Al denotes Aromatase Inhibitor, and FEC denotes 5-FU epirubicine cyclophosphamide. IHC staining of HER2 was performed using the HercepTest. BC1732, HBCx- 246, HBCx-139palbo+fulvR5, HBCx-3, HBCx-134palboR31, and vBC1220OvaRPalboR were treated with vehicle (Ctrl), onvansertib alone, T-DXd alone, or a combination of onvansertib and T-DXd. Tumor volume, percentage of tumor volume change and event-free survival rate were monitored for up to 90 days.Onvansertib and T-DXd combination in a HR+ breast cancer PDX model resistant to abemaciclib and fulvestrant

[0178] HBCx-246 PDX model scored HER2-low in HER2 IHC and resistant to abemaciclib and fulvestrant was treated with vehicle (Ctrl), onvansertib alone (Onv; oral,45mg / kg, 5 times / week for 29 days), T-DXd alone (IV, 4 mg / kg, on days 1, 22, 43 and 64, indicated by black arrows) or a combination of onvansertib and T-DXd (O+T; T-DXd administrated on days 1, 22, 43 and 64 and onvansertib 5 times / week for up to 82 days). Tumor volumes (TV) were measured twice a week. Percentage tumor volume change was calculated as (TV on measured day- TV on day 0) / (TV on day 0)*100. Tumor growth inhibition on day 22 was calculated as 100% x (TVcontrol-TVtreated) / TVcontrol). Statistical differences in tumor volume change at Day 29 was assessed using the Student’ s t-test. Event-free survival (EFS) was analyzed using Kaplan-Meier curves, and comparisons were performed using the log-rank (Mantel-Cox) test.

[0179] FIG. 2 are plots showing an immunohistochemistry (IHC) image of HER2 staining (panel A), tumor volume overtime (panel B), tumor volume change at Day 29 (panel C), and event-free survival rate (panel D) in HBCx-246 PDX model treated with vehicle (Ctrl), onvansertib, T-DXd, or onvansertib + T-DXd (O+T). Panel (A) IHC image of HER2 staining using HER2 IHC Dako HercepTest, showing HBCx-246 is HER2-low. Panel (B) Mean ± SEM of tumor volumes overtime. Panel (C) Percentage tumor volume change at Day 29. Unpaired t- test was used to compare tumor volume change between O+T and T-DXd. Panel (D) Kaplan- Meier analysis of event-free survival (EFS, time for RTV = 10). Log-rank Mantel-Cox test was employed for survival analyses. Significance levels are indicated as **** p < 0.0001.

[0180] 89% tumor growth inhibition was observed in the combination group versus54% in T-DXd monotherapy group and 75% in onvansertib group. The combination induced robust and durable efficacy, resulting in significant tumor growth inhibition and increased event- free survival compared to monotherapies. The data suggests that onvansertib and T-DXd combination can induce robust and durable anti-tumor activity in a HR+, HER2-low breast cancer PDX model resistant to abemaciclib and fulvestrant.Onvansertib and T-DXd combination in a HR+ breast cancer PDX model resistant to palbociclib and fulvestrant

[0181] HBCx-139palbo+fulvR5 PDX model that is HR+ / HER2 negative and resistant to palbocilcib and fulvestrant was treated with vehicle (Ctrl), Onv alone (oral, 45mg / kg, 5 times / week for 39 days), T-DXd alone (IV, 10 mg / kg, on days 1 and 22, as indicated by black arrows) or a combination of both (O+T, same dosing schedule as monotherapies). Tumor volumes (TV) were measured twice a week. Percentage tumor volume change was calculated as (TV on measured day- TV on day 0) / (TV on day 0)*100. Tumor growth inhibition on day 39 was calculated as 100% x (TVcontrol - TVtreated) / TVcontrol). Statistical differences in tumor volume change at Day 39 was assessed using the Student’ s t-test. Event-free survival (EFS) was analyzed using Kaplan-Meier curves, and comparisons were performed using the log-rank (Mantel-Cox)

[0182] FIG. 3 are plots showing an IHC image of HER2 staining (panel A), tumor volume overtime (panel B), tumor volume change (panel C), and event-free survival rate (panel D) in HBCx-139palbo+fulvR5 PDX model treated with vehicle (Ctrl), onvansertib, T-DXd, or O+T. Panel (A) IHC image of HER2 staining using HER2 IHC Dako HercepTest, showing HBCx- 139palbo+fulvR5 is HER2-negative. (B) Mean± SEM oftumor volumes overtime. (C) Percentage tumor volume change on Day 39. Unpaired t-test was used to compare tumor volume changes between O+T and T-DXd. (D) Kaplan-Meier analysis of EFS (time for RTV = 4). Log-rank Mantel-Cox test was employed for survival analyses. Significance levels are indicated as ** p < 0.01, ***p < 0.001.

[0183] 88% tumor growth inhibition was observed in the combination group versus69% in T-DXd monotherapy group and 54% in onvansertib monotherapy group. The combination exhibited increased tumor growth inhibition and delayed tumor progression compared to monotherapies. The data suggests that onvansertib and T-DXd combination can exhibit robust anti-tumor activity and delays tumor progression in a HR+, HER2 negative breast cancer PDX model resistant to palbociclib and fulvestrant.Onvansertib and T-DXd combination in a HR+ breast cancer PDX model resistant to palbociclib and fulvestrant

[0184] HBCx-3 PDX model that is HR+ / HER2 low and resistant to palbociclib and fulvestrant was treated with vehicle (Ctrl), Onv alone (oral, 45mg / kg, 5 times / week for 43 days), T-DXd alone (IV, 4 mg / kg, on days 1 and 22, indicated by black arrows) or a combination of both (O+T, same dosing schedule as monotherapies). Tumor volumes (TV) were measured twice a week. Percentage tumor volume change (TVC) was calculated as (TV on measured day- TV on day 0) / (TV on day 0)*100. Tumor regression is reported if TVC < -50% in at least one tumor measurement and a complete response is indicated when tumor volume is less than 10 mm3in at least one measurement. Statistical differences in tumor volume change at Day 43 was assessed using the Student’ s t-test.

[0185] FIG. 4 are plots showing an IHC image of HER2 staining (panel A), tumor volume overtime (panel B), and tumor volume change (panel C) in HBCx-3 PDX model treated with onvansertib, T-DXd, or both. Panel (A) IHC image of HER2 staining using HER2 IHC Dako HercepTest, showing HBCx-3 is HER2-low. (B) Mean ± SEM of tumor volumes overtime. (C) Percentage tumor volume change on Day 43. Unpaired t-test was used to compare tumor volume changes between O+T and T-DXd. Significance levels are indicated as ****p < 0.0001.

[0186] All mice (8 / 8) in the combination group showed tumor regression while none was observed in the monotherapy groups. Additionally, complete response was observed in 2 outof 8 mice in the combination group versus none in the monotherapy groups. Overall, the combination exhibited a more robust anti -tumor activity in the HR+ HER2 low breast cancer PDX model resistant to palbociclib and fulvestrant compared to single agents.Onvansertib and T-DXd combination in a HR+ breast cancer PDX model resistant to palbociclib and alpelisib + fulvestrant

[0187] HBCx-134palboR31 PDX model that is HR+ / HER2 low and resistant to alpelisib and fulvestrant was treated with vehicle (Ctrl), Onv alone (oral, 45mg / kg, 5 times / week for 43 days), T-DXd alone (IV, 4 mg / kg, on Day 1, indicated by black arrow) or a combination of both (O+T, same dosing schedule as monotherapies). Tumor volumes (TV) were measured twice a week. Percentage tumor volume change (TVC) was calculated as (TV on measured day- TV on day 0) / (TV on day 0)*100. Tumor regression is reported if TVC < -50% in at least one tumor measurement and a complete response is indicated when tumor volume is less than 10 mm3in at least one measurement. Statistical differences in tumor volume change at Day 43 was assessed using the Student’ s t-test.

[0188] FIG. 5 are plots showing an H4C image of HER2 staining (panel A), tumor volume overtime (panel B), and tumor volume change on Day 43 (panel C) in HBCx- 134palboR31 PDX model treated with onvansertib, T-DXd, or both. Panel (A) IHC image of HER2 staining using HER2 IHC Dako HercepTest, showing HBCx-134palboR31 is HER2-low. Panel (B) Mean ± SEM of tumor volumes overtime. Panel (C) Percentage tumor volume change on Day 43. Unpaired t-test was used to compare tumor volume changes between O+T and T-DXd. Significance levels are indicated as nsmon-significant.

[0189] All mice (7 / 7) in the combination group showed tumor regression, compared to 43% and 71% for monotherapy groups. Complete response was observed in 5 out of 7 mice (71%) in the combination group versus 28% and 57% in the two monotherapy groups. The data indicates that the combination exhibits increased anti-tumor activity in the HR+ HER2 low breast cancer PDX model resistant to alpelisib and fulvestrant compared to single agents.Onvansertib and T-DXd combination in a HR+ breast cancer PDX model vBC 1220QvaRPalboR

[0190] vBC1220OvaRPalboR PDX model is resistant to palbociclib. The mice were treated with vehicle (Ctrl), Onv alone (oral, 45 mg / kg, 5 times / week for 32 days), T-DXd alone (IV, 4 mg / kg, on days 1, 22 and 43) or a combination of both (O+T, T-DXd was administrated on days 1, 22 and 43, and onvansertib 5 times / week for 54 days). Tumor volumes (TV) were measured twice a week. Percentage tumor volume change (TVC) was calculated as (TV on measured day- TV on day 0) / (TV on day 0)*100. Tumor regression is reported if TVC < -50% in at least one tumor measurement and a complete response is indicated when tumor volume is less than 10 mm3in at least one measurement.

[0191] FIG. 14 depicts data showing tumor volume overtime (panel A), percentage of tumor volume change on Day 50 (panel B), and tumor regression and complete response rates (panel C) in vBC1220OvaRPalboR PDX model treated with onvansertib, T-DXd, or both. Tumor regression was defined as tumor volume change < -50% at any timepoint. Complete response was reported if tumor volume was < 10 mm3in at least one measurement. (A) Mean ± SEM of tumor volumes overtime. (B) Percentage tumor volume change on Day 50. Unpaired t-test was used to compare tumor volumes between O+T and T-DXd. Significance levels are indicated as **p < 0.01.

[0192] Six of the 8 mice (75%) in the combination group showed tumor regression compared to none in the onvansertib group and 4 out of 8 (50%) in the T-DXd group. Additionally, complete response was observed in 5 out of 8 mice (62%) in the combination group versus none in the monotherapy groups. Overall, the combination exhibited a more robust anti-tumor activity in the HR+ HER2 low breast cancer PDX model resistant to palbociclib compared to single agents. Onvansertib and T-DXd combination in a triple negative breast cancer PDX model BC1732

[0193] BC1732 TNBC PDX model was treated with onvansertib (Onv, oral, 45 mg / kg,5 times / week for 47 Days), T-DXd (IV, 4 mg / kg, Days 1, 22, 43) or the combination (O+T, same dosing schedule as monotherapies). FIG. 12 is a flowchart showing the patient treatment history of the BC1732 TNBC PDX model. FIG. 13 depicts data showing the tumor volume overtime (panel A), tumor volume change on Day 46 (panel B), and tumor regression and complete response rates (panel C). Tumor regression is reported if TVC < -50% in at least one tumor measurement and a complete response is indicated when tumor volume is less than 10 mm3in at least one measurement. Statistical differences in tumor volume change at Day 46 was assessed using the Student’ s t-test. Significance levels are indicated as *** / ? < 0.001. As suggested by the data, while onvansertib and T-DXd monotherapies resulted in tumor stasis, the combination induced more profound response with tumor regression observed in all mice and complete responses in 40% of mice.In vivo tolerance evaluation

[0194] Following the treatments, body weights of the treated mice were measured. FIG. 6 are plots showing the percentage change in body weight (BW) of mice treated with vehicle (Ctrl), Onv, T-DXd or a combination of both (O+T) as indicated in FIGs. 2-5. Panels: (A) HBCx- 246 (B) HBCx-139palbo+fulvR5 (C) HBCx-3 and (D) HBCx-134palboR31 PDX models. The data suggests that the onvansertib and T-DXd combination is well tolerated in vivo.

[0195] Collectively, the results demonstrate that the onvansertib and T-DXd combination enhanced antitumor activity compared with either onvansertib or T-DXd monotherapy, with significant deeper tumor inhibition and longer event-free survival in therapy-resistant, HER2-low breast cancer PDX models, including HR+ breast cancer and TNBC models. The combination overcame T-DXd resistance and delayed tumor progression in an abemaciclib- and ET-resistant HER2-low HR+ breast cancer PDX model, displayed enhanced anti-tumor activity compared to single agents across the 3 ET- and palbociclib-resistant HER2-low HR+ breast cancer PDX models, and displayed robust anti-tumor activity in an alpelisib-resistant HER2-low HR+ breast cancer PDX model. Therefore, combining onvansertib with T-DXd demonstrates an enhanced or synergistic efficacy in its anti-tumor activity, and can represent a promising therapeutic strategy for HER2-low breast cancer patients resistant to first-line therapies.Example 2Onvansertib and T-DXd combination in breast cancer cell lines

[0196] HER2 expression was assessed in selected triple negative breast cancer (TNBC) and HR+ HER2-low breast cancer cell lines by flow cytometry using an anti-HER2 antibody. FIG. 7 are plots showing the HER2 expression levels in selected breast cancer cell lines. Table 1 below provides the HER2 expression data for each selected breast cancer cell line. SK- BR-3 cell line is a HER2+ breast cancer cell line and was used as a positive control. All TNBC and HR+ BC cell lines tested showed low HER2 expression, including the palbociclib-resistant (MCF7 / PalboR) and fulvestrant-resistant (MCF7 / FulvR) cell lines.

[0197] The effect of onvansertib and T-DXd on cell viability was tested in 3 HER2- low TNBC cell lines (HCC1187, BT-20, and HCC38). FIG. 8 are plots showing the dose matrix (9x9) evaluation of T-DXd and onvansertib combination in these HER2-low TNBC cell lines. Cell viability was assessed using the CellTiter-Glo® assay after 6 days of treatment at the indicated concentrations. Synergy was determined using the Bliss synergy model and synergistic interactions are indicated in blue on the heatmap (n=3). The results suggest that the combination was synergistic in the 3 cell lines, as shown by high Bliss synergy scores.

[0198] The effect of onvansertib and T-DXd on cell viability was also tested in 5 HER2-low HR+ breast cancer cell lines (ZR-75-1, EFM-19, MCF7), including palbociclib- resistant (MCF7 / PalboR) and fulvestrant-resistant (MCF7 / FulvR) cell lines. FIGs. 9A-B are plotsshowing the dose matrix (9x9) evaluation of T-DXd and onvansertib combination in HER2-low HR+ breast cancer cell lines. Cell viability was assessed using the CellTiter-Glo® assay after 6 days of treatment at the indicated concentrations. Synergy was determined using the Bliss synergy model and synergistic interactions are indicated in blue on the heatmap (n=3). The results suggest that the combination was synergistic in the 5 cell lines, as shown by high Bliss synergy scores.

[0199] The effect of onvansertib and DXd (T-DXd payload) on cell viability was also tested in 1 TNBC (HCC38) and 3 HR+ breast cancer cell lines (EFM-19, MCF7), including a palbociclib-resistant (MCF7 / PR) cell line. FIGs. 10A-10B are plots showing the dose matrix (9x9) evaluation of DXd (T-DXd payload) and onvansertib combination in the HER2-low TNBC and HR+ breast cancer cell lines. Cell viability was assessed using the CellTiter-Glo® assay after 6 days of treatment at the indicated concentrations. Synergy was determined using the Bliss synergy model and synergistic interactions are indicated in blue on the heatmap (n=3). The results suggest that the combination of onvansertib and DXd was synergistic in the 4 cell lines, as shown by high Bliss synergy scores.

[0200] Apoptosis was then evaluated using the TUNEL assay, which detects DNA fragmentation in apoptotic cells. FIG. 11 are plots showing the percentage of TUNEL-positive cells in selected HER2-low breast cancer cell lines treated with DMSO (Ctrl), onvansertib, T-DXd or the combination (Onv+T-DXd). The results show that the combination of onvansertib and T- DXd markedly increased TUNEL-positive cells compared with DMSO and monotherapy in both triple negative and HR+ HER2-low breast cancer cell lines, indicating that onvansertib and T- DXd combination can induce significantly higher levels of apoptosis than either single agent in HER2-low BC cell lines.Example 3Onvansertib and T-DXd combination induces DNA damage in HER2-low breast cancer cell lines

[0201] In this example, DNA damage was first assessed by flow cytometry using the yH2AX marker (phosphorylated H2AX) which typically accumulates at sites of DNA doublestrand breaks (DSBs).

[0202] FIG. 15 depicts data showing percent of yH2AX+ cells in HER2-low breast cancer cell lines MCF7 / PR and ZR-75-1 treated with DMSO (Ctrl), onvansertib, T-DXd or the combination (O+T). The data shows that the combination treatment induced significantly higher levels of DNA damage compared to either single agent in HER2-low HR+ breast cancer cell lines.

[0203] yH2AX and 53BP1 foci were further evaluated by immunofluorescence. A skilled person would understand that following DSBs, RAD51 is recruited to DSBs to promote homologous recombination (HR) repair, whereas 53BP1 is recruited to suppress HR and therebypromote non-homologous end joining (NHEJ).

[0204] MCF7 cells were seeded in coverslips, then treated with DMSO (Ctrl), onvansertib (Onv), T-DXd, or the combination (Onv + T-DXd). The treated cells were allowed to recover for 24 h, prior to immunofluorescence staining using antibodies against yH2AX or 53BP 1.

[0205] FIG. 16 are immunofluorescence images showing nuclear localization of yH2AX (red) and 53BP1 (green) as bright foci within nuclei. Nuclei were stained using the DNA marker DAPI. The data shows that combination treatment induced elevated and persistent doublestrand break signaling compared to either single agent in the HER2-low HR+ breast cancer cell line MCF7.

[0206] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0207] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0208] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includesthe introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms.

[0209] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0210] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0211] While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating hormone receptor positive (HR+) breast cancer, the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor and deruxtecan (DXd) to a subject with the HR+ breast cancer, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject.

2. A method of treating hormone receptor positive (HR+) breast cancer, the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor to a subject with HR+ breast cancer and being treated with DXd, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject.

3. A method of treating hormone receptor positive (HR+) breast cancer, the method comprising: administering an antibody drug conjugate (ADC) capable of targeting breast cancer cells and a Polo-like kinase 1 (PLK1) inhibitor to a subject with the HR+ breast cancer, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject.

4. A method of treating hormone receptor positive (HR+) breast cancer, the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor to a subject with HR+ breast cancer and being treated with an antibody drug conjugate (ADC) capable of targeting breast cancer cells, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject.

5. The method of claim 1 or 4, wherein the HR+ breast cancer is estrogen receptor positive (ER+).

6. The method of any one of claims 1-5, wherein the HR+ breast cancer is progesterone receptor positive (PR+).

7. The method of any one of claims 1-6, wherein the HR+ breast cancer is progesterone receptor negative (PR-).

8. The method of any one of claims 1-7, wherein the HR+ breast cancer has a histological or cytological profile with ER > 1%, 10%, 20%, or higher.

9. A method of treating triple negative breast cancer (TNBC), the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor and deruxtecan (DXd) to a subject with the TNBC, thereby inhibiting or reducing progression of the TNBC in the subject.

10. A method of treating triple negative breast cancer (TNBC), the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor to a subject with the TNBCand being treated with DXd, thereby inhibiting or reducing progression of the TNBC in the subject.

11. A method of treating triple negative breast cancer (TNBC), the method comprising: administering an antibody drug conjugate (ADC) capable of targeting breast cancer cells and a Polo-like kinase 1 (PLK1) inhibitor to a subject with the TNBC, thereby inhibiting or reducing progression of the TNBC in the subject.

12. A method of treating triple negative breast cancer (TNBC), the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor to a subject with TNBC and being treated with an antibody drug conjugate (ADC) capable of targeting breast cancer cells, thereby inhibiting or reducing progression of the TNBC in the subject.

13. A method of treating HER2 -negative, low, or ultralow (HER2- / low / ultralow) breast cancer, the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor and deruxtecan (DXd) to a subject with the HER2- / low / ultralow breast cancer, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject.

14. A method of treating HER2-negative, low, or ultralow (HER2- / low / ultralow) breast cancer, the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor to a subject with the HER2- / low / ultralow breast cancer and being treated with DXd, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject.

15. A method of treating HER2 -negative, low, or ultralow (HER2- / low / ultralow) breast cancer, the method comprising: administering an antibody drug conjugate (ADC) capable of targeting breast cancer cells and a Polo-like kinase 1 (PLK1) inhibitor to a subject with the HER2- / low / ultralow breast cancer, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject.

16. A method of treating HER2 -negative, low, or ultralow (HER2- / low / ultralow) breast cancer, the method comprising: administering a Polo-like kinase 1 (PLK1) inhibitor to a subject with HER2- / low / ultralow breast cancer and being treated with an antibody drug conjugate (ADC) capable of targeting breast cancer cells, thereby inhibiting or reducing progression of the HER2- / low / ultralow breast cancer in the subject.

17. The method of any one of claims 1-16, wherein the HR+ breast cancer or TNBC is HER2 negative, low or ultralow.

18. The method of any one of claims 1-8, wherein the HR+ breast cancer or TNBCpatient has an HER2 immunohistochemistry (IHC) score of 0 or 1+.

19. The method of any one of claims 1-18, wherein the HR+ breast cancer or TNBC patient has an HER2 IHC score of 2+ and a negative in situ hybridization (ISH) score.

20. The method of any one of claims 1-18, wherein the HR+ breast cancer or TNBC is resistant to or does not respond effectively to first line treatments for HR+ breast cancer or TNBC, optionally the HR+ breast cancer or TNBC is HER2 negative, low or ultralow.

21. The method of any one of claims 1-20, wherein the subject with the HR+ breast cancer is resistant to or does not respond effectively to a hormone therapy, or wherein the subject is resistant to chemotherapy and optionally the chemotherapy comprises first-line treatment, or wherein the subject is resistant to chemotherapy, and optionally the chemotherapy comprises IL treatment and the subject suffers from TNBC).

22. The method of claim 21, wherein the hormone therapy comprises a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), an aromatase inhibitor, or a combination thereof.

23. The method of any one of claims 1-22, wherein the subject with the HR+ breast cancer is resistant to a kinase inhibitor, optionally the kinase inhibitor is a CDK inhibitor, further optionally the CDK inhibitor is a CDK 4 / 6 inhibitor.

24. The method of any one of claims 1-23, wherein the subject with the HR+ breast cancer develops stable disease, progressive disease, or resistance to a CDK4 / 6 inhibitor and / or a SERD.

25. The method of any one of claims 1-24, wherein the subject with the HR+ breast cancer is resistant to or develops stable or progressive disease following treatment with palbociclib, abemaciclib, fulvestrant, or a combination thereof.

26. The method of any one of claims 1-25, wherein the subject with the HR+ breast cancer is resistant to or does not respond effectively to alpelisib alone or in combination with a SERD, optionally the SERD is fulvestrant.

27. The method of any one of claims 1-26, wherein the subject with the TNBC is resistant to or does not respond effectively to chemotherapy.

28. The method of any one of claims 20-26, wherein the resistance is acquired resistance or intrinsic resistance.

29. The method of any one of claims 1-28, wherein the subject is known to be resistant to the anti-HER2 ADC alone, wherein the subject is known to be or is resistant to the treatment with the ADC alone, or wherein the subject is known to develop or has developed stable disease following the treatment with the ADC alone.

30. The method of any one of claims 3-4, 11-12 and 15-16, wherein the ADC capableof targeting breast cancer cells and the PLK1 inhibitor are co-administered simultaneously.

31. The method of any one of claims 3-4, 11-12 and 15-16, wherein the ADC capable of targeting breast cancer cells and the PLK1 inhibitor are administered sequentially.

32. The method of any claims 3-4, 11-12, 15-16 and 30-31, wherein the ADC capable of targeting breast cancer cells, the PLK1 inhibitor, or both are administered to the subject in a cycle of 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, or 49 days.

33. The method of any one of claims 3-4, 11-12, 15-16 and 30-32, wherein the ADC capable of targeting breast cancer cells is administered to the subject about once every two weeks and the PLK1 inhibitor is administered to the subject about 5 days a week.

34. The method of any one of claims 1-33, wherein each cycle of treatment is at least about 14 days.

35. The method of any one of claims 1-33, wherein each cycle of treatment is from about 14 days to about 28 days.

36. The method of any one of claims 1-35, wherein the PLK1 inhibitor is administered on at least five days, at least ten days, or at least fifteen days in a cycle.

37. The method of any one of claims 1-35, wherein the PLK1 inhibitor is not administered on at least one day, at least three days, or at least seven days in a cycle.

38. The method of any one of claims 1-37, wherein the ADC capable of targeting breast cancer cells is administered once weekly, once every two weeks, once every three weeks, or once every four weeks.

39. The method of any one of claims 1-38, wherein the ADC capable of targeting breast cancer cells is administered once every three weeks for once, twice, three, or four times in a cycle.

40. The method of any one of claims 1-39, wherein the ADC capable of targeting breast cancer cells is administered to the subject about once every three weeks and the PLK1 inhibitor is administered to the subject about 5 days a week.

41. The method of any one of claims 1-40, wherein the subject undergoes at least two cycles of the administration of the ADC capable of targeting breast cancer cells and the PLK1 inhibitor.

42. The method of any one of claims 1-41, wherein the PLK1 inhibitor is onvansertib (NMS-P937), BI2536, volasertib (BI 6727), GSK461364, adavosertib (AZD1775), CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, GTPL 10072, Ro3280; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and any combinations thereof.

43. The method of any one of claims 1-42, wherein the PLK1 inhibitor is onvansertib.

44. The method of any one of claims 1-43, wherein the ADC capable of targeting breastcancer cells comprises an anti-HER2 antibody or an anti-TROP2 antibody.

45. The method of any one of claim 1-43, wherein the ADC capable of targeting breast cancer cells comprising a chemotherapy drug conjugated to an antibody targeting breast cancer cells, and optionally the antibody targeting breast cancer cells is an anti-HER2 antibody or an anti- TR0P2 antibody.

46. The method of any one of claims 1-43, wherein the ADC capable of targeting breast cancer cells comprising a DNA damaging agent conjugated to an antibody targeting breast cancer cells, and optionally the antibody targeting breast cancer cells is an anti-HER2 antibody or an anti- TR0P2 antibody.

47. The method of claim 46, wherein the DNA damaging agent is selected from the group consisting of radiomimetic neocarzinostatin, a platinating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an antimetabolite, an alkylating agent, an antibiotic, and a combination thereof.

48. The method of claim 47, wherein the DNA damaging agent is a topoisomerase inhibitor.

49. The method of any one of claims 44-48, wherein the DNA damaging agent is selected from a group consisting of Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone, Teniposide, and Topotecan, Camptothecin, Rubitecan, Belotecan, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin, and a combination thereof.

50. The method of any one of claims 1-49, wherein the ADC capable of targeting breast cancer cells comprises deruxtecan (DXd) conjugated to an anti-HER2 antibody or an anti-TROP2 antibody.

51. The method of any one of claims 1-49, wherein the ADC capable of targeting breast cancer cells is trastuzumab deruxtecan (T-DXd).

52. The method of any one of claims 1-50, wherein the ADC capable of targeting breast cancer cells is datopotamab deruxtecan (Dato-DXd).

53. The method of any one of claims 1-50, wherein the subject has received at least one prior cancer treatment.

54. The method of claim 52, where the prior treatment does not comprise the use of an ADC, a PLK1 inhibitor, or both; and optionally the PLK1 inhibitor is onvansertib.

55. The method of any one of claims 1-54, wherein the PLK1 inhibitor is administered orally; and optionally the PLK1 inhibitor is administered every day or five times a week.

56. The method of any one of claims 1-55, wherein the ADC capable of targeting breast cancer cells is administered intravenously; and optionally wherein the ADC capable of targetingbreast cancer cells is administered every two weeks, three weeks, or four weeks.

57. The method of any one of claim 1-56, wherein the PLK1 inhibitor is administered at about 5 mg to about 80 mg; optionally at about 10 mg to about 30 mg; and further optionally at 10 mg, 15 mg, 20 mg, 25 mg, or 30 mg.

58. The method of any one of claims 1-57, wherein the ADC capable of targeting breast cancer cells is administered at about 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg or 9 mg / kg; optionally the ADC capable of targeting breast cancer cells is administered at 5.4 mg / kg.

59. The method of any one of claims 1-57, wherein the subject was in remission for cancer.

60. The method of any one of claims 1-59, wherein the subject in remission for cancer was in complete remission (CR) or in partial remission (PR).

61. The method of any one of claims 1-60, further comprising determining cancer status of the subject.

62. The method of any one of claims 1-61, further comprising determining responsiveness of the subject to the treatment of the ADC capable of targeting breast cancer cells and / or the PLK1 inhibitor.

63. The method of any one of claims 1-62, further comprising administering one or more additional cancer therapeutics or therapies for the cancer.

64. The method of any one of claims 1-63, the subject is human.

65. The method of any one of claims 1-64, wherein the subject achieves a complete response.

66. A kit, comprising: a PLKl inhibitor; and a manual providing instructions for co-administering the PLK1 inhibitor in combination with an ADC capable of targeting breast cancer cells to a subject in need thereof for treating hormone receptor positive (HR+) breast cancer.

67. A kit, comprising: a PLKl inhibitor; and a manual providing instructions for co-administering the PLK1 inhibitor in combination with deruxtecan to a subject in need thereof for treating HR+ breast cancer.

68. A kit, comprising: a PLKl inhibitor; and a manual providing instructions for co-administering the PLK1 inhibitor in combination with an ADC capable of targeting breast cancer cells to a subject in needthereof for treating triple negative breast cancer (TNBC).

69. A kit, comprising: a PLKl inhibitor; and a manual providing instructions for co-administering the PLK1 inhibitor in combination with deruxtecan to a subject in need thereof for treating TNBC breast cancer.

70. A kit, comprising: a PLKl inhibitor; and a manual providing instructions for co-administering the PLK1 inhibitor in combination with an ADC capable of targeting breast cancer cells to a subject in need thereof for treating HER2- / low / ultralow breast cancer.

71. A kit, compri sing : a PLKl inhibitor; and a manual providing instructions for co-administering the PLK1 inhibitor in combination with deruxtecan to a subject in need thereof for treating HER2- / low / ultralow breast cancer.

72. The kit of any one of claims 66-71, wherein the PLK1 inhibitor is onvansertib (NMS-P937), BI2536, volasertib (BI 6727), GSK461364, adavosertib (AZD1775), CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, GTPL 10072, Ro3280; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and any combinations thereof.

73. The kit of any one of claims 66-71, wherein the PLK1 inhibitor is onvansertib.

74. The kit of any one of claims 66-73, wherein the instructions comprise instructions for co-administrating the ADC capable of targeting breast cancer cells and the PLK1 inhibitor simultaneously.

75. The kit of any one of claims 66-73, wherein the instructions comprise instructions for co-administrating the ADC capable of targeting breast cancer cells and the PLK1 inhibitor sequentially.

76. The kit of any one of claims 66-75, wherein the instructions comprise instructions for administering to a subject that did not respond to treatment with the ADC capable of targeting breast cancer cells alone.

77. The kit of any one of claims 66-76, wherein the instructions comprise instructions for administering to a subject resistant to a CDK 4 / 6 inhibitor, hormone therapy, alpelisib, or a combination thereof.

78. The kit of claim 77, wherein the CDK 4 / 6 inhibitor is palbociclib or abemaciclib.

79. The kit of claim 77, wherein the hormone therapy comprise a selective estrogenreceptor degrader (SERD), a selective estrogen receptor modulator (SERM), an aromatase inhibitor, or a combination thereof, optionally the SERD is fulvestrant.

80. The kit of any one of claims 66-79, further comprising the ADC capable of targeting breast cancer cells.

81. The kit of any one of claims 66-80, wherein the ADC comprises an anti-HER2 antibody or an anti-TROP2 antibody and a DNA damaging agent selected from the group consisting of radiomimetic neocarzinostatin, a platinating agent, a topoisomerase I inhibitor, a topoisomerase II inhibitor, an antimetabolite, an alkylating agent, an antibiotic, and a combination thereof.

82. The kit of claim 81, wherein the DNA damaging agent is a topoisomerase inhibitor, optionally, the DNA damaging agent is selected from a group consisting of Etoposide, Irinotecan, Irinotecan liposomal, Mitoxantrone, Teniposide, and Topotecan, Camptothecin, Rubitecan, Belotecan, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin, and a combination thereof.

83. The kit of any one of claims 66-82, wherein the ADC capable of targeting breast cancer cells comprises deruxtecan conjugated to an anti-HER2 antibody or an anti-TROP2 antibody.

84. The kit of any one of claims 66-82, wherein the ADC capable of targeting breast cancer cells is T- trastuzumab deruxtecan (T-DXd).

85. The kit of any one of claims 68-83, wherein the ADC capable of targeting breast cancer cells is datopotamab deruxtecan (Dato-DXd).

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