PLK1 inhibitors in combination with anti-angiogenic agents for treatment of metastatic cancer

Abstract

Provided are methods, compositions, and kits for treating metastatic cancer in a subject. In some embodiments, methods may include administering a treatment including inhibition of angiogenesis and a PLK1 inhibitor (e.g., onvansertib) to a subject that has not yet undergone past anti-angiogenesis treatment in a manner sufficient to reduce or inhibit metastatic cancer progression.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 405,466, filed Sep. 11, 2022, and U.S. Provisional Patent Application No. 63 / 515,831, filed Jul. 26, 2023, under 35 U.S.C. § 119(e). The content of these related applications is hereby incorporated by reference in its entirety for all purposes.

[0003] Background

[0004] Field

[0005] The present disclosure generally relates to the field of the treatment of cancer.

[0006] Description of Related Art

[0007] Polo-like kinase 1 (PLK1) is a serine / threonine kinase and is the most well-characterized member of a family of five closely related regulatory proteins. PLK-1 is a master regulator of mitosis, controlling cell entry into and progression through mitosis via it. PLK1 performs several important functions during the mitotic (M) phase of the cell cycle, including regulating centrosome maturation and spindle assembly, removing cohesin from chromosome arms, inactivating anaphase-promoting complex / cyclosome (APC / C) inhibitors, and regulating mitotic exit and cytokinesis. PLK1 plays a key role in centrosome function and the assembly of bipolar spindles. PLK1 controls the interaction of kinetochores with spindle microtubules, which is necessary for the successful segregation and separation of chromatids into appropriate mother and daughter cells. PLK1 also acts as a negative regulator of p53 family members, resulting in the ubiquitination and subsequent degradation of p53 / TP53, the inhibition of p73 / TP73-mediated pro-apoptotic functions, and the phosphorylation / degradation of the cofactor bora of aurora kinase A. During different stages of mitosis, PLK1 localizes to the centrosome, kinetochores, and central spindle. PLK1 is aberrantly overexpressed in a variety of human cancers and is associated with cell proliferation and poor prognosis.

[0008] The most advanced stage of cancer is stage IV, which is defined as cancer spreading to body parts far from the original site of cancer origin (e.g., metastasis). Angiogenesis is necessary for metastasis to occur, and several anti-angiogenic agents have been approved for human use to treat metastatic cancer. However, according to the Centers for Disease Control and Prevention, in the United States, approximately one-fifth of deaths are still due to cancer. There is a need for more effective treatments for advanced (e.g., metastatic) cancer.

[0009] Overview

[0010] This disclosure relates to methods for treating cancer, including metastatic cancer. In some embodiments, a method for treating cancer can include administering a PLK1 inhibitor and an anti-angiogenic agent to a subject having metastatic cancer, thereby reducing or inhibiting the progression of metastatic cancer, wherein the subject has not received any prior treatment that includes inhibiting angiogenesis. In some embodiments, a method for treating cancer can include administering a PLK1 inhibitor and an anti-angiogenic agent to a subject, thereby reducing or inhibiting the progression of metastatic cancer, wherein the subject has not received any prior cancer treatment or the subject has not received any prior treatment that includes inhibiting angiogenesis. In some embodiments, a method for treating cancer can include administering a PLK1 inhibitor and an anti-angiogenic agent to a subject, thereby reducing or inhibiting the progression of metastatic cancer, wherein it is known that the subject has not received any prior cancer treatment or it is known that the subject has not received any prior treatment that includes inhibiting angiogenesis. In some embodiments, a method for treating cancer can include identifying a subject having metastatic cancer and who has not received any prior cancer treatment; and administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby reducing or inhibiting the progression of metastatic cancer. In some embodiments, the method includes: identifying a subject having metastatic cancer and who has not received any prior treatment that includes inhibiting angiogenesis; and administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby reducing or inhibiting the progression of metastatic cancer.

[0011] In some embodiments, the method includes administering chemotherapy, a PLK1 inhibitor, and an anti-angiogenic agent to the subject. In some embodiments, the subject has not received prior chemotherapy treatment. In some embodiments, the subject has not received prior chemotherapy treatment for metastatic cancer. Chemotherapy can include treatment with FOLFIRI, abiraterone, FOLFOX, an anti-EGFR agent, a KRAS-directed inhibitor, gemcitabine, abraxane, nanoliposomal irinotecan, 5-FU, FOLFIRINOX, FOLFOXIRI, or a combination thereof.

[0012] In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent synergistically reduces or inhibits the progression of metastatic cancer relative to the additive effect of treatment with the PLK1 inhibitor alone, treatment with the anti-angiogenic agent alone, and / or treatment with the PLK1 inhibitor alone and the anti-angiogenic agent alone. In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent improves one or more treatment outcomes in the subject relative to a control or baseline. In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent reduces the oncogenic allele burden in the subject relative to a subject who has received prior treatment that includes inhibiting angiogenesis.

[0013] The present disclosure also provides methods for improving objective response rate (ORR), progression-free survival (PFS), or both in the treatment of metastatic cancer. In some embodiments, the methods include administering a PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, thereby improving the ORR and / or PFS of the subject, wherein the subject has not received any prior cancer treatment or the subject has not received any prior treatment that includes inhibition of angiogenesis. In some embodiments, the methods include administering a PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, thereby improving the ORR and / or PFS of the subject, wherein it is known that the subject has not received any prior cancer treatment or it is known that the subject has not received any prior treatment that includes inhibition of angiogenesis. In some embodiments, the methods include identifying a subject with metastatic cancer who has not received any prior cancer treatment. In some embodiments, the methods include identifying a subject with metastatic cancer who has not received any prior treatment that includes inhibition of angiogenesis. In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent synergistically improves the ORR and / or PFS of the subject, relative to the additive effect of treatment with the PLK1 inhibitor alone, treatment with the anti-angiogenic agent alone, and / or treatment with the PLK1 inhibitor alone and the anti-angiogenic agent alone. In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent improves one or more treatment effects in the subject, relative to a control or baseline; and optionally, the one or more treatment effects include the size of a tumor originating from metastatic cancer, objective response rate (ORR), duration of response, time to response, progression-free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allele burden, or a combination thereof. In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent improves the ORR of the subject, improves the PFS of the subject, improves the OS of the subject, improves the DCR of the subject, reduces the oncogenic allele burden of the subject, or a combination thereof, relative to a subject who has received prior treatment that includes inhibition of angiogenesis. In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent improves the ORR, PFS, or both of the subject by at least 50% relative to a subject who has received prior treatment that includes inhibition of angiogenesis. In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent to a subject results in an ORR difference of 50%, 55%, 65%, 70%, 75% or higher in the subject, or an ORR difference of a number or range between any two of these values. In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent to a subject results in an mPFS of 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months or longer in the subject, or an mPFS of a number or range between any two of these values.As described herein, administering a PLK1 inhibitor and an anti-angiogenic agent to a subject can include administering to the subject a PLK1 inhibitor, an anti-angiogenic agent, and one or more anti-cancer therapeutic agents or therapies (e.g., one or more chemotherapies).

[0014] Non-limiting examples of metastatic cancers include metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic renal cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or combinations thereof.

[0015] In some embodiments, the PLK1 inhibitor is selective and / or specific for PLK1. 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 subject has not received any prior treatment that includes administering an angiogenesis inhibitor, and optionally wherein the angiogenesis inhibitor is the same as the anti-angiogenic agent. In some embodiments, the anti-angiogenic agent is bevacizumab. In some embodiments, the angiogenesis inhibitor is bevacizumab.

[0016] The PLK1 inhibitor and the anti-angiogenic agent can be administered simultaneously or sequentially. In some embodiments, the PLK1 inhibitor is administered before the anti-angiogenic agent, and optionally, wherein on each day of administering the PLK1 inhibitor and the anti-angiogenic agent to the subject, the PLK1 inhibitor is administered before the anti-angiogenic agent. In some embodiments, on a given day, the PLK1 inhibitor is administered about 30 minutes to about 5 hours before the anti-angiogenic agent. In some embodiments, the administration of the PLK1 inhibitor is oral administration, and the administration of the anti-angiogenic agent is intravenous administration or oral administration. In some embodiments, the anti-angiogenic agent and the PLK1 inhibitor are each administered to the subject in a cycle of at least twice or at least five times within a week. In some embodiments, the anti-angiogenic agent, the PLK1 inhibitor, or both are administered in a cycle of at least 7 days; optionally, each treatment cycle is at least about 21 days; and further optionally, each treatment cycle is from about 21 days to about 28 days.

[0017] In some embodiments, the PLK1 inhibitor is administered for at least four days in a cycle. In some embodiments, the PLK1 inhibitor is not administered for at least one day in a cycle. In some embodiments, the anti-angiogenic agent is administered daily, weekly, bi-weekly, tri-weekly, monthly, or monthly. In some embodiments, the subject undergoes administration of the anti-angiogenic agent and the PLK1 inhibitor for at least two cycles. In some embodiments, the anti-angiogenic agent is bevacizumab, and the PLK1 inhibitor is onvansertib. Onvansertib can be administered at, for example, 12 mg / m 2 -90 mg / m 2 is administered. Bevacizumab can be administered at, for example, about 1 mg / kg - 20 mg / kg; optionally, wherein bevacizumab is administered at about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, or about 15 mg / kg.

[0018] In some embodiments, the subject has received at least one prior cancer treatment, and optionally wherein the prior treatment does not include the use of an anti-angiogenic agent, a PLK1 inhibitor, or both. In some embodiments, one or more subjects have a prior remission of cancer. The prior remission can be a complete remission (CR) or a partial remission (PR).

[0019] In some embodiments, the method further comprises one or more of the following: (1) determining the cancer status of one or more subjects, (2) determining the responsiveness of one or more subjects to PLK1 inhibitor treatment, and (3) administering one or more cancer therapeutics or therapies to one or more subjects. One or more subjects can be human.

[0020] In some embodiments, reducing or inhibiting cancer progression includes inhibiting the growth of one or more tumors in one or more subjects and / or reducing the number of cancer cells detected in one or more subjects by at least about 25%, 30%, 40%, 50%, 60%, or 70% relative to an untreated subject. In some embodiments, reducing or inhibiting cancer progression includes inhibiting the growth of one or more tumors in one or more subjects and / or reducing the number of cancer cells detected in one or more subjects by at least about 25%, 30%, 40%, 50%, 60%, or 70% relative to the one or more subjects prior to administration of a PLK1 inhibitor and an anti-angiogenic agent. In some embodiments, after one or more treatment cycles, the growth of at least one tumor among the one or more tumors in one or more subjects is reduced by at least about 25%, 30%, 40%, 50%, 60%, or 70%. In some embodiments, after one or more treatment cycles, the size / volume of at least one tumor among the one or more tumors in one or more subjects is reduced by at least about 25%, 30%, 40%, 50%, 60%, or 70%.

[0021] In some embodiments, one or more cancer therapeutics or therapies include FOLFIRI, abiraterone, FOLFOX, an anti-EGFR agent, a KRAS-directed inhibitor, gemcitabine, abraxane, nanoliposomal irinotecan, 5-FU, or a combination thereof; wherein the anti-EGFR agent is optionally cetuximab, and the KRAS-directed inhibitor is optionally a G12C inhibitor, a G12D inhibitor, or a combination thereof. In some embodiments, determining the responsiveness of one or more subjects includes determining whether a subject is a responder to treatment, whether one or more subjects are in or will be in complete remission (CR), or whether one or more subjects are in or will be in partial remission (PR). In some embodiments, determining the responsiveness of a subject includes determining the objective response rate (ORR), duration of response, time to response, progression-free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allele burden, or a combination thereof. In some embodiments, determining the responsiveness of one or more subjects includes determining whether one or more subjects have a partial response to treatment, whether a subject has a complete response to treatment, whether a subject has a stable disease (SD) status, or whether a subject has a progressive disease (PD) status.

[0022] Also disclosed herein are kits. In some embodiments, the kit comprises a PLK1 inhibitor; and a manual providing instructions for administering the PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, wherein the subject has not received any prior cancer treatment or the subject has not received any prior treatment including inhibition of angiogenesis. In some embodiments, the kit comprises a PLK1 inhibitor; and a manual providing instructions for administering the PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, wherein it is known that the subject has not received any prior cancer treatment or it is known that the subject has not received any prior treatment including inhibition of angiogenesis.

[0023] In some embodiments, the instructions include instructions for co-administering the PLK1 inhibitor and the anti-angiogenic agent. In some embodiments, the instructions include instructions for sequential administration of the PLK1 inhibitor and the anti-angiogenic agent. In some embodiments, the instructions include (1) instructions for oral administration of the PLK1 inhibitor, (2) instructions for oral administration of the anti-angiogenic agent, (3) instructions for intravenous administration of the anti-angiogenic agent, or any combination thereof. In some embodiments, the instructions include instructions wherein the subject has not received any prior treatment including administration of an angiogenesis inhibitor, and optionally wherein the angiogenesis inhibitor is the same as the anti-angiogenic agent. In some embodiments, the instructions include instructions for administering each of the anti-angiogenic agent and the PLK1 inhibitor to the subject in a cycle of at least twice or at least five times within a week. In some embodiments, the instructions include instructions for administering the anti-angiogenic agent, the PLK1 inhibitor, or both in a cycle of at least 7 days; and optionally, wherein each treatment cycle is at least about 21 days, and further optionally, each treatment cycle is from about 21 days to about 28 days. In some embodiments, the instructions include instructions for administering the PLK1 inhibitor for at least four days in a cycle. In some embodiments, the instructions include instructions for not administering the PLK1 inhibitor for at least one day in a cycle.

[0024] In some embodiments, the instructions include instructions for administering the anti-angiogenic agent daily, weekly, bi-weekly, tri-weekly, monthly, or monthly. In some embodiments, the instructions include instructions for administering the anti-angiogenic agent and the PLK1 inhibitor for at least two cycles. The anti-angiogenic agent can be bevacizumab. The PLK1 inhibitor can be onvansertib. In some embodiments, the instructions include instructions for administering onvansertib at 12 mg / m 2 -90 mg / m 2 . In some embodiments, the kit further comprises the anti-angiogenic agent. Brief Description of the Drawings

[0026] Figure 1Depicts exemplary Kaplan-Meir survival curves of cancer patients treated with the methods and compositions provided herein, with or without prior anti-angiogenic agent treatment.

[0027] Figure 2 Depicts the number of event patients and censored (not reported) patients for the study described herein.

[0028] Figure 3 Depicts the objective response rate of a cohort of patients treated with the methods and compositions provided herein, subdivided by patients who have or have not received prior anti-angiogenic agent treatment. The numbers in the boxes are the total number of patients in the designated groups.

[0029] Figures 4A - 4B Shows the best radiological response and duration of response (as of June 16, 2023) for 66 evaluable patients. *Radiological response determined according to RECIST 1.1. The waterfall plot and table reflect interim data from ongoing trials and unlocked databases as of June 16, 2023. Patients 02-008 and 07-029 were classified as not bev in the data on July 25, 2022, but are now determined to have been exposed to bev. mDOR CI: "-" means not reached. After an external review of tumor measurements completed on May 12, 2023, patients 02-028 and 04-038 were determined to have confirmed PRs.

[0030] Figures 5A - 5B Is a swim plot showing the responses of 66 evaluable patients (as of June 16, 2023). *The swim plot / table reflects interim data from ongoing trials and unlocked databases as of June 16, 2023. After an external review of tumor measurements completed on May 12, 2023, patients 02-028 and 04-038 were determined to have confirmed PRs.

[0031] Figure 6 Depicts a plot showing the progression-free survival of 66 evaluable patients (as of June 16, 2023). *Onvansertib mPFS is interim data from ongoing trials and unlocked databases as of June 16, 2023.

[0032] Figure 7 Shows a plot displaying the change in tumor size relative to baseline. *The spider plot reflects interim data from ongoing trials and unlocked databases as of June 16, 2023.

[0033] Figure 8Shows the changes in tumor volume in a KRAS mutant xenograft model when treated with vehicle, onvansertib, bevacizumab, or a combination of onvansertib and bevacizumab. 8 - 9 mice / group. Mean ± SEM is shown on the graph. Unpaired t - test was used to test the difference in the change of tumor volume between the combination treatment group and the most effective control group on the last day of treatment. *p<0.05, ***p<0.001, ****p<0.0001.

[0034] Figure 9 Shows the changes in tumor vascularization in a KRAS mutant xenograft model when treated with vehicle, onvansertib, bevacizumab, or a combination of onvansertib and bevacizumab. Detailed Description

[0035] In the following detailed description, reference is made to the accompanying drawings that form a part hereof. In the drawings, like symbols generally identify like components, unless the 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 aspects of the present disclosure, as generally described herein and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in many different configurations, all of which are explicitly contemplated herein and form a part of the disclosure herein.

[0036] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety for the relevant art.

[0037] The present disclosure includes methods for treating cancer. In some embodiments, the methods include: administering a PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, thereby reducing or inhibiting the progression of metastatic cancer, wherein the subject has not received any prior treatment that includes inhibiting angiogenesis, or wherein the subject has not received any cancer treatment (e.g., treatment for metastatic cancer). In some embodiments, the methods may include administering a PLK1 inhibitor and an anti-angiogenic agent to a subject, thereby reducing or inhibiting the progression of metastatic cancer, wherein it is known that the subject has not received any prior cancer treatment or it is known that the subject has not received any prior treatment that includes inhibiting angiogenesis. In some embodiments, the methods for treating cancer may include identifying a subject with metastatic cancer who has not received any prior cancer treatment; and administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby reducing or inhibiting the progression of metastatic cancer. In some embodiments, the methods include: identifying a subject with metastatic cancer who has not received any prior treatment that includes inhibiting angiogenesis; and administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby reducing or inhibiting the progression of metastatic cancer. Any of the methods disclosed herein can be used for first-line treatment of metastatic cancer, second-line treatment of metastatic cancer, or both. It is known that for a subject receiving second-line cancer treatment, the subject will have received at least one prior cancer treatment that has failed, stopped working, and / or has intolerable side effects. As disclosed herein, in some embodiments, the subject treated by the methods disclosed herein experiences treatment failure or is intolerant to the treatment (e.g., chemotherapy treatment, including oxaliplatin-based chemotherapy). In some embodiments, the chemotherapy treatment is chemotherapy with fluoropyrimidine and oxaliplatin.

[0038] The present disclosure includes methods for improving the objective response rate (ORR), progression-free survival (PFS), or both in the treatment of metastatic cancer. In some embodiments, the methods include administering a PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, thereby improving the subject's ORR and / or PFS, wherein the subject has not received any prior cancer treatment (e.g., treatment for metastatic cancer) or the subject has not received any prior treatment that includes inhibiting angiogenesis. In some embodiments, the methods include administering a PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, thereby improving the subject's ORR and / or PFS, wherein it is known that the subject has not received any prior cancer treatment (e.g., treatment for metastatic cancer) or it is known that the subject has not received any prior treatment that includes inhibiting angiogenesis. The method can be used for first-line treatment of metastatic cancer, second-line treatment of metastatic cancer, or both.

[0039] Disclosed herein are compositions and kits for treating cancer. In some embodiments, the kit comprises: a PLK1 inhibitor; and a manual providing instructions for administering the PLK1 inhibitor and an anti-angiogenic agent to a subject for treating metastatic cancer, wherein the subject has not received any prior treatment including angiogenesis inhibition, or wherein the subject has not received any cancer treatment (e.g., treatment for metastatic cancer).

[0040] Definitions

[0041] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 the purposes of this disclosure, the following terms are defined below.

[0042] As used herein, "subject" refers to an animal that is the object of treatment, observation, or experiment. "Animal" includes cold-blooded and warm-blooded vertebrates and invertebrates such as fish, crustaceans, reptiles and especially mammals. "Mammal" includes but is not limited to mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates such as monkeys, chimpanzees and apes and especially humans.

[0043] As used herein, "patient" refers to a subject being treated by a medical professional such as a doctor or veterinarian in an 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. A patient can be an animal. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human.

[0044] As used herein, "administration" or "administering" refers to a method of giving a vertebrate a dose of a pharmaceutically active ingredient.

[0045] As used herein, "dose" refers to the combined amount of an active ingredient (e.g., a PLK1 inhibitor (e.g., onvansertib) or an anti-angiogenic agent (e.g., bevacizumab)).

[0046] As used herein, "unit dose" refers to the amount of a therapeutic agent administered to a patient in a single dose.

[0047] As used herein, the terms "daily dose" or "daily dosage" refer to the total amount of a pharmaceutical composition or therapeutic agent to be taken within 24 hours.

[0048] As used herein, the term "delivery" refers to methods, formulations, techniques, and systems for delivering a pharmaceutical composition or therapeutic agent into a patient's body 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 patient's bloodstream.

[0049] As used herein, the terms "formulated" or "formulation" refer to the process of combining different chemical substances including one or more pharmaceutically active ingredients to produce a dosage form. In some embodiments, two or more pharmaceutically active ingredients may be co-formulated into a single dosage form or a combined dosage unit, or may be formulated separately and then combined into a combined dosage unit. Sustained release formulations are formulations designed to slowly release a therapeutic agent in the body over an extended period of time, while immediate release formulations are formulations designed to rapidly release a therapeutic agent in the body over a shortened period of time.

[0050] As used herein, the term "pharmaceutically acceptable" indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner, considering the disease or condition to be treated and the corresponding route of administration, to avoid administering the material to a patient. For example, such materials are typically required to be substantially sterile.

[0051] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, which is involved in carrying or transporting any supplement or composition or its components from one organ or part of the body to another organ or part of the body, or in delivering an agent to diseased tissue or tissue adjacent to diseased tissue. A carrier or excipient may be used to produce a composition. A carrier or excipient may be selected to facilitate the administration of a drug or prodrug. Examples of carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or various types of starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and physiologically compatible solvents. Examples of physiologically compatible solvents include water for injection (WFI), saline solutions, and sterile solutions of dextrose.

[0052] As used herein, the term "pharmaceutically acceptable salt" refers to any acid addition salt or base addition salt, the counterions of which are non-toxic to a patient at the dosage of the salt. Many pharmaceutically acceptable salts are well known in the pharmaceutical art. If pharmaceutically acceptable salts of the compounds of the present disclosure are used in these compositions, these salts are preferably derived from inorganic or organic acids and bases. Such acid salts include the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, hydrohalides (e.g., hydrochloride and hydrobromide), sulfate, phosphate, nitrate, sulfamate, malonate, salicylate, methylene-bis-b-hydroxynaphthoate, gentisate, hydroxyethanesulfonate, ditoluoyl tartrate, ethanesulphonate, cyclohexylaminosulfonate, quinate, etc. Pharmaceutically acceptable base addition salts include, but are not limited to, those salts derived from alkali metal bases 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 salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, salts of organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine salts, and salts of amino acids such as arginine, lysine, etc.

[0053] As used herein, each of the terms "partial response", "partial remission", or "PR" refers to an improvement in the cancer status as measured, for example, by tumor size and / or cancer marker levels, in response to treatment. In some embodiments, "partial response" means that the tumor or a blood marker indicative of the tumor has decreased in size or level by about 50% in response to treatment. The treatment can be any treatment for cancer, including but not limited to chemotherapy, radiotherapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The size of the tumor can be detected by clinical or radiological means. Markers indicative of the tumor can be detected by means well known to those skilled in the art, e.g., ELISA or other antibody-based tests.

[0054] As used herein, each of the terms "complete response", "complete remission", "complete recovery", or "CR" means that the cancer status, as measured by, for example, tumor size and / or cancer marker levels, has disappeared after treatment, which includes but is not limited to chemotherapy, radiotherapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The presence of a tumor can be detected by clinical or radiological means. Markers indicative of a tumor can be detected by means well known to those skilled in the art, such as ELISA or other antibody-based tests. However, "complete response" does not necessarily indicate that the cancer has been cured. Recurrence may occur after a complete response. Complete response of target lesions includes the disappearance of all target lesions and any pathological lymph nodes (whether target or non-target), with the short axis of the lymph nodes reduced to <10 mm. Complete response of non-target lesions includes the disappearance of all non-target lesions and normalization of the cancer marker levels (all lymph nodes must be non-pathological in size (<10 mm short axis)). If the cancer marker was initially above the upper limit of normal, it needs to be normalized for the patient to be considered to be in a complete clinical response of non-target lesions. The duration of overall CR is measured from the time when the CR measurement criteria are first met until the first day of objectively recorded disease progression or death from any cause. Participants with no reported events are censored at the last disease assessment.

[0055] As used herein, the term "stable disease" or "SD" means that there has been neither sufficient shrinkage to meet PR nor sufficient increase to meet progressive disease (PD), with the smallest sum diameter at the time of study as the reference. The duration of stable disease is measured from the start of treatment until the progression criteria are met, with the smallest measurement recorded since the start of treatment (including the baseline measurement) as the reference.

[0056] As used herein, the term "progressive disease" or "PD", when referring to target lesions, means that the sum of the diameters of the target lesions has increased by at least 20%, with the smallest sum in the study as the reference (if the baseline sum is the smallest sum in the study, this includes the baseline sum). In addition to the 20% relative increase, 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 progression). When progressive disease or PD refers to non-target lesions, it means the appearance of one or more new lesions and / or definite progression of existing non-target lesions. Definite progression generally should not exceed the status of target lesions. It must represent a change in the overall disease state, rather than an increase in a single lesion.

[0057] As used herein, the term "overall survival" or "OS" means the time from randomization (or enrollment) to death from any cause. Surviving participants are censored at the last known survival date.

[0058] As used herein, the term "progression-free survival" or "PFS" means the time from randomization (or enrollment) to the earlier of progression or death due to any cause. Participants who survive without disease progression are censored at the date of the last disease assessment.

[0059] As used herein, the term "best overall response" means the best response recorded from the start of treatment until disease progression / recurrence (using the smallest measurement recorded since the start of treatment as the reference for progressive disease). The assignment of a patient's best response depends on the achievement of both measurement criteria and confirmation criteria. The duration of the overall response is measured from the time that the measurement criteria for CR or PR are met (whichever is recorded first) until the day of objective documentation of recurrent disease or progressive disease (using the smallest measurement recorded since the start of treatment as the reference for progressive disease) or death due to any cause. Participants with no reported events are censored at the last disease assessment.

[0060] As used herein, the term "hydrate" refers to a complex formed by the combination of water molecules with the molecules or ions of a solute. As used herein, the term "solvate" refers to a complex formed by the combination of solvent molecules with the molecules or ions of a solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Solvates are intended to include hydrates, hemihydrates, channel hydrates, etc. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water.

[0061] As used herein, "therapeutically effective amount" or "pharmaceutically effective amount" means an amount of a therapeutic agent that has a therapeutic effect. The dose of a pharmaceutically active ingredient that is useful in treatment when administered alone or in combination with one or more additional therapeutic agents is a therapeutically effective amount. Thus, as used herein, a therapeutically effective amount means an amount of a therapeutic agent that produces a desired therapeutic effect as judged from 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 dose can be conveniently administered, for example, in separate doses up to four times a day or in a sustained release form.

[0062] As used herein, the terms "treat", "treatment", or "treating" refer to the administration of a therapeutic agent or pharmaceutical composition to a subject for prophylactic and / or therapeutic purposes. The term "prophylactic treatment" refers to treating a subject who has not yet exhibited symptoms of a disease or condition but is susceptible to or otherwise at risk of a particular disease or condition, such that 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 who already has a disease or condition. As used herein, a "therapeutic effect" alleviates one or more symptoms of a disease or disorder to some extent. For example, a therapeutic effect can be observed by a reduction in subjective discomfort communicated by the subject (e.g., reduced discomfort recorded in a self-administered patient questionnaire).

[0063] As used herein, the terms "prophylaxis", "prevent", "preventing", "prevention" and their grammatical variations refer to prophylactic treatment of a subclinical disease state in a subject (e.g., a mammal including a human) to reduce the probability of occurrence of a clinical disease state. The method can partially or completely delay or preclude the onset or recurrence of one or more of a disorder or condition and / or its attendant symptoms, or prevent a subject from acquiring or reacquiring a disorder or condition, or reduce the risk that a subject will acquire or reacquire a disorder or condition or one or more of its attendant symptoms. Subjects are selected for prophylactic therapy based on factors known to increase the risk of developing a clinical disease state compared to the general population. "Prophylactic" therapy can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment of a subject who has not yet presented a clinical disease state, while secondary prevention is defined as preventing a second occurrence of the same or a similar clinical disease state.

[0064] As used herein, each of the terms "partial response" and "partial remission" can refer to an improvement in a cancerous state, as measured, for example, by tumor size and / or cancer marker levels, in response to treatment. In some embodiments, "partial response" means that the tumor or a blood marker indicative of the tumor has decreased in size or level by about 50% in response to treatment. The treatment can be any treatment for cancer, including but not limited to chemotherapy, radiotherapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The size of the tumor can be detected by clinical or radiological means. Markers indicative of the tumor can be detected by means well known to those skilled in the art, e.g., ELISA or other antibody-based tests.

[0065] As used herein, each of the terms "complete response" or "complete remission" means that the cancer status, as measured by, for example, tumor size and / or cancer marker levels, has disappeared after treatment, the treatment including, but not limited to, chemotherapy, radiotherapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The presence and / or size of a tumor can be detected by clinical means or by radiological means. Markers indicative of a tumor can be detected by means well known to those of skill in the art, e.g., ELISA or other antibody-based tests. However, "complete response" does not necessarily indicate that the cancer has been cured, as recurrence may occur after complete response.

[0066] Cancer

[0067] The methods, compositions, and kits disclosed herein can be used to treat cancer, including metastatic cancer. In some embodiments, methods for treating cancer include administering an anti-angiogenic agent (e.g., bevacizumab) and a PLK1 inhibitor (e.g., onvansertib) or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof to a subject in need thereof (e.g., a subject having metastatic cancer). In some embodiments, the subject has not received any prior treatment for inhibiting angiogenesis (e.g., the subject has not received any prior treatment with bevacizumab alone, or the subject has not received any prior treatment with bevacizumab in combination with one or more anti-cancer agents or one or more anti-cancer therapies (e.g., chemotherapy)). In some embodiments, the subject has not received any prior cancer treatment. The subject can be, for example, a subject without a prior cancer diagnosis. In some embodiments, the subject has not received any prior treatment with bevacizumab and / or chemotherapy.

[0068] The methods, compositions, and kits disclosed herein can be used for various types of cancer. The cancer can be a solid tumor, a liquid tumor, or a combination thereof. In some embodiments, the cancer is a solid tumor, including but not limited to melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gallbladder cancer, laryngeal cancer, liver cancer, thyroid cancer, gastric cancer, salivary gland cancer, prostate cancer, pancreatic cancer, Merkel cell carcinoma, brain and central nervous system cancers, and any combination thereof. In some embodiments, the cancer is a liquid tumor. In some embodiments, the cancer is a hematological cancer. Non-limiting examples of hematological cancers include diffuse large B-cell lymphoma ("DLBCL"), Hodgkin lymphoma ("HL"), non-Hodgkin lymphoma ("NHL"), follicular lymphoma ("FL"), acute myeloid leukemia ("AML"), and multiple myeloma ("MM"). Additionally, the diseases or conditions provided herein include refractory or recurrent malignancies, the growth of which can be inhibited using the methods and compositions disclosed herein.

[0069] In some embodiments, the cancer is metastatic cancer. As used herein, "metastatic cancer" can refer to cancer that has spread (metastasized) from its original site to another area of the body. In fact, all cancers have the potential to spread in this way. Whether metastasis occurs depends on a complex interplay of many tumor cell factors, including the type of cancer, the degree of maturation (differentiation) of the tumor cells, the location of the cancer and how long the cancer has been present, as well as other factors that are not fully understood. As used herein, the term "metastasis" can refer to the formation of secondary tumor foci of progressive growth at sites discontinuous with the primary lesion. The metastatic process is a multi-step mechanism in which metastatic cancer cells escape the primary tumor, enter the circulation, invade distant tissue sites and grow into macroscopic tumors at the target sites. Metastatic cancer can be metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof.

[0070] PLK1 inhibitor

[0071] Polo-like kinase (PLK) is 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 mitotic entry, centrosome maturation, bipolar spindle formation, chromosome segregation, and cytokinesis. PLK1 has been shown to be overexpressed in solid tumors and hematological malignancies, including AML. PLK1 inhibition induces G2-M phase arrest and subsequent apoptosis in cancer cells and has emerged as a promising targeted therapy. Several PLK inhibitors have been studied in clinical trials. In a randomized phase II study of patients with untreated but ineligible for induction therapy AML, the pan-PLK inhibitor volasertib (BI6727), administered intravenously in combination with LDAC, showed a significant increase in OS when compared to LDAC alone. A subsequent randomized phase III study did not find a benefit of the combination and described an increased risk of severe infections. PLK1 promotes HR during double-strand DNA break (DSB) repair. PLK1 phosphorylates Rad51 and BRCA1, promoting their recruitment to DSB sites and thus HR-mediated DNA repair. PLK1 inhibitors can be selective and / or specific for PLK1.

[0072] The PLK1 inhibitor can be dihydropteridinone, pyridopyrimidine, aminopyrimidine, substituted thiazolinone, pteridine derivative, dihydroimidazo[1,5-f]pteridine, meta-substituted thiazolinone, benzyl styryl sulfone analog, stilbene derivative or any combination thereof. The PLK1 inhibitor can be onvansertib, BI2536, Volasertib (BI6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960 or Ro3280.

[0073] onvansertib (also known as PCM-075, NMS-1286937, NMS-937, "Compound of formula (I)" in US8,927,530; IUPAC name 1-(2-hydroxyethyl)-8-{[5-(4-methylpiperazin-1-yl)-2-(trifluoromethoxy)phenyl]amino}-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide) is a selective ATP-competitive PLK1 inhibitor. Biochemical assays have shown that onvansertib has high specificity for PLK1 in a panel of 296 kinases (including other PLK members). In models of both solid and hematological malignancies, onvansertib has strong in vitro and in vivo antitumor activity. onvansertib is the first orally administered PLK1-specific ATP-competitive inhibitor to enter clinical trials with proven antitumor activity in different preclinical models. onvansertib inhibits cell proliferation in AML cell lines and tumor growth in AML xenograft models at nanomolar concentrations. onvansertib also significantly increases the antitumor activity of cytarabine in a disseminated AML model.

[0074]

[0075] Onvansertib demonstrated high potency in proliferation assays, with low nanomolar activity against a large number of cell lines from both solid tumors and hematological malignancies. After oral administration at well-tolerated doses in mice, onvansertib effectively induced mitotic cell cycle arrest and subsequent apoptosis in cancer cell lines and inhibited xenograft tumor growth, with a well-defined PLK1-related mechanism of action. In addition, onvansertib showed activity in combination therapies with approved cytotoxic drugs such as irinotecan, where enhanced tumor regression was present in HT29 human colon adenocarcinoma xenografts compared to each agent alone, and in combination therapy with cytarabine showed prolonged animal survival in a disseminated model of AML. Onvansertib has favorable pharmacology parameters and good oral bioavailability in rodent and non-rodent species, as well as demonstrated anti-tumor activity in different non-clinical models using multiple dosing regimens, which may provide a high degree of flexibility in dosing schedules, warranting studies in the clinical setting. Onvansertib has several advantages over volasertib (BI6727, another PLK1 inhibitor), including a higher degree of potency and specificity for PLK1 isoforms and a higher degree of oral bioavailability.

[0076] A Phase I, first-in-human, dose-escalation study of onvansertib in patients with advanced / metastatic solid tumors identified neutropenia and thrombocytopenia as the major dose-limiting toxicities. These hematological toxicities were expected based on the mechanism of action of the drug and were reversible, with recovery occurring within 3 weeks. The half-life of onvansertib was determined to be between 20 and 30 hours. The oral bioavailability of onvansertib combined with its short half-life provides the opportunity for a convenient, controlled, and flexible dosing schedule, potentially minimizing toxicity and improving the therapeutic window. Pharmacodynamic and biomarker studies have been conducted, including baseline genomic profiling, serial monitoring of mutant allele fractions in plasma, and the extent of PLK1 inhibition in circulating blasts, to identify biomarkers associated with clinical response and are described in WO2021 / 146322, the content of which is incorporated herein by reference in its entirety.

[0077] The cancer treatment of the present disclosure can include administering a PLK1 inhibitor (e.g., onvansertib) to a subject with cancer for a desired duration in one cycle, two cycles, or more cycles. The desired duration in each cycle can independently be one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, or more days. The length of the cycle can be, for example, at least 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, or more days. For example, a single cycle of treatment can include administering a PLK1 inhibitor (e.g., onvansertib) 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, or more days in one cycle (e.g., a cycle of at least 21 days (e.g., 21 to 28 days)). In some embodiments, the treatment can include administering a PLK1 inhibitor (e.g., onvansertib) for the following or for at least the following in one cycle (e.g., a cycle of at least 21 days (e.g., 21 to 28 days)): 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. Administering the PLK1 inhibitor (e.g., onvansertib) in a single cycle of treatment can be continuous or have one or more intervals (e.g., interrupted by one or two days). In some embodiments, the treatment includes administering a PLK1 inhibitor (e.g., onvansertib) for 5 days in a cycle of 21 to 28 days. In some embodiments, the duration of administering the PLK1 inhibitor in one cycle can be different from the duration of administering the PLK1 inhibitor in one or more other cycles. For example, the PLK1 inhibitor can be administered to the subject for 10 days in the first cycle (e.g., days 1 to 5 of the first 14 days and days 1 to 5 of the last 14 days in a 28-day cycle), and for 14 days in the second cycle (e.g., days 1 to 7 of the first 14 days and days 1 to 7 of the last 14 days in a 28-day cycle). The length of each cycle can vary. For example, cycle 1 can be 28 days, and cycle 2 can be 21 days.

[0078] The cancer treatment disclosed herein can include at 12 mg / m 2 -90 mg / m 2 or at about 12 mg / m 2 -90 mg / m 2, for example, as a daily dose, administer a PLK1 inhibitor (e.g., onvansertib). For example, treatment can include daily administration of a PLK1 inhibitor (e.g., onvansertib) at the following or at about the following: 8 mg / m 2 , 10 mg / m 2 , 12 mg / m 2 , 14 mg / m 2 , 15 mg / m 2 , 16 mg / m 2 , 18 mg / m 2 , 20 mg / m 2 , 23 mg / m 2 , 27 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , 50 mg / m 2 , 55 mg / m 2 , 60 mg / m 2 , 65 mg / m 2 , 70 mg / m 2 , 80 mg / m 2 , 85 mg / m 2 , 90 mg / m 2 , a range between any two of these values or any value between 8 mg / m 2 –90 mg / m 2 . In some embodiments, for a subject, the daily dose of the PLK1 inhibitor (e.g., onvansertib) can be adjusted (e.g., increased or decreased within a range) during treatment or during a single cycle of treatment (e.g., the first cycle, the second cycle, the third cycle, and subsequent cycles). In some embodiments, the daily dose of the PLK1 inhibitor (e.g., onvansertib) is 12 mg / m 2 , 15 mg / m 2 , 18 mg / m 2 or 24 mg / m 2 . In some embodiments, the daily dose of the PLK1 inhibitor (e.g., onvansertib) is 15 mg / m 2 . The daily dose of the PLK1 inhibitor (e.g., onvansertib) can vary for each cycle of treatment. For example, the daily dose of the PLK1 inhibitor (e.g., onvansertib) for the first cycle can be 12 mg / m 2, and the daily dose of the PLK1 inhibitor (e.g., onvansertib) for the second cycle can be increased to, for example, 15 mg / m 2 . In some embodiments, the daily dose of the PLK1 inhibitor (e.g., onvansertib) for the second cycle can then be increased to, for example, 18 mg / m 2 . Without being bound by any particular theory, it is believed that the mg / m 2 dose herein is a dose based on body surface area (BSA) and corresponds to a flat dose in the range of 20 mg to 45 mg.

[0079] When the PLK1 inhibitor is administered alone or in combination with one or more additional cancer therapeutics (e.g., FOLFIRI and bevacizumab), the maximum concentration (C max ) of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject (during or after treatment) can be from about 100 nmol / L to about 1500 nmol / L. For example, when the PLK1 inhibitor is administered alone or in combination with one or more additional cancer therapeutics (e.g., FOLFIRI and bevacizumab), the C max of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject can be the following or about the following: 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 and 1500 nmol / L.

[0080] When the PLK1 inhibitor is administered alone or in combination with one or more additional cancer therapeutics (e.g., FOLFIRI and bevacizumab), the area under the curve (AUC) of the graph of the concentration of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject over time (e.g., the AUC for the first 24 hours after administration 0-24) can be from about 1000 nmol / L·hour to about 400000 nmol / L·hour. For example, when the PLK1 inhibitor is administered alone or in combination with one or more additional cancer therapeutics (e.g., FOLFIRI and bevacizumab), the AUC of the graph of the concentration of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject over time (e.g., the AUC in the first 24 hours after administration) 0-24 ) can be the following or about the following: 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, the range between any two of these values, or any value between 1000 nmol / L·hour and 400000 nmol / L·hour.

[0081] When the PLK1 inhibitor is administered alone or in combination with one or more additional cancer therapeutics (e.g., FOLFIRI and bevacizumab), the time (T max ) to reach the maximum concentration of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject can be from about 1 hour to about 5 hours. For example, when the PLK1 inhibitor is administered alone or in combination with one or more additional cancer therapeutics (e.g., FOLFIRI and bevacizumab), the time (T max ) to reach the maximum concentration of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject can be the following or about the following: 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, the range between any two of these values, or any value between 1 hour and 5 hours.

[0082] When the PLK1 inhibitor is administered alone or in combination with one or more additional cancer therapeutics (e.g., FOLFIRI and bevacizumab), the elimination half-life (T 1 / 2 ) of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject can be from about 10 hours to about 60 hours. For example, when the PLK1 inhibitor is administered alone or in combination with one or more additional cancer therapeutics (e.g., FOLFIRI and bevacizumab), the elimination half-life (T 1 / 2) may be the following or about the following: 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.

[0083] Anti - angiogenic Agent

[0084] Angiogenesis is known to be the inappropriate formation of new blood vessels and is generally necessary for cancer metastasis. "Angiogenesis" typically occurs in tumors when endothelial cells secrete a set of growth factors that are mitogenic for endothelial cells, causing elongation and proliferation of endothelial cells, which results in the generation of new blood vessels. Inhibition of angiogenesis can cause tumor regression in animal models and has also been found to be effective in the treatment of metastatic cancer in humans. The terms "anti-angiogenic agent" and "angiogenesis inhibitor" shall have their ordinary meanings and may also be used interchangeably herein to refer to any agent that can inhibit angiogenesis. Several anti-angiogenic agents have been approved for use in humans to treat cancer. In some embodiments of the methods disclosed herein, the subject has not received any prior treatment that includes administration of an angiogenesis inhibitor.

[0085] An angiogenesis inhibitor and / or anti-angiogenic agent may be capable of inhibiting VEGF-A, VEGFR-1, VEGFR-2, VEGFR-3, EGFR, HER2, PDGFR family proteins, RAF, Kit (or c-Kit), FLT3, CSF-1R, RET, Abl, Itk, LcK, c-FMS, FGFR family proteins, c-Met, PlGF, TNF-α, IFN, ILs, bFGF, mTOR, or any combination thereof. An anti-angiogenic agent can be afatinib axitinib bevacizumab cabozantinib cetuximab erlotinib everolimus gefitinib imatinib lapatinib lenalidomide lenvatinib mesylate necitumumab (Portrazza TM )、neratinib panitumumab pazopanib pertuzumab ramucirumab regorafenib sorafenib Sunitinib Thalidomide (Synovir, )、Trastuzumab Vandetanib or ziv-aflibercept The angiogenesis inhibitor can be afatinib Axitinib Bevacizumab Cabozantinib Cetuximab Erlotinib Everolimus Gefitinib Imatinib Lapatinib Lenalidomide Lenvatinib mesylate Necitumumab (Portrazza TM )、Neratinib Panitumumab Pazopanib Pertuzumab Ramucirumab Regorafenib Sorafenib Sunitinib Thalidomide (Synovir, )、Trastuzumab Vandetanib or ziv-aflibercept

[0086] Bevacizumab (also referred to herein as "bev") can be used in combination with chemotherapy in both first-line and second-line therapies for treating cancer. Other anti-angiogenic agents, such as ramucirumab and aflibercept, can be used in the second-line setting. Prior to the methods disclosed herein, overall survival (OS) and median progression-free survival (mPFS) benefits in the second-line have been shown to be independent of whether bevacizumab was given in the first-line (e.g., the subject received prior therapy including anti-angiogenesis). For example, in a study examining OS and PFS in patients with and without prior bevacizumab treatment, the OS was found to be 13.9 months for those without prior bevacizumab, compared to 12.5 months for those with prior bevacizumab. For PFS, the mPFS was 6.9 months for those without prior bevacizumab, compared to 6.7 months for those with prior bevacizumab. Anti-angiogenic therapy progressively improved the response rate in patients with prior bevacizumab compared to patients without prior bevacizumab. The objective response rate for patients who had received prior bevacizumab was 5% to 13%, compared to an objective response rate of ~25% for patients without prior bevacizumab.

[0087] As disclosed herein, combination therapies of anti-angiogenic agents and PLK1 inhibitors (including onvansertib) can surprisingly result in significantly enhanced potency against metastatic cancer (e.g., metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma or combinations thereof) in subjects who have not received prior therapy including anti-angiogenesis, resulting in tumor regression and cancer survival. The tumor regression and cancer survival rate / duration resulting from the combination can surprisingly be synergistic (i.e., beyond additive, superior to the cumulative anti-tumor potency caused by the anti-angiogenic agent alone and the PLK1 inhibitor alone). The PLK1 inhibitor can be onvansertib. Methods, compositions, and kits are provided herein for treating metastatic cancer in a subject (e.g., a human patient having metastatic cancer). Methods, compositions, and kits are provided herein for treating cancer in a subject (e.g., a human patient having cancer), wherein the subject has not received any prior therapy including anti-angiogenesis. The methods include administering an anti-angiogenic agent and a PLK1 inhibitor to the patient in a manner sufficient to inhibit cancer progression. For example, the anti-angiogenic agent and the PLK1 inhibitor can be administered to the subject having cancer simultaneously, separately, or sequentially.

[0088] In some embodiments, administration of a PLK1 inhibitor and an anti-angiogenic agent synergistically reduces or inhibits the progression of metastatic cancer relative to the additive effects of treatment with a PLK1 inhibitor alone, treatment with an anti-angiogenic agent alone, and / or treatment with a PLK1 inhibitor alone and an anti-angiogenic agent alone.

[0089] The inhibition or reduction of cancer progression that can be achieved using a PLK1 inhibitor (e.g., onvansertib) and an anti-angiogenic agent (e.g., bevacizumab) by the methods disclosed herein is not merely additive, but rather enhanced or synergistic (i.e., the inhibition is greater than the combined inhibition of progression caused by the anti-angiogenic agent alone plus the PLK1 inhibitor alone). The enhanced or synergistic potency or inhibition of any combination of the anti-angiogenic agents and PLK1 inhibitors of the present disclosure can be different in different embodiments. In some embodiments, the enhanced or synergistic potency or inhibition of any combination of the anti-angiogenic agents and PLK1 inhibitors of the present disclosure is higher than the combined inhibition of progression caused by the anti-angiogenic agent alone plus the PLK1 inhibitor alone by less than, by about less than, by at least less than, by at least about less than, by at most less than, or by at most about less than: 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 range between any two of these values.

[0090] The molar ratio of a PLK1 inhibitor (e.g., onvansertib) to an anti-angiogenic agent (e.g., bevacizumab) can be, for example, about 1:200, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 100:1, 1000:1, 2000:1, or 5000:1 or a number or range between any two of these values. In some embodiments, the enhanced or synergistic potency or inhibition of cancer progression caused by the combination of an anti-angiogenic agent and a PLK1 inhibitor (e.g., onvansertib) is lower than, about lower than, at least lower than, at least about lower than, at most lower than, or at most about lower than the combined inhibition of progression caused by the anti-angiogenic agent alone plus the PLK1 inhibitor (e.g., onvansertib) alone by: 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300% or a number or range between any two of these values. For example, the combination of an anti-angiogenic agent and a PLK1 inhibitor can cause 50%, 60%, 70%, 80%, 90% or more inhibition of cancer progression (cancer cell viability is 50%, 40%, 30%, 20%, 10% or less), while under the same conditions, the combined inhibition of the anti-angiogenic agent alone plus the PLK1 inhibitor alone can be 10%, 20%, 25%, 30% or less inhibition of cancer progression (cancer cell viability is 90%, 80%, 75%, 70% or more). Thus, the enhanced or synergistic potency or inhibition of cancer progression caused by the combination of an anti-angiogenic agent and a PLK1 inhibitor is higher than the combined inhibition of progression caused by the anti-angiogenic agent alone plus the PLK1 inhibitor alone by, for example, 50%, 60%, 70%, 80%, 90%, 100% or more. In some embodiments, the anti-angiogenic agent is bevacizumab and the PLK1 inhibitor is onvansertib.

[0091] The anti-angiogenic agent and the PLK1 inhibitor can be administered to a patient in any manner considered effective for treating cancer. The anti-angiogenic agent can be administered together with the PLK1 inhibitor or separately from the PLK1 inhibitor. When administered separately, the anti-angiogenic agent can be administered before or after the PLK1 inhibitor or in different dosing cycles. The administration of the PLK1 inhibitor can be oral administration. The administration of the anti-angiogenic agent can be intravenous administration or oral administration.

[0092] A PLK1 inhibitor and an anti-angiogenic agent can be administered simultaneously or sequentially. In some embodiments, it may be advantageous to administer a PLK1 inhibitor (e.g., onvansertib) to a subject before administering an anti-angiogenic agent (e.g., bevacizumab) to the subject, e.g., on one or more days or daily on the days of administering the PLK1 inhibitor and the anti-angiogenic agent to the subject. The time interval between administering the PLK1 inhibitor and administering the anti-angiogenic agent can be, for example, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, a range between any two of these values, or any value between 30 minutes and 12 hours. In some embodiments, both the PLK1 inhibitor (e.g., onvansertib) and the anti-angiogenic agent (e.g., bevacizumab) are administered to the subject on 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the days in a cycle (e.g., each cycle during combination therapy), or on at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the days, and optionally, on each day of administering both, the PLK1 inhibitor is administered to the subject before the anti-angiogenic agent, e.g., the PLK1 inhibitor is administered 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, a range between any two of these values, or any value between 30 minutes and 12 hours before the anti-angiogenic agent is administered.

[0093] The anti-angiogenic agent and the PLK1 inhibitor can each be administered on any schedule, such as daily or once or more than once a week; once, twice, three times, four times, five times, six times, or seven times (daily) a week; for one week or more than one week; and so on. In some embodiments, the anti-angiogenic agent and the PLK1 inhibitor are each administered to the patient in a cycle of at least twice a week. In other embodiments, the anti-angiogenic agent and the PLK1 inhibitor are each administered to the patient in a cycle of at least five times a week. In some embodiments, the PLK1 inhibitor is administered daily, and the anti-angiogenic agent is administered daily, weekly, bi-weekly, every four weeks, every five weeks, or monthly. In additional embodiments, the patient undergoes at least two cycles of administration. The patient can undergo one cycle or more than one cycle of administration, e.g., two cycles, three cycles, three cycles, four cycles, five cycles, or more cycles. The administration of two adjacent cycles can be continuous, i.e., there is no interruption between the last day of the first cycle and the first day of the second cycle. In some embodiments, the administration of two adjacent cycles has an interruption therebetween, i.e., an interval between the last day of the first cycle and the first day of the second cycle. The interruption (i.e., the interval) can be the following or at least the following: one day, two days, three days, five days, seven days, ten days, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or a number or range between any two of these values. In some embodiments, the patient undergoes three or four cycles of administration, where each cycle includes at least five times a week (e.g., 5 days a week). Each cycle in a multi-cycle administration can have the same or different dosing schedules. For example, one cycle in a multi-cycle administration can be daily administration of the PLK1 inhibitor and the anti-angiogenic agent for five consecutive days and interruption for two days of the week, for four weeks, and one or more other cycles in the same multi-cycle administration can be daily administration of the PLK1 inhibitor and the anti-angiogenic agent for 28 consecutive days in a four-week period. The PLK1 inhibitor can be administered for at least four days in a cycle. In some embodiments, the PLK1 inhibitor is not administered for at least one day in a cycle.

[0094] An anti-angiogenic agent can be administered to a patient at any suitable dose, e.g., a dose of about, at least, or at most: 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, 1500 mg / kg, 2000 mg / kg, or a number between any two of these values. A dose unit based on body weight (mg / kg) can be converted to another unit (e.g., mg / m 2 ) as will be understood by those skilled in the art. An anti-angiogenic agent (e.g., bevacizumab) can be administered at about 1 mg / kg - 20 mg / kg. In some embodiments, the anti-angiogenic agent is bevacizumab, which is administered at a dose of about, at least, or at most: 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 200 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1000 mg / kg, or a number between any two of these values. Bevacizumab can be administered at about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, or about 15 mg / kg.

[0095] An anti-angiogenic agent can be administered to a patient once daily, twice daily, or three times daily. The anti-angiogenic agent can be administered daily, weekly, bi-weekly, tri-weekly, monthly, or monthly. In some embodiments, the anti-angiogenic agent is administered daily, weekly, bi-weekly, tri-weekly, monthly, or monthly in a cycle of 7 - 56 days. In some embodiments, the anti-angiogenic agent is administered in a cycle of 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 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, 29 days, 30 days, 32 days, 35 days, 42 days, 49 days, or 56 days. In some embodiments, the anti-angiogenic agent is administered for 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, 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, 29 days, 30 days, 32 days, 35 days, 42 days, 49 days, or 56 days in a cycle. In some embodiments, the anti-angiogenic agent is administered on the 1st day, 2nd day, 3rd day, 4th day, 5th day, 6th day, 7th day, 8th day, 9th day, 10th day, 11th day, 12th day, 13th day, 14th day, 15th day, 16th day, 17th day, 18th day, 19th day, 20th day, 21st day, 22nd day, 23rd day, 24th day, 25th day, 26th day, 27th day, 28th day, 29th day, 30th day, 31st day, 32nd day, 33rd day, 34th day, 35th day, 36th day, 37th day, 38th day, 39th day, 40th day, 41st day, 42nd day, 43rd day, 44th day, 45th day, 46th day, 47th day, 48th day, 49th day, 50th day, 51st day, 52nd day, 52nd day, 53rd day, 54th day, 55th day, and / or 56th day. In some embodiments, the anti-angiogenic agent is not administered on the 1st day, 2nd day, 3rd day, 4th day, 5th day, 6th day, 7th day, 8th day, 9th day, 10th day, 11th day, 12th day, 13th day, 14th day, 15th day, 16th day, 17th day, 18th day, 19th day, 20th day, 21st day, 22nd day, 23rd day, 24th day, 25th day, 26th day, 27th day, 28th day, 29th day, 31st day, 32nd day, 33rd day, 34th day, 35th day, 36th day, 37th day, 38th day, 39th day, 40th day, 41st day, 42nd day, 43rd day, 44th day, 45th day, 46th day, 47th day, 48th day, 49th day, 50th day, 51st day, 52nd day, 52nd day, 53rd day, 54th day, 55th day, and / or 56th day.

[0096] In some embodiments, the subject has not received any prior treatment that includes administration of an angiogenesis inhibitor. The angiogenesis inhibitor can be the same as an anti-angiogenic agent. Any agent that can inhibit angiogenesis, now known or later discovered, can be used in these methods (e.g., an anti-angiogenic agent), including an anti-angiogenic agent that inhibits the activity of one or more growth factors or any other protein that promotes angiogenesis. For example, the angiogenesis inhibitor and / or anti-angiogenic agent can be capable of inhibiting a vascular endothelial growth factor family protein or the signaling pathway of the protein, such as VEGF-A, VEGFR-1, VEGFR-2, VEGFR-3, and PlGF. Other proteins and pathways that can be targeted by anti-angiogenic agents include, but are not limited to: epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), platelet-derived growth factor receptor (PDGFR) family proteins, RAF kinase; tyrosine kinases, including Kit (or c-Kit), RET, Abl, Itk, LcK, c-Met, and FLT3; colony-stimulating factor 1 receptor (CSF-1R) and c-FMS, fibroblast growth factor receptor (FGFR) family proteins, tumor necrosis factor alpha family proteins, tumor), interferon (IFN), interleukin (IL), basic fibroblast growth factor (bFGF), mammalian target of rapamycin (mTOR), or any combination thereof. The anti-angiogenic agent can be a small molecule, an antibody or fragment thereof, an aptamer, RNA (e.g., miRNA), or any other agent capable of inhibiting angiogenesis. In some embodiments, the anti-angiogenic agent or angiogenesis inhibitor is bevacizumab (e.g., ).

[0097] Similarly, any PLK1 inhibitor, now known or later discovered, can be used in these methods, including a PLK1 inhibitor that is selective for PLK1 and a PLK1 inhibitor that also inhibits the activity of other proteins. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, pyridopyrimidine, aminopyrimidine, substituted thiazolinone, pteridine derivative, dihydroimidazo[1,5-f]pteridine, meta-substituted thiazolinone, benzyl styryl sulfone analog, stilbene derivative, or a combination thereof. In some of these embodiments, the PLK1 inhibitor is onvansertib, BI2536, Volasertib (BI6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, or Ro3280.

[0098] The PLK1 inhibitor can be onvansertib. In these embodiments, onvansertib is administered to a patient at any suitable dose, such as a dose less than 12 mg / m 2 2, a dose less than or equal to 24 mg / m 2 2, or a dose greater than 24 mg / m 2 2. In some embodiments, onvansertib is administered to a patient at about 12 mg / m 2 , at about 15 mg / m 2 or at about 18 mg / m 2 2. In some embodiments, onvansertib is administered to a patient daily. In additional embodiments, onvansertib is administered in cycles of daily onvansertib administration for 3 - 10 days, wherein onvansertib is not administered for 2 - 16 days. In some embodiments, onvansertib is administered to a patient in cycles of at least five times within a week. The patient may undergo two, three, or four cycles of administration. In some embodiments, the patient undergoes four cycles of administration in cycles of at least five days of daily onvansertib administration, wherein onvansertib is not administered for 1 - 2 days.

[0099] In some embodiments, the PLK1 inhibitor, alone or in combination with an anti - angiogenic agent, is administered to a patient who takes a drug holiday after undergoing one or more cycles of administration. As used herein, a drug holiday refers to the period of time when the patient stops taking the PLK1 inhibitor and / or the anti - angiogenic agent. The drug holiday can be from several days to several months. In some embodiments, the drug holiday can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or any value or range between any two of these values.

[0100] As can be understood by those skilled in the art, the amount and timing of administration of the anti - angiogenic agent and the PLK1 inhibitor can depend on the type (species, sex, age, weight, etc.) and condition of the treated subject and the severity of the disease or condition being treated. The anti - angiogenic agent and the PLK1 inhibitor can be formulated as a single pharmaceutical composition or as two separate pharmaceutical compositions. The active ingredients can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as encapsulated in hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules respectively, encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macro - droplet emulsions.

[0101] The methods, compositions, kits, and systems disclosed herein can be applied to different types of subjects. For example, the subject can be a subject undergoing cancer treatment, a subject in cancer remission, a subject who has received one or more cancer treatments, or a subject suspected of having cancer. The subject can have stage I cancer, stage II cancer, stage III cancer, and / or stage IV cancer. In some embodiments, the subject has stage IV cancer. In some embodiments, the subject has metastatic cancer. In some embodiments, the subject has not received any prior treatment that includes inhibition of angiogenesis.

[0102] Treatment according to the present disclosure may include administering a PLK1 inhibitor (onvansertib) for a desired duration in a cycle. Administration of the PLK1 inhibitor (and / or anti-angiogenic agent) may be daily, or with one or more breaks between administration days. The break may 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. When administering a PLK1 inhibitor (and / or anti-angiogenic agent) to a patient, the administration may be once, twice, three times, four times or more per day. The administration may be, for example, once every two days, once every three days, once every four days, once every five days, once every six days or once every seven days. The length of the desired duration may vary, 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, 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. Each treatment cycle may 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 treatment may include administering a PLK1 inhibitor (e.g., onvansertib) and / or anti-angiogenic agent for 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 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 a cycle (e.g., in a cycle of at least 21 days (e.g., 21 days to 28 days)). In some embodiments, treatment may include administering a PLK1 inhibitor (e.g., onvansertib) and / or anti-angiogenic agent for the following or at least the following in a cycle (e.g., a cycle of at least 21 days (e.g., 21 days to 28 days)): 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or a range between any two of these values. Administration of a PLK1 inhibitor (e.g., onvansertib) and / or anti-angiogenic agent in a single cycle of treatment may be continuous or have one or more intervals (e.g., a break of one day or two days). In some embodiments, treatment includes administering a PLK1 inhibitor (e.g., onvansertib) for 5 days in a cycle of 21 days to 28 days.

[0103] In some embodiments, a PLK1 inhibitor (e.g., onvansertib) is administered to a subject in need thereof for 20 days (e.g., days 1-10 and days 15-24) during a 28-day cycle. The 20 days can be, for example, 10 consecutive daily administrations for 10 days (e.g., days 1-10) and another 10 consecutive daily administrations (e.g., days 15-24) for 10 days, or 10 consecutive daily administrations for four groups of 5 days (e.g., days 1-5, days 8-12, days 15-19, and days 22-26). In some embodiments, for example when a patient is identified as having low tolerance to the PLK1 inhibitor, the PLK1 inhibitor is administered to a subject in need thereof for 10 days (e.g., days 1-5 and days 15-19) during a 28-day cycle. The 10 days can be, for example, 10 consecutive daily administrations for 10 days (e.g., days 1-10) or two 10 consecutive daily administrations each for 5 days (e.g., days 1-5 and days 15-19). In some embodiments, the PLK1 inhibitor is administered daily to a subject in need thereof throughout the cycle (e.g., daily for 28 days in a 28-day cycle). Depending on the need to inhibit / reverse cancer progression in the subject, the subject can receive one, two, three, four, five, six, or more cycles of treatment. For combination therapy, the administration cycles, dosing schedules, and / or amounts of the doses of the anti-angiogenic agent and the PLK1 inhibitor can be the same or different. For combination therapy, the administration cycles, dosing schedules, and / or amounts of the doses of the anti-angiogenic agent can be adjusted according to the administration cycles, dosing schedules, and / or amounts of the doses of the PLK1 inhibitor. For example, an anti-angiogenic agent (e.g., bevacizumab) can be administered in four 7-day cycles (e.g., dosed daily on days 1-5 and not dosed on days 6-7, repeated for 4 weeks), which corresponds to the 28-day cycle of the PLK1 inhibitor (e.g., onvansertib) administration.

[0104] Treatment can include administering a PLK1 inhibitor (e.g., onvansertib) at 6 mg / m 2 -90 mg / m 2 or at about 12 mg / m 2 -90 mg / m 2 , for example, as a daily dose. For example, treatment can include administering a PLK1 inhibitor (e.g., onvansertib) at or at about the following daily: 6 mg / m 2 , 8 mg / m 2 , 10 mg / m 2 , 12 mg / m 2 , 14 mg / m 2 , 16 mg / m 2 , 18 mg / m 2 , 20 mg / m2 , 23 mg / m 2 , 27 mg / m 2 , 30 mg / m 2 , 35 mg / m 2 , 40 mg / m 2 , 45 mg / m 2 , 50 mg / m 2 , 55 mg / m 2 , 60 mg / m 2 , 65 mg / m 2 , 70 mg / m 2 , 80 mg / m 2 , 85 mg / m 2 , 90 mg / m 2 , A number or range between any two of these values, or any value between 8 mg / m 2 –90 mg / m 2 . In some embodiments, for a subject, the daily dose of a PLK1 inhibitor (e.g., onvansertib) can be adjusted (e.g., increased or decreased within a range) during treatment or during a single cycle of treatment (e.g., the first cycle, the second cycle, the third cycle, and subsequent cycles). In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered at 12 mg / m 2 for 20 days (e.g., days 1 - 10 and days 15 - 24) during a 28 - day cycle. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered at 15 mg / m 2 for 10 days (e.g., days 1 - 5 and days 15 - 19) during a 28 - day cycle. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered at 8 mg / m 2 or 10 mg / m 2 daily (e.g., days 11 - 28) during a 28 - day cycle. In some embodiments, for a subject, the daily dose of a PLK1 inhibitor (e.g., onvansertib) can be adjusted (e.g., increased or decreased within a range) during treatment or during a single cycle of treatment (e.g., the first cycle, the second cycle, the third cycle, and subsequent cycles). In some embodiments, the PLK1 inhibitor is administered at 12 mg / m 2 or at approximately 12 mg / m 2 . In some embodiments, the PLK1 inhibitor is administered at 15 mg / m 2 or at approximately 15 mg / m 2 . In some embodiments, the PLK1 inhibitor is administered at 18 mg / m 2 or at approximately 18 mg / m2 Administration.

[0105] When the PLK1 inhibitor is administered alone or in combination with an anti-angiogenic agent, the maximum concentration (C max ) of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject (during or after treatment) can be from about 100 nmol / L to about 1500 nmol / L. For example, when the PLK1 inhibitor is administered alone or in combination with an anti-angiogenic agent, the C max of the PLK1 inhibitor in the blood of the subject can be the following or about the following: 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, the range between any two of these values, or any value between 200 nmol / L and 1500 nmol / L.

[0106] When the PLK1 inhibitor is administered alone or in combination with an anti-angiogenic agent, the area under the curve (AUC) of the graph of the concentration of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject over time (e.g., the AUC 0-24 ) can be from about 1000 nmol / L·h to about 400000 nmol / L·h. For example, when the PLK1 inhibitor is administered alone or in combination with an anti-angiogenic agent, the AUC of the graph of the concentration of the PLK1 inhibitor in the blood of the subject over time (e.g., the AUC 0-24 ) can be the following or about the following: 1000 nmol / L·h, 5000 nmol / L·h, 10000 nmol / L·h, 15000 nmol / L·h, 20000 nmol / L·h, 25000 nmol / L·h, 30000 nmol / L·h, 35000 nmol / L·h, 40000 nmol / L·h, the range between any two of these values, or any value between 1000 nmol / L·h and 400000 nmol / L·h.

[0107] When the PLK1 inhibitor is administered alone or in combination with an anti-angiogenic agent, the time (T max) can be from about 1 hour to about 5 hours. For example, when the PLK1 inhibitor is administered alone or in combination with an anti-angiogenic agent, the time (T max ) can be the following or about the following: 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.

[0108] When the PLK1 inhibitor is administered alone or in combination with an anti-angiogenic agent, the elimination half-life (T 1 / 2 ) of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject can be from about 10 hours to about 60 hours. For example, when the PLK1 inhibitor is administered alone or in combination with an anti-angiogenic agent, the elimination half-life (T 1 / 2 ) of the PLK1 inhibitor in the blood of the subject can be the following or about the following: 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.

[0109] As described herein, the methods, compositions, and kits described herein can reduce or inhibit the progression of cancer. In some embodiments, relative to an untreated subject, reducing or inhibiting includes inhibiting the growth of one or more tumors in the subject and / or reducing the number of cancer cells detected in the subject by at least about 25%, 30%, 40%, 50%, 60%, or 70%. In some embodiments, relative to the subject prior to administration of the PLK1 inhibitor and the anti-angiogenic agent, reducing or inhibiting includes inhibiting the growth of one or more tumors in the subject and / or reducing the number of cancer cells detected in the subject by at least about 25%, 30%, 40%, 50%, 60%, or 70%. After one or more treatment cycles, the growth of at least one of the one or more tumors in the subject can be reduced, for example, by at least about 25%, 30%, 40%, 50%, 60%, or 70%. In some embodiments, after one or more treatment cycles, the size / volume of at least one of the one or more tumors in the subject is reduced by at least about 25%, 30%, 40%, 50%, 60%, or 70%.

[0110] Additional Cancer Therapeutic Agent or Therapy

[0111] The methods, compositions, and kits disclosed herein can be used to treat cancer. In some embodiments, methods for treating cancer (e.g., mCRC) include administering an anti-angiogenic agent and a PLK1 inhibitor (e.g., onvansertib) to a subject (e.g., a patient) in need thereof. The method can include administering a therapeutically effective amount of the anti-angiogenic agent and a therapeutically effective amount of the PLK1 inhibitor. The treatment can include administering at least one additional cancer therapeutic agent or cancer therapy. In some embodiments, the subject has not received any prior treatment that includes inhibition of angiogenesis. In some embodiments, the subject has not received any prior treatment for inhibition of angiogenesis (e.g., the subject has not received any prior treatment with bevacizumab alone, or the subject has not received any prior treatment with bevacizumab in combination with one or more anti-cancer agents or one or more anti-cancer therapies (e.g., chemotherapy)). In some embodiments, the subject has not received any prior cancer treatment. The subject can be, for example, a subject without a prior cancer diagnosis. In some embodiments, the subject has not received any prior treatment with bevacizumab and / or chemotherapy.

[0112] Non-limiting examples of additional cancer therapeutic agents or cancer therapies can include surgery, chemotherapy, radiotherapy (including external beam, stereotactic, and intraoperative radiotherapy and brachytherapy), bone marrow transplantation, immunotherapy, targeted drug therapy, cryoablation, or radiofrequency ablation. In the case where the cancer is colorectal cancer, examples of treatment include surgery, radiofrequency ablation, cryoablation, radiotherapy, chemotherapy (including drugs comprising capecitabine, 5-fluorouracil (5-FU), irinotecan, oxaliplatin, trifluridine / tipiracil), targeted therapies (including anti-angiogenic therapies using, for example, bevacizumab, regorafenib, ziv-aflibercept, or ramucirumab; immunotherapies using, for example, pembrolizumab, nivolumab, or ipilimumab; and PLK1 inhibitors).

[0113] Additional cancer therapeutics or cancer therapies can be chemotherapy, such as FOLFIRI, FOLFOX, XELOX (CAPOX), FOLFOXIRI, or combinations thereof. Chemotherapy regimens using fluorouracil are standard treatments for advanced colorectal cancer. Fluorouracil is a pyrimidine analogue and antimetabolite that incorporates into DNA molecules and halts synthesis, thus preventing cancer cell replication. Examples of these regimens include FOLFOX and FOLFIRI. Additional cancer therapeutics or cancer therapies can include a therapeutically effective amount of FOLFIRI. FOLFIRI is a chemotherapy mixture containing leucovorin (folinic acid), fluorouracil, and irinotecan hydrochloride. Leucovorin is a vitamin B derivative and increases the cytotoxicity of fluorouracil in this combination. Irinotecan is a topoisomerase inhibitor that prevents DNA unwinding and replication. FOLFIRI is often used in combination with other therapeutic agents, such as bevacizumab, to increase efficacy and response rates.

[0114] FOLFOX is a chemotherapy regimen used to treat colorectal cancer and consists of leucovorin, fluorouracil, and oxaliplatin (Eloxatin). Depending on how these three drugs are administered, FOLFOX can be broken down into other subtypes, such as FOLFOX-4, FOLFOX-6, and FOLFOX-7. FOLFOX is commonly used to treat colorectal cancer. It can also be used to treat pancreatic cancer and certain other cancers. FOLFOX is typically used as adjuvant therapy (in addition to the primary therapy) for advanced cancers. However, FOLFOX can also be used as a first-line therapy for colorectal adenocarcinoma, the most common type of colon cancer. In this combination, oxaliplatin shows a synergistic effect with fluorouracil with little toxicity overlap.

[0115] For decades, fluorouracil has been the only drug proven to have anti - colorectal cancer activity and is typically used in combination with leucovorin. Oxaliplatin and capecitabine are two relatively new drugs used to treat colorectal cancer. These drugs have been found to act synergistically both in vivo and in vitro. The chemotherapy combination of oxaliplatin and capecitabine is called XELOX, which is highly active in metastatic colorectal cancer (mCRC). Capecitabine has been shown to be highly potent as a first - line treatment for MCRC. It is an oral fluoropyrimidine rationally designed to preferentially produce FU at the tumor site via a three - step enzymatic process that takes advantage of the significantly higher activity of thymidine phosphorylase (TP) in tumors compared to healthy tissues. Compared to combination therapies such as FOLFIRI and FOLFOX with leucovorin and fluorouracil, capecitabine causes fewer adverse reactions such as diarrhea, stomatitis, nausea, alopecia, and neutropenia, resulting in a lower incidence of neutropenic fever / sepsis and related hospitalizations, although hand - foot syndrome (HFS) occurs more frequently in the case of capecitabine administration. Oxaliplatin is a third - generation cisplatin analogue and an organoplatinum complex and is generally classified as an alkylating agent, although it does not actually add an alkyl group to DNA. Oxaliplatin is an integral part of various fluorouracil regimens such as FOLFOX, which has become the standard treatment for metastatic and node - positive colorectal cancer.

[0116] Another combination therapy of oxaliplatin and fluorouracil is called FOLFOXIRI, which includes leucovorin, fluorouracil, oxaliplatin, and irinotecan. FOLFOXIRI is also an approved chemotherapy regimen for the treatment of advanced colorectal cancer and is usually given in combination with bevacizumab.

[0117] Additional cancer therapeutics can include FOLFIRI, bevacizumab, abiraterone, FOLFOX, anti - EGFR agents, KRAS - directed inhibitors, gemcitabine, abraxane, nanoliposomal irinotecan, 5 - FU, or combinations thereof. The PLK1 inhibitor and the cancer therapeutic or cancer therapy can be administered simultaneously or sequentially. In some embodiments, the additional cancer therapeutic or therapy includes FOLFIRI, abiraterone, FOLFOX, anti - EGFR agents, KRAS - directed inhibitors, gemcitabine, abraxane, nanoliposomal irinotecan, 5 - FU, or combinations thereof. In some embodiments, the anti - EGFR agent is optionally cetuximab. In some embodiments, the KRAS - directed inhibitor is optionally a G12C inhibitor, a G12D inhibitor, or combinations thereof. In some embodiments, the additional cancer therapy is FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), or combinations thereof.

[0118] Methods for Predicting / Determining Cancer Treatment Efficacy and Status

[0119] Disclosed herein are methods for treating cancer. In some embodiments, the methods include: administering a PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, thereby reducing or inhibiting the progression of metastatic cancer, wherein the subject has not received any prior treatment that includes inhibiting angiogenesis. It is contemplated that the methods described herein using a combination of an anti-angiogenic agent and a PLK1 inhibitor are effective against various metastatic cancers, such as metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or combinations thereof.

[0120] The methods can include one or more of the following: (1) determining the cancer status of the subject, (2) determining the responsiveness of the subject to PLK1 inhibitor treatment, and (3) administering one or more cancer therapeutic agents or therapies for cancer.

[0121] In some embodiments, administration of a PLK1 inhibitor and an anti-angiogenic agent improves one or more treatment effects in the treated subject relative to a control or baseline. One or more treatment effects can include the size of the tumor arising from metastatic cancer, objective response rate (ORR), duration of response, time to response, progression-free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allele burden, or combinations thereof. In some embodiments, administration of a PLK1 inhibitor and an anti-angiogenic agent improves the ORR of the subject, improves the PFS of the treated subject, improves the OS of the treated subject, improves the DCR of the treated subject, reduces the oncogenic allele burden of the treated subject, or combinations thereof, relative to a subject who has received prior treatment that includes inhibiting angiogenesis.

[0122] Disclosed herein are methods for improving objective response rate (ORR), progression-free survival (PFS), or both, in a subject with metastatic cancer. In some embodiments, the methods include administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby improving the ORR and / or PFS of the subject, wherein the subject has not received any prior treatment that includes inhibiting angiogenesis.

[0123] In some embodiments, administration of a PLK1 inhibitor and an anti-angiogenic agent synergistically improves the ORR and / or PFS of the treated subject relative to treatment with a PLK1 inhibitor alone, treatment with an anti-angiogenic agent alone, and / or the additive effect of treatment with a PLK1 inhibitor alone and treatment with an anti-angiogenic agent alone.

[0124] Tumor evaluation and assessment of tumor burden and treatment efficacy can be based on the RECIST criteria (Therasse et al. 2000, New Guidelines to Evaluate the Response to Treatment in Solid Tumors, Journal of National Melanoma Institute, Vol. 92; 205-16), and in some embodiments, are conducted in the methods disclosed herein according to the Revised RECIST Guidelines (version 1.1) (Eisenhauer et al. 2009, New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Melanoma, 45(2):228-47.), which are hereby incorporated by reference in their entirety.

[0125] In some embodiments, treatment results in a durable response in the subject after treatment is discontinued. "Durable response" can refer to a continued effect on reducing tumor growth after treatment is discontinued. For example, the tumor size can remain the same or smaller compared to the size at the start of the administration phase. In some embodiments, the durable response has a duration that is at least as long as the treatment duration, at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times the length of the treatment duration.

[0126] The treatment methods disclosed herein can result in a partial response or a complete response. As used herein, "complete response" or "CR" can refer to the disappearance of all target lesions; "partial response" or "PR" can refer to a reduction of at least 30% in the sum of the longest diameters (SLD) of the target lesions, referenced to the baseline SLD; and "stable disease" or "SD" can refer to target lesions that have neither shrunk sufficiently to meet PR nor increased sufficiently to meet PD, referenced to the smallest SLD since the start of treatment. As used herein, "objective response rate" (ORR) can refer to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0127] The treatment methods disclosed herein can lead to an increase in progression-free survival (PFS) and overall survival (OS) of subjects administered the combination therapy. As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment that the disease being treated (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient experiences a complete response or a partial response, as well as the amount of time a patient experiences stable disease. As used herein, "overall survival rate" refers to the percentage of subjects in a group who may be alive after a specific duration. As used herein, "disease control rate" refers to complete response (CR) plus partial response (PR) plus stable disease (SD).

[0128] In some embodiments, relative to subjects who have received prior treatment including anti-angiogenesis inhibition, administration of a PLK1 inhibitor and an anti-angiogenic agent improves the ORR of the subjects by less than, about less than, at least less than, or at least about less than: 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or a number or range between any two of these values). In some embodiments, relative to subjects who have received prior treatment including anti-angiogenesis inhibition, administration of a PLK1 inhibitor and an anti-angiogenic agent improves the PFS of the subjects by less than, about less than, at least less than, or at least about less than: 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or a number or range between any two of these values).

[0129] In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent to a subject who has not received any prior treatment including anti-angiogenesis can result in an ORR of the following, at least the following, about the following, or at least about the following: 69% (e.g., 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or a number or range between any two of these values). In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent to a subject who has received any prior treatment including anti-angiogenesis can result in an ORR of about 23% or less (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23% or a number or range between any two of these values). In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent to a subject who has not received any prior treatment including anti-angiogenesis can result in a median PFS of the following, at least the following, about the following, or at least about the following: 13.5 months (e.g., 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years). In some embodiments, administering a PLK1 inhibitor and an anti-angiogenic agent to a subject who has received any prior treatment including anti-angiogenesis can result in a median PFS of about 7.8 months or less (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months or a number or range between any two of these values).

[0130] The term "inhibition of tumor growth" or "reduced tumor growth" can refer to a reduction in or complete cessation of tumor growth and / or a regression in tumor size (e.g., diameter and / or volume). The term "tumor volume" or "tumor size" can refer to the total size of the tumor, which can include the tumor itself plus any affected lymph nodes (if applicable). The presence or absence of a tumor and tumor size can be determined by a variety of methods known in the art, such as by measuring the size of the tumor using calipers, computed tomography (CT) or magnetic resonance imaging (MRI) scans, mammography, and x-rays. Volumes can be calculated using equations based on, for example, the z-axis diameter or based on standard shapes such as spheres, ellipsoids, or cubes. Tumor size can be evaluated at any time before, during, or after at least one cycle of treatment with onvansertib and / or an antiangiogenic agent. Tumor size can be evaluated at a first time point and at one or more additional time points. In some embodiments, tumor size can be evaluated in a subject and, for example, in an untreated subject at equivalent time points (e.g., at a first time point and at one or more additional time points). Tumor growth can be determined, for example, by measuring tumor size at a first time point and at one or more additional time points. In some embodiments, an increase in the inhibition of tumor growth in a subject (e.g., improved ORR) indicates that the subject is responsive to cancer treatment.

[0131] After one or more treatment cycles, the inhibition of growth of at least one of one or more tumors in a subject can be the following fold, can be about the following fold, can be at least the following fold, or can be at least about the following fold of the growth inhibition caused by onvansertib and an anti-angiogenic agent in a subject who has received a prior treatment for inhibiting angiogenesis: 1.1-fold greater, 1.2-fold greater, 1.3-fold greater, 1.4-fold greater, 1.5-fold greater, 1.6-fold greater, 1.7-fold greater, 1.8-fold greater, 1.9-fold greater, 2-fold greater, or a number or range between any two of these values, or more fold greater. After one or more treatment cycles, the inhibition of growth of at least one of one or more tumors in a subject can be increased by the following, about the following, at least the following, or at least about the following relative to a subject who has received a prior treatment for inhibiting angiogenesis: 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a number or range between any two of these values. After treating with the combination for one or more cycles, the growth of at least one of one or more tumors in a subject can be inhibited by the following, about the following, at least the following, or at least about the following relative to an untreated subject or a subject who has received prior anti-angiogenic treatment: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a number or range between any two of these values. After one or more treatment cycles, the growth of at least one of one or more tumors in a subject can be inhibited by the following, about the following, at least the following, or at least about the following relative to an untreated subject: 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a number or range between any two of these values. A subject can be tumor-free after one or more treatment cycles.

[0132] After one or more treatment cycles, the inhibition of the growth of at least one of one or more tumors in a subject can be the following fold, can be about the following fold, can be at least the following fold, or can be at least about the following fold of the inhibition of growth caused by onvansertib alone or an anti-angiogenic agent alone: 1.1-fold greater, 1.2-fold greater, 1.3-fold greater, 1.4-fold greater, 1.5-fold greater, 1.6-fold greater, 1.7-fold greater, 1.8-fold greater, 1.9-fold greater, 2-fold greater, or a number or range between any two of these values, or more fold greater. After one or more treatment cycles, relative to subjects treated with onvansertib alone or an anti-angiogenic agent alone, the inhibition of the growth of at least one of one or more tumors in a subject can be increased by the following, about the following, at least the following, or at least about the following: 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a number or range between any two of these values. After one or more treatment cycles, relative to untreated subjects, the growth of at least one of one or more tumors in a subject can be inhibited by the following, about the following, at least the following, or at least about the following: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a number or range between any two of these values. After one or more treatment cycles, relative to untreated subjects, the growth of at least one of one or more tumors in a subject can be inhibited by the following, about the following, at least the following, or at least about the following: 70%, 75%, 80%, 85%, 90%, 95%, 100%, or a number or range between any two of these values. The subject can be tumor-free after one or more treatment cycles.

[0133] In some embodiments, the first time point is before or immediately before the combination treatment, and at least one of the one or more additional time points is at or after the end of at least one 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 before or immediately before the first cycle of the combination treatment, and the one or more additional time points are at or after the end of the second cycle of the combination treatment.

[0134] In some embodiments, the first cycle of the combination treatment is immediately before the second cycle of the combination treatment. In some embodiments, the method includes continuing the combination treatment of the subject if the subject is indicated as responsive to the combination treatment. In some embodiments, the method includes stopping the combination treatment of the subject and / or starting a different combination treatment for the subject if the subject is not indicated as responsive to the combination treatment.

[0135] The first time point can be before or immediately before the combination therapy, and one or more additional time points are at or after the end of at least one cycle of the combination therapy. Optionally, the cycle of the combination therapy is the first cycle of the combination therapy. In some embodiments, the first time point is before or immediately before the first cycle of the combination therapy, and one or more additional time points are at or after the end of the second cycle of the combination therapy. Optionally, the first cycle of the combination therapy is immediately before the second cycle of the combination therapy.

[0136] Determining the responsiveness of a subject can include determining whether the subject is a responder to the treatment, whether the subject is in or will be in complete remission (CR), or whether the subject is in or will be in partial remission (PR). Determining the responsiveness of a subject can include determining the objective response rate (ORR), duration of response, time to response, progression-free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allele burden, or a combination thereof in the subject. Determining the responsiveness of a subject can include determining whether the subject has a partial response to the treatment, whether the subject has a complete response to the treatment, whether the subject has a stable disease (SD) status, or whether the subject has a progressive disease (PD) status. In some embodiments, the method includes initiating additional treatment for the subject if the subject is indicated to be in cancer recurrence. The additional treatment can be the same as or different from the current combination therapy or a previous combination therapy.

[0137] Additional methods for assessing the cancer status of a subject include determining the ECOG status. As used herein, the ECOG status refers to the Eastern Cooperative Oncology Group (ECOG) performance status (Oken M et al., Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 1982; 5(6):649-655), as shown below: 0, fully active, able to carry out all pre-disease performance without restriction; 1, restricted in physical activity but ambulatory and able to perform light or sedentary work, such as light housework, office work; 2, ambulatory and able to take care of oneself completely but unable to perform any work activities; up to and approximately more than 50% of waking hours; 3, only able to perform limited self-care; more than 50% of waking hours are restricted to bed or chair; 4, completely disabled; unable to perform any self-care; completely restricted to bed or chair; and 5, dead.

[0138] As described herein, after treatment with an anti-angiogenic agent and a PLK1 inhibitor, a patient can achieve a complete response or a partial response. In some embodiments, the patient achieves a complete response. In some embodiments, the patient achieves a partial response. In some embodiments, the patient has not received any prior anti-angiogenic agent treatment.

[0139] Disclosed herein are methods, compositions, kits, and systems for predicting / determining the clinical outcome of combination therapy for cancer of the present disclosure, monitoring combination therapy, predicting / determining the responsiveness of a subject to combination therapy, determining the status of cancer in a subject, and improving the outcome of combination therapy. The methods, compositions, kits, and systems can be used to guide combination therapy, provide combination therapy recommendations, and reduce or avoid unnecessary ineffective combination therapy for patients. In some embodiments, the combination therapy disclosed herein can improve the oncogenic allele burden of a subject. As used herein, "allele burden" can refer to the ratio between mutant (e.g., oncogenic) alleles and wild-type alleles in a clinical sample used for genotyping. In some embodiments, the sample can include circulating tumor DNA (ctDNA). CtDNA can be analyzed to predict / determine the clinical outcome of cancer treatment, monitor cancer treatment, predict / determine the responsiveness of a subject to cancer treatment, determine the status of cancer in a subject, improve the outcome of cancer treatment, guide cancer treatment, provide treatment recommendations, and / or reduce or avoid ineffective cancer treatment. Such ctDNA analysis has been described in WO2021146322, the content of which is incorporated herein by reference in its entirety.

[0140] A method for determining the responsiveness of a subject to combination therapy comprising an anti-angiogenic agent and a PLK1 inhibitor of the present disclosure can include, for example, analyzing the ctDNA of a subject with cancer, where the subject is undergoing treatment and / or has received combination therapy, to determine the responsiveness of the subject to the combination therapy. In some embodiments, determining the responsiveness of the subject includes determining whether the subject is a responder to the treatment, whether the subject is in or will be in CR, or whether the subject is in or will be in partial remission (PR). For example, analyzing ctDNA can include: detecting the variant allele frequency in ctDNA in a first sample obtained from the subject at a first time point, detecting the variant allele frequency in ctDNA obtained from the subject in one or more additional samples at one or more additional time points, and determining the difference in the variant allele frequency in ctDNA between the first sample 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 indicating that the subject is responsive to cancer treatment.

[0141] In some embodiments, the first time point is before or immediately before the combination therapy, and at least one of the one or more additional time points is at or after the end of at least one cycle of the combination therapy. In some embodiments, the cycle of the combination therapy is the first cycle of the combination therapy. In some embodiments, the first time point is before or immediately before the first cycle of the combination therapy, and the one or more additional time points are at or after the end of the second cycle of the combination therapy.

[0142] In some embodiments, the first cycle of the combination therapy is immediately before the second cycle of the combination therapy. In some embodiments, the method includes continuing the combination therapy for the subject if the subject is indicated as responsive to the combination therapy. In some embodiments, the method includes stopping the combination therapy for the subject and / or starting a different combination therapy for the subject if the subject is not indicated as responsive to the combination therapy.

[0143] Methods disclosed herein for determining a subject's cancer status include analyzing the subject's ctDNA to determine the subject's cancer status. The subject can be a subject who is undergoing a current combination therapy including an anti-angiogenic agent and a PLK1 inhibitor of the present disclosure, a subject who has received a previous combination therapy of the present disclosure, and / or a subject in cancer remission. A subject in cancer remission can be in complete remission (CR) or partial remission (PR).

[0144] In some embodiments, analyzing the ctDNA includes detecting the variant allele frequency in the ctDNA. In some embodiments, analyzing the ctDNA includes: detecting the variant allele frequency in the ctDNA obtained from the subject in a first sample at a first time point, detecting the variant allele frequency in the ctDNA obtained from the subject in one or more additional samples at one or more additional time points, and determining the difference in the variant allele frequency in the ctDNA between at least one of the first sample and the one or more additional samples, an increase in the variant allele frequency in the one or more additional samples relative to the first sample indicating that the subject is at risk of cancer recurrence or is in cancer recurrence.

[0145] In some embodiments, the first time point is before or immediately before the combination therapy, and the one or more additional time points are at or after the end of at least one cycle of the combination therapy, optionally, the cycle of the combination therapy is the first cycle of the combination therapy. In some embodiments, the first time point is before or immediately before the first cycle of the combination therapy, and the one or more additional time points are at or after the end of the second cycle of the combination therapy, optionally, the first cycle of the combination therapy is immediately before the second cycle of the combination therapy.

[0146] In some embodiments, the method includes initiating additional treatment of the subject if the subject is indicated to be at cancer recurrence. The additional treatment can be the same as or different from the current combination therapy or a previous combination therapy.

[0147] The variant allele frequency in ctDNA can be determined, for example, by the total mutation count in ctDNA of each of the first sample and the one or more additional samples, or by the average variant allele frequency in ctDNA of each of the first sample and the one or more additional samples. In some embodiments, the variant allele frequency is the mutant allele frequency (MAF) of a driver mutation of a cancer (e.g., metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof). In some embodiments, the variant allele frequency is the MAF of one or more driver mutations of a cancer (e.g., metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof). In some embodiments, Log2(C1 / C0) < MAF threshold indicates a decrease in ctDNA MAF. C0 is the ctDNA MAF in the first sample, and C1 is the ctDNA MAF in one of the additional samples. In some embodiments, the MAF threshold is from 0.01 to -0.10 or is about 0.01 to -0.10. In some embodiments, the MAF threshold is 0.06 or is about 0.06. In some embodiments, the MAF threshold is 0.05 or is about 0.05.

[0148] In some embodiments, the first sample comprises ctDNA from a subject before treatment, and one of the additional samples comprises ctDNA from the subject after treatment.

[0149] Driver mutations can be or can include a mutation in one of the following 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 driver mutations is a mutation in one of these 75 genes. In some embodiments, the one or more driver mutations are mutations in one of these 75 genes. The driver mutation or at least one of the one or more driver mutations can be located in a gene selected from: 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 located 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 the variant allele frequency in one or more of the subject's ctDNA, PBMC, and BMMC.

[0150] ctDNA can be analyzed using, for example, polymerase chain reaction (PCR), next-generation sequencing (NGS), and / or droplet digital PCR (ddPCR). The samples disclosed herein can be derived from, for example, whole blood of a subject, plasma of a subject, serum of a subject, or a combination thereof. In some embodiments, the ctDNA is from whole blood of a subject, plasma of a subject, serum of a subject, or a combination thereof.

[0151] In some embodiments, the method includes analyzing the ctDNA of a subject prior to treatment. In some embodiments, the treatment includes one or more cycles, and the ctDNA is analyzed before, during, and after each cycle of the treatment. Each cycle of the treatment can be at least 21 days. In some embodiments, each cycle of the treatment is from about 21 days to about 28 days. In some embodiments, the subject is human.

[0152] Disclosed herein are methods for improving cancer treatment outcomes. The methods can include: detecting a variant allele frequency in ctDNA obtained from a subject in a first sample at a first time point prior to the subject undergoing combination treatment comprising an anti-angiogenic agent and a PLK1 inhibitor of the present disclosure; detecting a variant allele frequency in ctDNA obtained from the subject in one or more additional samples at one or more additional time points after the subject undergoes the combination treatment; determining a difference in the variant allele frequency in the ctDNA between the first sample and at least one of the one or more additional samples, a decrease in the oncogenic allele burden and / or variant allele frequency in at least one of the additional samples relative to the first sample indicating that the subject is responsive to the combination treatment; and if the subject is indicated as being responsive to the combination treatment, continuing the combination treatment for the subject, or if the subject is not indicated as being responsive to the combination treatment, stopping the combination treatment for the subject and / or initiating a different cancer treatment for the subject.

[0153] Also disclosed herein are methods of treating cancer. The methods can include: administering to a subject in need thereof a combination therapy comprising an anti-angiogenic agent and a PLK1 inhibitor of the present disclosure; determining a reduction in variant allele frequency in a second sample of the subject obtained at a second time point after the subject has received the combination therapy, relative to the oncogenic allele burden and / or variant allele frequency in a first sample of the subject obtained at a first time point prior to the subject receiving the combination therapy; and continuing the combination therapy. In some embodiments, the subject is a subject newly diagnosed with cancer, such as a subject who has not received any prior cancer treatment prior to the combination therapy. In some embodiments, the subject has received prior cancer treatment and is in cancer remission, such as a subject who is in complete remission (CR) or partial remission (PR) after receiving prior combination therapy. In some embodiments, the prior treatment does not include the use of an anti-angiogenic agent, a PLK1 inhibitor, or both.

[0154] The first time point can be, for example, prior to or immediately prior to the combination therapy. At least one of one or more additional time points can be, for example, at or after the end of at least one cycle of the combination therapy. In some embodiments, the cycle of the combination therapy is the first cycle of the combination therapy. In some embodiments, the first time point is prior to or immediately prior to the first cycle of the combination therapy, and one or more additional time points are at or after the end of the second cycle of the combination therapy. In some embodiments, the first cycle of the combination therapy is immediately prior to the second cycle of the combination therapy.

[0155] The variant allele frequency in ctDNA can be determined, for example, by the total mutation count in ctDNA of each of the first sample and one or more additional samples, and / or by the average variant allele frequency in ctDNA of each of the first sample and one or more additional samples. In some embodiments, the variant allele frequency is the mutant allele frequency (MAF) of a driver mutation of cancer (e.g., metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof). In some embodiments, the variant allele frequency is the MAF of one or more driver mutations of cancer (e.g., metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof). In some embodiments, Log2(C1 / C0) < MAF threshold indicates a decrease in ctDNA MAF. C0 is the ctDNA MAF in the first sample, and C1 is the ctDNA MAF in one of the additional samples. In some embodiments, the MAF threshold is -0.05.

[0156] The method can further include determining the variant allele frequency in one or more of the subject's ctDNA, PBMC, and BMMC. For example, the variant allele frequency in ctDNA can be detected 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). At least one of the first sample, one or more additional samples, and the second sample can be derived from the subject's whole blood, the subject's plasma, the subject's serum, or a combination thereof. In some embodiments, the ctDNA is from the subject's whole blood, the subject's plasma, the subject's serum, or a combination thereof.

[0157] In some embodiments, the subject whose ctDNA is analyzed is undergoing or will undergo cancer treatment. The method can include analyzing the subject's ctDNA before treatment. The treatment can include one or more cycles, and the ctDNA can be 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 cycles of the treatment, three or more cycles of the treatment, or each cycle of the treatment. Each cycle of the treatment can be at least 21 days, e.g., 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 the treatment is about 21 days to about 28 days. In some embodiments, each cycle of the treatment is 21 days to 28 days. In some embodiments, the subject is human.

[0158] Compositions and Kits

[0159] Compositions and kits for treating cancer are disclosed herein. In some embodiments, the kit comprises: a Polo-like kinase 1 (PLK1) inhibitor; and a manual providing instructions for administering the PLK1 inhibitor and an anti-angiogenic agent to a subject for treating metastatic cancer, wherein the subject has not received any prior cancer treatment or wherein the subject has not received any prior treatment including inhibition of angiogenesis. In some embodiments, the kit comprises an anti-angiogenic agent. The cancer can be, for example, metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof.

[0160] In some embodiments, the instructions include instructions for co-administering the PLK1 inhibitor and the anti-angiogenic agent. In some embodiments, the instructions include instructions for sequentially administering the PLK1 inhibitor and the anti-angiogenic agent. In some embodiments, the instructions include instructions for orally administering the PLK1 inhibitor. In some embodiments, the instructions include instructions for orally administering the anti-angiogenic agent. In some embodiments, the instructions include instructions for intravenously administering the anti-angiogenic agent.

[0161] The instructions can include instructions wherein the subject has not received any prior cancer treatment (e.g., any prior cancer treatment including administration of an angiogenesis inhibitor (e.g., bev)).

[0162] The instructions may include instructions that the subject has received at least one prior treatment for cancer. In some embodiments, the prior treatment does not include the use of an anti-angiogenic agent, a PLK1 inhibitor, or both. In some embodiments, the instructions include instructions that the subject is in cancer remission. In some embodiments, a subject in cancer remission is in complete remission (CR) or partial remission (PR).

[0163] In some embodiments, the instructions include instructions for administering each of an anti-angiogenic agent and a PLK1 inhibitor to the subject in a cycle of at least twice a week. In some embodiments, the instructions include instructions for administering each of an anti-angiogenic agent and a PLK1 inhibitor to the subject in a cycle of at least five times a week. In some embodiments, the instructions include instructions for administering an anti-angiogenic agent, a PLK1 inhibitor, or both in a cycle of at least 7 days. In some embodiments, each cycle of treatment is at least about 21 days. In some embodiments, each cycle of treatment is from about 21 days to about 28 days, such as 28 days. In some embodiments, the instructions include instructions for administering a PLK1 inhibitor for at least four days in a cycle. In some embodiments, the instructions include instructions for not administering a PLK1 inhibitor for at least one day in a cycle. In some embodiments, the instructions include instructions for administering an anti-angiogenic agent daily. In some embodiments, the instructions include instructions for administering an anti-angiogenic agent and a PLK1 inhibitor for at least two cycles. The instructions may include instructions for administering an anti-angiogenic agent daily, weekly, bi-weekly, tri-weekly, monthly, or monthly.

[0164] The anti-angiogenic agent may be capable of inhibiting VEGF-A, VEGFR-1, VEGFR-2, VEGFR-3, EGFR, HER2, PDGFR family proteins, RAF, Kit (or c-Kit), FLT3, CSF-1R, RET, Abl, Itk, LcK, c-FMS, FGFR family proteins, c-Met, PlGF, TNF-α, IFN, ILs, bFGF, mTOR, or any combination thereof. The anti-angiogenic agent (e.g., an angiogenesis inhibitor) may be afatinib axitinib bevacizumab cabozantinib cetuximab erlotinib everolimus gefitinib imatinib lapatinib lenalidomide lenvatinib mesylate necitumumab (PortrazzaTM ) Neratinib Panitumumab Pazopanib Pertuzumab Ramucirumab Regorafenib Sorafenib Sunitinib Thalidomide (Synovir, ) Trastuzumab Vandetanib or ziv-Aflibercept

[0165] The PLK1 inhibitor can be selective and / or specific for PLK1. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, pyridopyrimidine, aminopyrimidine, substituted thiazolinone, pteridine derivative, dihydroimidazo[1,5-f]pteridine, meta-substituted thiazolinone, benzyl styryl sulfone analog, stilbene derivative, or any combination thereof. In some embodiments, the PLK1 inhibitor is onvansertib, BI2536, Volasertib (BI6727), 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. In some embodiments, the anti-angiogenic agent is bevacizumab and the PLK1 inhibitor is onvansertib. In some embodiments, the instructions include administering the PLK1 inhibitor at 12 mg / m 2 -90 mg / m 2 Instructions for administering the PLK1 inhibitor. In some embodiments, the instructions include administering the anti-angiogenic agent at 20 mg - 1200 mg.

[0166] Examples

[0167] Some aspects of the embodiments discussed above are further disclosed in detail in the following examples, which are not intended to limit the scope of the present disclosure in any way.

[0168] Example 1

[0169] Clinical Trial of Onvansertib in Combination with Bevacizumab and FOLFIRI

[0170] This example describes the results (as of July 25, 2022) of a Phase 1b / 2 clinical trial (NCT03829410) to determine the safety and efficacy of onvansertib in combination with FOLFIRI and bevacizumab, which is the current standard of care for second-line treatment in patients with disease recurrence or progression after first-line treatment with oxaliplatin and fluoropyrimidines. Patients received standard doses and schedules of FOLFIRI and bevacizumab ("bev") on Days 1 and 15 of a 28-day treatment cycle, and onvansertib orally once daily on Days 1 to 5 and 15 to 19 of a 28-day cycle. Patients were scanned every eight weeks at baseline and during treatment for disease response assessment. This trial was conducted to show that onvansertib safely supplements and improves the efficacy of the standard of care.

[0171] The three efficacy endpoints for the Phase 2 part of this trial were: (1) Primary efficacy endpoint: objective response rate (or ORR); (2) Secondary endpoints: other measures of disease response, including progression-free survival and duration of response; and (3) For one of the exploratory endpoints, we evaluated the correlation between changes in KRAS mutation burden in circulating tumor DNA and radiologic disease response.

[0172] Table 1 shows the inclusion and patient characteristics of this study.

[0173] Table 1. Patient Inclusion

[0174]

[0175] Table 2. Patient Characteristics

[0176] Total Number of Patients N = 50 Median n(%) Liver Metastasis None 13(26％) Liver and Others 27(54％) Liver Only 10(20％) Number of Metastatic Organs 1 16(32％) ≥2 34(68％) Previous Bevacizumab Treatment Yes 35(70％) No 15(30％)

[0177] Table 3. Achieved Strong, Durable Responses in Patients

[0178] All Doses RP2D Objective Response Rate*(CR + PR) 35％(17 / 48) 34％(12 / 35) Disease Control Rate(CR + PR + SD) 92％(44 / 48) 94％(33 / 35) Durability Median Duration of Response 11.7 months 12.5 months

[0179] An initial PR lasting up to 8 months of treatment was observed. The combination of onvansertib with FOLFIR-bev was well tolerated. Neutropenia was the most commonly reported TEAE, and more than half of the observed events were Grade 3 or 4. Although it is part of standard FOLFIRI, the 5FU bolus was considered to only increase the toxicity of the regimen without increasing efficacy. Early in the Phase 1b part of the trial, based on feedback from our investigators, we revised the protocol to allow interruption of the 5FU bolus from the treatment regimen if Grade 2 or higher neutropenia was observed. Subsequently, it was shown that interruption of the 5FU bolus improved the severity of observed neutropenia with or without the use of growth factors.

[0180] In previous clinical trials, the first-line use of bev had the least impact on the efficacy of second-line use. In those previous trials (e.g., Hansen et al., Cancers 2021, 13, 1031; Tabernaro et al., Eur J Cancer, 2014, 50, 320 - 332; Bennouna et al., Lancet Oncol. 2013, 14, 29 - 37; Van Cutsem et al., J. Clin. Oncol. 2012, 30, 3499 - 3506; Tabenaro et al., Lancet Oncol 2015; 16:499 - 508; Beretta et al., Med Oncol (2013) 30:486; Moriwakij et al., Med Oncol (2012) 29:2842 - 2848), for patients who had received prior bev treatment (“prior bev”) compared to patients who had not received prior bev treatment (“bev-naïve”), the mPFS (mo) was 6.7 months and 6.9 months, respectively, and the mOS (mo) was 12.5 months and 13.9 months, respectively. The conclusion of the previous trials was that mPFS and mOS were similar in the second line regardless of whether the patient was bev-naïve or had prior bev in the first line. For the ORR, there was an incremental increase of 12% to 20%. Surprisingly, in this trial (NCT03829410 “Onvansertib Combined with FOLFIRI and Bevacizumab for Second-Line Treatment of Metastatic Colorectal Cancer Patients with Kras Mutations”), it was found that the “no prior Bev” subgroup had a much larger ORR and mPFS than expected (see Figures 1 - 2 ). For the ORR of 33 patients with prior bevacizumab treatment, there were 8 / 35 responders (22.9%) ORR (CI: 10.4% - 40.1%). The ORR for 15 patients without prior bevacizumab was 9 / 13 responders (69.2%) ORR (CI: 38.6% - 90.9%). Thus, the ORR of patients without prior anti-angiogenic treatment was twice as large as that of patients with prior anti-angiogenic treatment, with an odds ratio of 0.14 (0.03 - 0.56, p = 0.0049). The PFS of patients with prior bevacizumab compared to patients without prior bevacizumab also showed a surprising improvement in patients without prior bevacizumab treatment. In this trial, the mPFS of patients with prior bevacizumab treatment was 7.8 months, and the mPFS of patients without prior bevacizumab treatment was 13.5 months (p = 0.14)( Figure 1 ).

[0181] Table 4: Events

[0182]

[0183] It was found that the ORR of bev-naïve patients with different characteristics was consistently higher. No single patient characteristic explained the observed ORR differences. As Figure 3 shown in Table 5, the lowest ORR in the no prior Bev group was 50%, and the highest ORR at the RP2D was 80%. The ORR ranged from 14% to 31.3%, depending on the variables of patients with prior bevacizumab treatment.

[0184] Table 5. Observed ORR

[0185]

[0186] The results presented herein show a surprising improvement in multiple outcomes (e.g., ORR, PFS) of metastatic cancer patients treated with the combination of onvansertib and bevacizumab in patients without prior bev treatment. Without being bound by any particular theory, it is believed that there is a synergistic effect between onvansertib and bevacizumab in the bev-naïve setting.

[0187] Example 2

[0188] Treatment Benefit for Patients Not Exposed to Bev

[0189] Figure 4A Depicts the best radiological response and duration of response for 66 evaluable patients (as of June 16, 2023). Figures 4A - 4B Shows that bev-naïve patients achieved a higher response rate with the combination of onvansertib with FOLFIRI and bevacizumab, which is the current standard of care (SoC) for patients with metastatic colorectal cancer. In Figure 4AAmong them, the historical controls were from Bennouna et al., Lancet Oncol 2013; 14:29–37; Giessen et al., Acta Oncologica, 2015, 54:187-193; Cremolini et al., Lancet Oncol 2020, 21:497–507; Antoniotti et al., Correspondence Lancet Oncol June 2020; Giantonio et al., 2007, J Clin Oncol 25:1539-1544; Moriwaki et al., Med Oncol, 2012, 29:2842–2848; Beretta et al., Med Oncol 2013, 30:486. In addition, bev-naïve patients experienced more durable responses( Figures 5A - 5B ).

[0190] It was found that the PFS of the treated patients exceeded the historical controls of the SoC, especially in bev-naïve patients. The progression-free survival (as of June 16, 2023) of 66 evaluable patients is shown in Figure 6 In Figure 6 Among them, the historical controls were from Bennouna et al., Lancet Oncol 2013; 14:29–37; Giessen et al., Acta Oncologica, 2015, 54:187-193; Cremolini et al., Lancet Oncol 2020, 21:497–507; Antoniotti et al., Correspondence Lancet Oncol June 2020; Giantonio et al., 2007, J Clin Oncol 25:1539-1544; Moriwaki et al., Med Oncol, 2012, 29:2842–2848; Beretta et al., Med Oncol 2013, 30:486.).

[0191] It was also observed that bev-naïve patients experienced deeper tumor regression. Figure 7 Shows the change in tumor size relative to baseline* - all doses (as of June 16, 2023).

[0192] Example 3

[0193] Onvansertib in Combination with Bevacizumab is an Effective Combination in KRAS Mutant CRC Xenograft Model

[0194] Treat three KRAS-mutant xenograft models (HCT116, LoVo, and SW620) with vehicle, onvansertib, bevacizumab, or a combination of onvansertib and bevacizumab. As Figure 8 shown, the combination of onvansertib and bevacizumab demonstrated anti-tumor activity in the 3 models, including: (1) tumor stabilization was observed after treatment with the combination; (2) the combination had significantly superior anti-tumor activity compared to single agents. Figure 9 Shown is that the combination of onvansertib and bevacizumab reduced tumor vascularization, as demonstrated by observing that tumors from mice treated with the combination of onvansertib and bevacizumab appeared smaller and paler (less vascularized).

[0195] Without being bound by any particular theory, it is believed that prior bev therapy modulation may confer gene pathways that confer resistance to bev and onvansertib. Figure 10 The non-limiting methods shown in can be used to identify potential mechanisms of treatment resistance in bev-exposed KRAS-mutant mCRC patients.

[0196] In at least some of the previously described embodiments, one or more of the elements used in an embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art will understand 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 alterations are intended to fall within the scope of the subject matter defined by the appended claims.

[0197] Regarding the use of generally any plural and / or singular terms herein, those skilled in the art may translate the plural to the singular and / or the singular to the plural according to the context and / or application. Various singular / plural permutations may be explicitly set forth herein for clarity. As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Unless otherwise stated, any reference to "or" herein is intended to include "and / or".

[0198] Those skilled in the art will understand that, generally, the terms used herein, and especially the terms used in the appended claims (e.g., the subject matter of the appended claims), generally mean "open" terms (e.g., the term "including" should be understood as "including but not limited to", the term "having" should be understood as "having at least", the term "include" should be understood as "including but not limited to", etc.). Those skilled in the art will also understand that if a specific number of introduced claim recitations are intended, such an intention will be explicitly recited in the claims, and in the absence of such a recitation, such an intention does not exist. For example, for the sake of understanding, the appended claims may contain the use of introductory phrases such as "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to mean that any particular claim containing such an introduced claim recitation is limited to an embodiment containing only one such recitation due to the introduction of the indefinite article "a" or "an", even when the same claim includes an introductory phrase such as "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"); this also applies to the use of definite articles used to introduce claim recitations. Additionally, even if a specific number of introduced claim recitations are explicitly recited, those skilled in the art will recognize that such recitations should be understood to mean at least the recited number (e.g., a bare recitation of "two recitations" without any other modifiers means at least two recitations, or means two or more recitations). Further, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally, such a construction is intended in the sense that those skilled in the art will understand the meaning of the convention (e.g., a "system having at least one of A, B, and C" will include, but not be limited to, a system having only A, only B, only C, having A and B together, having A and C together, having B and C together, and / or having A, B, and C together, etc.). In those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally, such a construction is intended in the sense that those skilled in the art will understand the meaning of the convention (e.g., a "system having at least one of A, B, or C" will include, but not be limited to, a system having only A, only B, only C, having A and B together, having A and C together, having B and C together, and / or having A, B, and C together, etc.). Those skilled in the art should also understand that, whether in the specification, the claims, or the drawings, any disjunctive word and / or phrase that actually represents two or more alternative terms should be understood as contemplating the possibility of including one of the terms, any one of the terms, or both terms.

[0199] In addition, when features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is thereby also described in terms of any individual member or subgroup of members of the Markush group.

[0200] As will be understood by those 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 subranges and combinations of subranges thereof. Any listed range can be readily identified as fully describing and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of 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 those skilled in the art, all language such as "up to", "at least", "greater than", "less than", etc. includes the recited numbers and refers to ranges that can then be broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 - 3 members refers to a group having 1, 2, or 3 members. Similarly, a group having 1 - 5 members refers to a group having 1, 2, 3, 4, or 5 members, and so on.

[0201] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and not intended to be limiting, with the true scope and spirit being indicated by the appended claims.

Claims

1. A method for treating metastatic cancer in a subject, the method comprising: Administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby reducing or inhibiting the progression of the metastatic cancer, wherein the subject has not received any prior cancer treatment or the subject has not received any prior treatment including inhibition of angiogenesis.

2. A method of treating metastatic cancer in a subject, the method comprising: Administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby reducing or inhibiting the progression of the metastatic cancer, wherein it is known that the subject has not received any prior cancer treatment or it is known that the subject has not received any prior treatment including inhibition of angiogenesis.

3. A method of treating metastatic cancer in a subject, the method comprising: Identifying a subject having metastatic cancer and who has not received any prior cancer treatment; and Administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby reducing or inhibiting the progression of the metastatic cancer.

4. A method of treating metastatic cancer in a subject, the method comprising: Identifying a subject having metastatic cancer and who has not received any prior treatment including inhibition of angiogenesis; and Administering a PLK1 inhibitor and an anti-angiogenic agent to the subject, thereby reducing or inhibiting the progression of the metastatic cancer.

5. The method according to any one of claims 1-4, comprising administering chemotherapy, the PLK1 inhibitor, and the anti-angiogenic agent to the subject.

6. The method according to any one of claims 1-4, wherein the subject has not received prior chemotherapy treatment.

7. The method according to any one of claims 1-4, wherein the subject has not received prior chemotherapy treatment for the metastatic cancer.

8. The method according to any one of claims 5-7, wherein the chemotherapy comprises treatment with FOLFIRI, abiraterone, FOLFOX, an anti-EGFR agent, a KRAS-directed inhibitor, gemcitabine, abraxane, nanoliposomal irinotecan, 5-FU, FOLFIRINOX, FOLFOXIRI, or a combination thereof.

9. The method according to any one of claims 1-8, wherein the administration of the PLK1 inhibitor and the anti-angiogenic agent synergistically reduces or inhibits the progression of the metastatic cancer relative to the additive effect of treatment with the PLK1 inhibitor alone, treatment with the anti-angiogenic agent alone, and / or treatment with the PLK1 inhibitor alone and the anti-angiogenic agent alone.

10. The method according to any one of claims 1-9, wherein the administration of the PLK1 inhibitor and the anti-angiogenic agent improves one or more treatment effects in the subject relative to a control or baseline.

11. The method according to claim 10, wherein the administration of the PLK1 inhibitor and the anti-angiogenic agent reduces the oncogenic allele burden in the subject relative to a subject who has received prior treatment including inhibition of angiogenesis.

12. A method of improving objective response rate (ORR), progression-free survival (PFS), or both in the treatment of metastatic cancer, the method comprising administering a PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, thereby improving the ORR and / or PFS of the subject, wherein the subject has not received any prior cancer treatment or the subject has not received any prior treatment including anti-angiogenesis.

13. A method of improving objective response rate (ORR), progression-free survival (PFS), or both in the treatment of metastatic cancer, the method comprising administering a PLK1 inhibitor and an anti-angiogenic agent to a subject with metastatic cancer, thereby improving the ORR and / or PFS of the subject, wherein it is known that the subject has not received any prior cancer treatment or it is known that the subject has not received any prior treatment including anti-angiogenesis.

14. The method according to any one of claims 12-13, comprising identifying a subject with the metastatic cancer who has not received any prior cancer treatment.

15. The method according to any one of claims 12-13, comprising identifying a subject with the metastatic cancer who has not received any prior treatment including anti-angiogenesis.

16. The method according to any one of claims 12-15, wherein the administration of the PLK1 inhibitor and the anti-angiogenic agent synergistically improves the ORR and / or PFS of the subject as compared to the additive effect of treatment with the PLK1 inhibitor alone, treatment with the anti-angiogenic agent alone, and / or treatment with the PLK1 inhibitor alone and the anti-angiogenic agent alone.

17. The method according to any one of claims 12-15, wherein the administration of the PLK1 inhibitor and the anti-angiogenic agent improves one or more treatment effects in the subject as compared to a control or baseline; and optionally, the one or more treatment effects include the size of a tumor originating from the metastatic cancer, objective response rate (ORR), duration of response, time to response, progression-free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allele burden, or a combination thereof.

18. The method according to any one of claims 12-15, wherein the administration of the PLK1 inhibitor and the anti-angiogenic agent improves the ORR of the subject, improves the PFS of the subject, improves the OS of the subject, improves the DCR of the subject, reduces the oncogenic allele burden of the subject, or a combination thereof as compared to a subject who has received prior treatment including anti-angiogenesis.

19. The method according to any one of claims 12-18, wherein the administration of the PLK1 inhibitor and the anti-angiogenic agent improves the ORR, PFS, or both of the subject by at least 50% as compared to a subject who has received prior treatment including anti-angiogenesis.

20. The method according to any one of claims 1-19, wherein the metastatic cancer is metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof.

21. The method according to any one of claims 1-20, wherein the PLK1 inhibitor is selective and / or specific for PLK1.

22. The method according to any one of claims 1-21, wherein 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.

23. The method according to any one of claims 1-22, wherein the subject has not received any prior treatment comprising administration of an angiogenesis inhibitor, and optionally wherein the angiogenesis inhibitor is the same as the anti-angiogenic agent.

24. The method according to any one of claims 1-23, wherein the anti-angiogenic agent is bevacizumab.

25. The method according to any one of claims 23-24, wherein the angiogenesis inhibitor is bevacizumab.

26. The method according to any one of claims 1-25, wherein the PLK1 inhibitor and the anti-angiogenic agent are administered simultaneously.

27. The method according to any one of claims 1-25, wherein the PLK1 inhibitor and the anti-angiogenic agent are administered sequentially.

28. The method according to claim 27, wherein the PLK1 inhibitor is administered before the anti-angiogenic agent, and optionally, wherein on each day of administration of the PLK1 inhibitor and the anti-angiogenic agent to the subject, the PLK1 inhibitor is administered before the anti-angiogenic agent.

29. The method according to claim 28, wherein on a given day, the PLK1 inhibitor is administered about 30 minutes to about 5 hours before the anti-angiogenic agent.

30. The method according to any one of claims 1-29, wherein the administration of the PLK1 inhibitor is oral administration, and the administration of the anti-angiogenic agent is intravenous administration or oral administration.

31. The method according to any one of claims 1-30, wherein the anti-angiogenic agent and the PLK1 inhibitor are each administered to the subject in a cycle of at least twice or at least five times within a week.

32. The method according to any one of claims 1-31, wherein the anti-angiogenic agent, the PLK1 inhibitor, or both are administered in a cycle of at least 7 days; optionally, each treatment cycle is at least about 21 days; and further optionally, each treatment cycle is from about 21 days to about 28 days.

33. The method according to any one of claims 31-32, wherein the PLK1 inhibitor is administered for at least four days during the cycle.

34. The method according to any one of claims 32-33, wherein the PLK1 inhibitor is not administered for at least one day during the cycle.

35. The method according to any one of claims 1-34, wherein the anti-angiogenic agent is administered daily, weekly, bi-weekly, tri-weekly, monthly, or monthly.

36. The method according to claims 31-35, wherein the subject undergoes at least two cycles of administration of the anti-angiogenic agent and the PLK1 inhibitor.

37. The method according to any one of claims 1-36, wherein the anti-angiogenic agent is bevacizumab and the PLK1 inhibitor is onvansertib.

38. The method according to claim 37, wherein onvansertib is administered at 12 mg / m 2 - 90 mg / m 2 body surface area.

39. The method according to any one of claims 37-38, wherein bevacizumab is administered at about 1 mg / kg - 20 mg / kg; optionally, wherein bevacizumab is administered at about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, or about 15 mg / kg.

40. The method according to any one of claims 1-39, wherein the subject has received at least one prior cancer treatment, and optionally, wherein the prior treatment does not include the use of an anti-angiogenic agent, a PLK1 inhibitor, or both.

41. The method according to any one of claims 1-40, wherein the one or more subjects have a prior remission of cancer.

42. The method according to claim 41, wherein the prior remission is a complete remission (CR).

43. The method according to claim 41, wherein the prior remission is a partial remission (PR).

44. The method according to any one of claims 1-43, further comprising one or more of the following: (1) determining the cancer status of the one or more subjects, (2) determining the responsiveness of the one or more subjects to PLK1 inhibitor treatment, and (3) administering to the one or more subjects one or more cancer therapeutic agents or therapies.

45. The method according to any one of claims 1-44, wherein the one or more subjects are human.

46. The method according to any one of claims 1-44, wherein reducing or inhibiting the progression of the cancer comprises inhibiting the growth of one or more tumors in the one or more subjects and / or reducing the number of cancer cells detected in the one or more subjects by at least about 25%, 30%, 40%, 50%, 60%, or 70% relative to untreated subjects.

47. The method according to any one of claims 1-44, wherein reducing or inhibiting the progression of the cancer comprises inhibiting the growth of one or more tumors in the one or more subjects and / or reducing the number of cancer cells detected in the one or more subjects by at least about 25%, 30%, 40%, 50%, 60% or 70% relative to the one or more subjects prior to administration of the PLK1 inhibitor and the anti-angiogenic agent.

48. The method according to claim 46 or 47, wherein after one or more treatment cycles, the growth of at least one of the one or more tumors in the one or more subjects is reduced by at least about 25%, 30%, 40%, 50%, 60% or 70%.

49. The method according to claim 46 or 47, wherein after one or more treatment cycles, the size / volume of at least one of the one or more tumors in the one or more subjects is reduced by at least about 25%, 30%, 40%, 50%, 60% or 70%.

50. The method according to any one of claims 44-49, wherein the one or more cancer therapeutics or therapies comprise FOLFIRI, abiraterone, FOLFOX, an anti-EGFR agent, a KRAS-directed inhibitor, gemcitabine, abraxane, nanoliposomal irinotecan, 5-FU, or a combination thereof; wherein the anti-EGFR agent is optionally cetuximab, and the KRAS-directed inhibitor is optionally a G12C inhibitor, a G12D inhibitor, or a combination thereof.

51. The method according to any one of claims 44-50, wherein determining the responsiveness of the one or more subjects comprises determining whether the subject is a responder to the treatment, whether the one or more subjects are in or will be in complete remission (CR), or whether the one or more subjects are in or will be in partial remission (PR).

52. The method according to any one of claims 44-51, wherein determining the responsiveness of the subject comprises determining the objective response rate (ORR), duration of response, time to response, progression-free survival (PFS), overall survival (OS), disease control rate (DCR), oncogenic allele burden, or a combination thereof.

53. The method according to any one of claims 44-52, wherein determining the responsiveness of the one or more subjects comprises determining whether the one or more subjects have a partial response to the treatment, whether the subject has a complete response to the treatment, whether the subject has a stable disease (SD) state, or whether the subject has a progressive disease (PD) state.

54. A kit comprising: a polo-like kinase 1 (PLK1) inhibitor; and A manual providing instructions for administering the PLK1 inhibitor and the anti-angiogenic agent to a subject with metastatic cancer, wherein the subject has not received any prior cancer treatment or the subject has not received any prior treatment including anti-angiogenesis.

55. A kit comprising: A polo-like kinase 1 (PLK1) inhibitor; and A manual providing instructions for administering the PLK1 inhibitor and the anti-angiogenic agent to a subject with metastatic cancer, wherein it is known that the subject has not received any prior cancer treatment or it is known that the subject has not received any prior treatment including anti-angiogenesis.

56. The kit according to claim 54, wherein the metastatic cancer is metastatic colorectal cancer, metastatic bladder cancer, metastatic breast cancer, metastatic kidney cancer, metastatic lung cancer, metastatic ovarian cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic gastric cancer, metastatic thyroid cancer, metastatic uterine cancer, metastatic kidney cancer, metastatic cervical cancer, metastatic recurrent glioblastoma, or a combination thereof.

57. The kit according to any one of claims 54-56, wherein the instructions include instructions for co-administering the PLK1 inhibitor and the anti-angiogenic agent.

58. The kit according to any one of claims 54-56, wherein the instructions include instructions for sequentially administering the PLK1 inhibitor and the anti-angiogenic agent.

59. The kit according to any one of claims 54-58, wherein the instructions include (1) instructions for orally administering the PLK1 inhibitor, (2) instructions for orally administering the anti-angiogenic agent, (3) instructions for intravenously administering the anti-angiogenic agent, or any combination thereof.

60. The kit according to any one of claims 54-59, wherein the instructions include that the subject has not received any prior treatment including administering an angiogenesis inhibitor, and optionally wherein the angiogenesis inhibitor is the same as the anti-angiogenic agent.

61. The kit according to any one of claims 54-60, wherein the instructions include instructions for administering each of the anti-angiogenic agent and the PLK1 inhibitor to the subject in a cycle of at least twice or at least five times within a week.

62. The kit according to any one of claims 54-61, wherein the instructions include instructions for administering the anti-angiogenic agent, the PLK1 inhibitor, or both in a cycle of at least 7 days; and optionally, wherein each treatment cycle is at least about 21 days, and further optionally, each treatment cycle is from about 21 days to about 28 days.

63. The kit according to claim 62, wherein the instructions include instructions for administering the PLK1 inhibitor for at least four days during the cycle.

64. The kit according to any one of claims 62-63, wherein the instructions include instructions for not administering the PLK1 inhibitor for at least one day during the cycle.

65. The kit according to any one of claims 54-64, wherein the instructions include instructions for administering the anti-angiogenic agent daily, weekly, bi-weekly, tri-weekly, quad-weekly, or monthly.

66. The kit according to any one of claims 61-65, wherein the instructions include instructions for administering the anti-angiogenic agent and the PLK1 inhibitor for at least two cycles.

67. The kit according to any one of claims 54-66, wherein the anti-angiogenic agent is bevacizumab.

68. The kit according to any one of claims 54-67, wherein the PLK1 inhibitor is selective and / or specific for PLK1.

69. The kit according to any one of claims 54-68, wherein the PLK1 inhibitor is onvansertib.

70. The kit according to claim 69, wherein the instructions include instructions for administering onvansertib at 12 mg / m 2 - 90 mg / m 2 .

71. The kit according to any one of claims 54-70, further comprising the anti-angiogenic agent.

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