Pharmaceutical kit for treating colon cancer and use thereof

Abstract

The present disclosure generally describes a pharmaceutical kit for treating colorectal cancer, the pharmaceutical kit including (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide and an anti-PD-1 antibody or an anti-PD-L1 antibody. The present disclosure also describes a method for treating colorectal cancer in a subject, the method comprising administering to the subject the pharmaceutical kit, wherein the (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide is administered to the subject at a dose ranging from about 10 μg / kg to 1 g / kg, and the anti-PD-1 antibody or anti-PD-L1 antibody is administered to the subject at a dose ranging from about 1 μg / kg to 100 mg / kg.

Description

[Technical Field]

[0001] This application claims priority to U.S. Application No. 63 / 444,242, filed February 8, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates generally to the field of cancer treatment. More specifically, the present disclosure relates to a pharmaceutical kit for treating colorectal cancer, comprising a combination of (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide (ABT-301) and an anti-PD-1 antibody or an anti-PD-L1 antibody. [Background technology]

[0003] Colorectal cancer (CRC) is a neoplastic disease localized within the colon or rectum, an integral part of the large intestine. CRC progresses gradually over several years and typically begins as benign polyps in the mucosal lining of the colon or rectum. Over time, these polyps can transform into cancerous cells, leading to colorectal cancer. There are various types of CRC, with adenocarcinoma accounting for the majority of cases. According to World Health Organization (WHO) statistics, CRC is the second leading cause of cancer-related deaths worldwide. In 2020, there were an estimated 1.9 million new cases of CRC and 930,000 deaths worldwide. Incidence rates were highest in Europe, Australia, and New Zealand, with mortality rates highest in Eastern Europe. By 2040, the burden of CRC is estimated to reach 3.2 million new cases per year (a 63% increase) and 1.6 million deaths per year (a 73% increase). These devastating cancers pose a significant health and economic burden to society.

[0004] With regard to medical intervention, modern approaches to treating colorectal cancer generally involve a multifaceted strategy combining surgery, radiation therapy, chemotherapy, and, in some cases, targeted therapy or immunotherapy. Various management strategies are employed for early-stage and advanced disease. For early-stage disease (i.e., tumors confined to the bowel or regional lymph nodes and without distant metastasis), surgical removal of the tumor and nearby lymph nodes is employed. In some cases, a temporary or permanent colostomy or ileostomy may be required to maintain drainage. For early-stage colorectal cancer, adjuvant chemotherapy may be recommended to kill any remaining cancer cells that cannot be identified or removed during surgery, or adjuvant chemotherapy may be administered before surgery to shrink the tumor (neoadjuvant chemotherapy). For rectal tumors, radiation therapy can be administered to reduce tumor size. For advanced-stage metastatic colorectal cancer, systemic therapy, such as chemotherapy, is the primary treatment. For patients with certain genetic mutations, such as KRAS or BRAF mutations, targeted therapy may be used in combination with chemotherapy. Patients with tumors that have certain genetic markers, such as microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR), may be considered for immunotherapy.

[0005] Although advances in medical interventions have improved the prognosis for many patients, current treatments have limitations. For example, surgical interventions are invasive procedures that directly harm patients, and the creation of temporary or permanent colostomies or ileostomies significantly impacts patients' quality of life. Chemotherapy can cause significant side effects, including nausea and vomiting, loss of appetite, fatigue, mouth sores and ulcers, and alopecia. Furthermore, the effectiveness of chemotherapy varies among individual patients, and resistance to chemotherapy develops over time, presenting additional challenges in long-term tumor management. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above, there is a need in the art for improved therapeutic methods in the treatment of colorectal cancer, with the aim of enhancing therapeutic efficacy, reducing side effects, and protecting the quality of life of patients. [Means for solving the problem]

[0007] The following presents a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an exhaustive overview of the disclosure and it does not identify key / critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0008] In light of the above, it is a primary object of the present disclosure to provide improved therapeutic methods for the treatment of colorectal cancer.As embodied and broadly described herein, one aspect of the present disclosure relates to a pharmaceutical kit for treating colorectal cancer in a subject, the pharmaceutical kit comprising: a first container containing (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide; and a second container containing an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0009] In some embodiments of the present disclosure, the anti-PD-1 antibody is selected from the group consisting of cemiplimab, pembrolizumab, nivolumab, and RMP1-14, and the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0010] Preferably, the colon cancer treatable with the pharmaceutical kit of the present invention is microsatellite-stable colon cancer or microsatellite-unstable colon cancer. Furthermore, the colon cancer may be primary colon cancer or metastatic colon cancer.

[0011] Another aspect of the present disclosure is a method for treating colorectal cancer in a subject using the pharmaceutical kit of the present invention, comprising administering the pharmaceutical kit of the present invention to the subject, wherein (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide is administered to the subject in an amount of about 10 μg / kg to 1 g / kg; The method relates to a method in which the anti-PD-1 antibody or anti-PD-L1 antibody is administered to the subject in an amount of about 1 μg / kg to 100 mg / kg.

[0012] More preferably, the (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide is administered to the subject in an amount of about 2.5 to 10 mg / kg, and the anti-PD-1 antibody or anti-PD-L1 antibody is administered to the subject in an amount of about 100 μg / kg to 1 mg / kg.

[0013] Examples of anti-PD-1 antibodies for use in the pharmaceutical kit of the present invention include cemiplimab, pembrolizumab, nivolumab, and RMP1-14. Alternatively, anti-PD-L1 antibodies suitable for use in the pharmaceutical kit of the present invention may be atezolizumab, avelumab, or durvalumab.

[0014] In a preferred embodiment, the colon cancer treatable with the pharmaceutical kit of the present invention is microsatellite-stable colon cancer or microsatellite-unstable colon cancer. Furthermore, the colon cancer may be primary colon cancer or metastatic colon cancer.

[0015] Subjects that can be treated by the pharmaceutical kit or method of the present invention are mammals, such as humans, mice, rats, guinea pigs, hamsters, monkeys, pigs, dogs, cats, horses, sheep, goats, cows, rabbits, etc. Preferably, the subject is a human.

[0016] Other aspects of the present disclosure include subject matter including pharmaceutical compositions comprising the pharmaceutical combinations described herein and pharmaceutically acceptable excipients, as well as pharmaceutical kits or pharmaceutical compositions for use in the manufacture of a medicament for treating colorectal cancer in a subject in need thereof.

[0017] Many of the attendant features and advantages of the present disclosure will be better understood by reference to the following detailed description considered in connection with the accompanying drawings.

[0018] These and other features, aspects and advantages of the present invention will become better understood with reference to the following description, appended claims and accompanying drawings. [Brief explanation of the drawings]

[0019] [Figure 1A] Figures 1A-1G show the results of treatment of mice bearing CT26 (a microsatellite stable (MSS) subtype colon cancer cell line) with a combination of ABT-301 and avelumab (an anti-PD-L1 antibody). Figure 1A shows the number of mice that achieved a complete response (CR) in the indicated treatment groups (n = 8 / group). [Figure 1B] FIG. 1B shows the number of mice that exhibited a complete response (CR) in the indicated treatment groups (n=8 / group). [Figure 1C] FIG. 1C shows the number of mice that exhibited a complete response (CR) in the indicated treatment groups (n=8 / group). [Figure 1D] FIG. 1D shows the number of mice that exhibited a complete response (CR) in the indicated treatment groups (n=8 / group). [Figure 1E] FIG. 1E shows the progression of tumor volume over 28 days. [Figure 1F] FIG. 1F shows the tumor volume on day 28. [Figure 1G] FIG. 1G shows the survival rate of mice after each treatment. [Figure 2A]Figures 2A-2J show the results of treatment of CT26-bearing mice with a combination of ABT-301 and avelumab. Figure 2A shows the number of mice in the indicated treatment groups (n=10 / group) that showed progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR). [Figure 2B] Figure 2B shows the number of mice that experienced progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR) in the indicated treatment groups (n=10 / group). [Figure 2C] FIG. 2C shows the number of mice in the indicated treatment groups (n=10 / group) that showed progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR). [Figure 2D] FIG. 2D shows the number of mice in the indicated treatment groups (n=10 / group) that showed progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR). [Figure 2E] FIG. 2E shows the number of mice that showed progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR) in the indicated treatment groups (n=10 / group). [Figure 2F] FIG. 2F shows the number of mice in the indicated treatment groups (n=10 / group) that showed progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR). [Figure 2G] FIG. 2G shows the number of mice in the indicated treatment groups (n=10 / group) that showed progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR). [Figure 2H] Figure 2H shows the number of mice that showed progressive disease (PD), stable disease (SD), partial response (PR), or complete response (CR) in the indicated treatment groups (n=10 / group). [Figure 2I] FIG. 2I shows the progression of tumor volume over 24 days in mice after the indicated treatments. [Figure 2J] Figure 2J shows tumor volumes on day 21 in mice after the indicated treatments. [Figure 3A]Figures 3A-3J show the results of treatment of CT26-bearing mice with a combination of ABT-301 and RMP1-14 (an anti-mouse PD-1 antibody). Figure 3A shows the number of mice that achieved PD, SD, PR, or CR in the indicated treatment groups (n = 10 / group). [Figure 3B] FIG. 3B shows the number of mice that exhibited PD, SD, PR, or CR in the indicated treatment groups (n=10 / group). [Figure 3C] FIG. 3C shows the number of mice that exhibited PD, SD, PR, or CR in the indicated treatment groups (n=10 / group). [Figure 3D] FIG. 3D shows the number of mice that exhibited PD, SD, PR, or CR in the indicated treatment groups (n=10 / group). [Figure 3E] FIG. 3E shows the number of mice that exhibited PD, SD, PR, or CR in the indicated treatment groups (n=10 / group). [Figure 3F] FIG. 3F shows the number of mice that exhibited PD, SD, PR, or CR in the indicated treatment groups (n=10 / group). [Figure 3G] FIG. 3G shows the number of mice that exhibited PD, SD, PR, or CR in the indicated treatment groups (n=10 / group). [Figure 3H] FIG. 3H shows the number of mice that exhibited PD, SD, PR, or CR in the indicated treatment groups (n=10 / group). [Figure 3I] FIG. 3I shows the progression of tumor volume over 27 days in mice after the indicated treatments. [Figure 3J] Figure 3J shows tumor volumes on day 17 in mice after the indicated treatments. [Figure 4A] Figures 4A-4I show the results of treatment of mice bearing MC-38 (a microsatellite instability-high (MSI-H) subtype colon cancer cell line) with ABT-301 in combination with avelumab or RMP1-14. Figure 4A shows the number of mice that achieved a complete response (CR) in the indicated treatment group (n = 8 / group). [Figure 4B] Figure 4B shows the number of mice that exhibited a complete response (CR) in the indicated treatment group (n=8 / group). [Figure 4C] FIG. 4C shows the number of mice that exhibited a complete response (CR) in the indicated treatment group (n=8 / group). [Figure 4D] FIG. 4D shows the number of mice that exhibited a complete response (CR) in the indicated treatment group (n=8 / group). [Figure 4E] FIG. 4E shows the number of mice that exhibited a complete response (CR) in the indicated treatment group (n=8 / group). [Figure 4F] FIG. 4F shows the number of mice that exhibited a complete response (CR) in the indicated treatment group (n=8 / group). [Figure 4G] Figure 4G shows the progression of tumor volume over 25 days, except for the vehicle group, which reached the humane endpoint and therefore only shows results up to day 20. [Figure 4H] Figure 4H shows tumor volumes on day 25, except for the vehicle group, which reached the humane endpoint and therefore shows results up to day 20. [Figure 4I] Figure 4I shows the survival rate of mice after the indicated treatments. [Figure 5A] Figures 5A-5K show the results of treating CT26-bearing mice with a combination of HDAC inhibitors (ABT-301, vorinostat, or citalinostat) and RMP1-14. Figure 5A shows the tumor volume for each mouse in the indicated treatment group (n = 8 or 9 / group). [Figure 5B] FIG. 5B shows the tumor volume for each mouse in the designated treatment group (n=8 or 9 / group). [Figure 5C] FIG. 5C shows the tumor volume for each mouse in the designated treatment group (n=8 or 9 / group). [Figure 5D] FIG. 5D shows the tumor volume for each mouse in the designated treatment group (n=8 or 9 / group). [Figure 5E] FIG. 5E shows the tumor volume for each mouse in the designated treatment group (n=8 or 9 / group). [Figure 5F] FIG. 5F shows the tumor volume for each mouse in the designated treatment group (n=8 or 9 / group). [Figure 5G]Figure 5G shows the tumor volume for each mouse in the designated treatment group (n=8 or 9 / group). [Figure 5H] FIG. 5H shows the tumor volume for each mouse in the indicated treatment group (n=8 or 9 / group). [Figure 5I] FIG. 5I shows the progression of tumor volume over 49 days in mice after the indicated treatments. [Figure 5J] FIG. 5J shows tumor volumes on day 22 in mice after the indicated treatments. [Figure 5K] FIG. 5K shows tumor volumes in mice after the indicated treatments at day 31 (when the indicated treatments were discontinued). DETAILED DESCRIPTION OF THE INVENTION

[0020] The detailed description provided below in conjunction with the accompanying drawings illustrates embodiments of the invention and is not intended to represent the only manner in which the embodiments of the invention may be constructed or used. The description sets forth functions of the embodiments and the sequence of steps for constructing and operating the embodiments, although the same or equivalent functions and steps may be accomplished by different embodiments.

[0021] I. Definition For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood and used by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall be understood to include the plural of the same term, and plural terms shall include the singular. Specifically, as used in the specification and claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Furthermore, as used in the specification and claims, the terms "at least one" and "one or more" have the same meaning and include one, two, three, or more. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of those of ordinary skill in the art. Such techniques are fully explained in the literature.

[0022] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors resulting from the standard deviation in their respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error of the mean value as considered by one of ordinary skill in the art. Except in the operating / working examples, or unless otherwise specified, all numerical ranges, amounts, values, and percentages disclosed herein (e.g., amounts of materials, lengths of time, temperatures, operating conditions, ratios of amounts, etc.) should be understood in all instances to be modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in this disclosure and the appended claims are approximations and can be modified as necessary. At the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0023] Colorectal cancer can be classified according to traditional histological subtypes defined by the WHO. The most common subtype is adenocarcinoma not otherwise specified, accounting for 85% of colorectal cancer cases worldwide. The second most common subtype is mucinous carcinoma, characterized by the presence of mucus lakes in at least 50% of the tumor area and accounting for 5–20% of colorectal cancer cases worldwide. Medullary carcinoma, characterized by solid growth accompanied by an inflammatory response, is estimated to account for 4% of colorectal cancer cases. Signet ring cell carcinoma accounts for less than 2% of colorectal cancer cases worldwide. Alternatively, colorectal cancer can be classified according to molecular classification, in which (1) DNA mismatch repair proficient / microsatellite stable (pMMR / MSS) or (2) DNA mismatch repair deficient / microsatellite instability-high (dMMR / MSI-H) are the primary criteria for colorectal cancer classification. MSI-H (or dMMR / MSI-H) subtype colorectal cancer accounts for only 8-10% of all colorectal cancer cases, while 90% of colorectal cancer patients are pMMR / MSS. Microsatellite instability (MSI) and its opposite, microsatellite stability (MSS), can be determined by pentaplex PCR using the mononucleotide MSI markers BAT-25, BAT-26, NR-21, NR-22, and NR-24. MSI is defined as positive when allele size variations are observed in three or more of the five markers.

[0024] As used herein, the terms "treatment" and "treating" may refer to curative or palliative measures. The term "treating" includes partially or completely preventing, ameliorating, alleviating, and / or managing symptoms, secondary diseases, or conditions associated with colorectal cancer. As used herein, the term "treating" refers to administering or administering the pharmaceutical kit and / or method of the present invention to a subject with a symptom, secondary disease, or condition associated with colorectal cancer, with the aim of partially or completely alleviating, ameliorating, mitigating, delaying the onset, inhibiting progression, reducing the severity, and / or reducing the incidence of one or more symptoms, secondary diseases, or characteristics associated with colorectal cancer. Symptoms, secondary diseases, and / or conditions associated with colorectal cancer include, but are not limited to, fever, weakness, fatigue, weight loss, pain, cough, bleeding, skin changes, diarrhea or constipation, nausea, vomiting, and loss of appetite. Treatment may be administered to subjects who exhibit only early signs of symptoms, secondary diseases, and / or conditions associated with colorectal cancer, with the intent of reducing the risk of developing such symptoms, diseases, and / or conditions. Treatment is generally "effective" if one or more symptoms, clinical manifestations, or markers are reduced, as defined herein. Alternatively, treatment is "effective" if the progression of a symptom, disease, or condition is reduced or halted.

[0025] As used herein, the terms "administered," "administering," or "administration" are used interchangeably to refer to the direct administration of the pharmaceutical kit of the present disclosure, including (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide and an anti-PD-1 antibody or anti-PD-L1 antibody.

[0026] The term "effective amount" as used herein refers to an amount of a component sufficient to produce a desired response. For therapeutic purposes, an effective amount is also an amount in which any toxic or harmful effects of the component are outweighed by the therapeutically beneficial effects. The specific effective or sufficient amount will vary depending on factors such as the specific condition being treated, the patient's physical condition (e.g., the patient's weight, age, and sex), the type of mammal or animal being treated, the duration of treatment, the nature of concomitant therapy (if any), and the specific formulation used, as well as the structure of the compound or its derivatives. An effective amount can be expressed, for example, in grams, milligrams, micrograms, or milligrams per kilogram of body weight (mg / kg). Alternatively, an effective amount can be expressed as the concentration of the active component (e.g., the drug ABT-301), for example, in molar concentration, mass concentration, volume concentration, molality, mole fraction, mass fraction, and mixture ratio. Specifically, the term "therapeutically effective amount" as used herein refers to an amount of drug sufficient to reduce or ameliorate symptoms associated with colorectal cancer in a subject. Those skilled in the art can calculate the human equivalent dose (HED) of a pharmaceutical (e.g., the drug ABT-301 of the present invention) based on the dose determined from the animal model. For example, when estimating the maximum safe dose for use in human subjects, the industry guidance issued by the U.S. Food and Drug Administration (FDA) entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" can be followed.

[0027] The term "subject" or "patient" refers to an animal, including a human, that can be treated by the pharmaceutical kits and / or methods of the present disclosure. The term "subject" or "patient" refers to both male and female genders unless otherwise indicated. Thus, the term "subject" or "patient" includes any mammal that may benefit from treatment for colon cancer. Examples of "subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and chickens. In an exemplary embodiment, the subject is a human.

[0028] Tumor response was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) as described herein. A complete response (CR) was defined as the complete disappearance of measurable or evaluable tumor lesions. A partial response (PR) was defined as a reduction in the size of measurable tumor lesions by at least 30% (≥30%) compared to the original tumor size. Stable disease (SD) was defined as a reduction in the size of measurable tumor lesions by less than 30% (<30%) or an increase in the size of measurable tumor lesions by less than 20% (<20%) compared to the original tumor size. Progressive disease (PD) was defined as an increase in tumor volume by more than 30% (>30%) compared to the original tumor size or the appearance of new lesions.

[0029] II. DESCRIPTION OF THE INVENTION

[0030] The present disclosure is based, at least in part, on the discovery that the compound (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide (ABT-301) exhibits efficacy against colorectal cancer. Furthermore, it is noteworthy that ABT-301 enhances the efficacy of immunotherapy in the treatment of colorectal cancer, not only of the MSI-H subtype but also of the difficult-to-treat, refractory MSS subtype. In accordance with the above, ABT-301 exerts synergistic effects in the treatment of colorectal cancer when administered in combination with immunotherapy, particularly immune checkpoint inhibitors such as anti-PD-1 antibodies and anti-PD-L1 antibodies.

[0031] 1. Medicine Kit Thus, a first aspect of the present disclosure relates to a pharmaceutical kit for treating colorectal cancer in a subject in need thereof. According to an embodiment of the present disclosure, the pharmaceutical kit of the present invention comprises: a first container containing (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide; and a second container containing an anti-PD-1 antibody or an anti-PD-L1 antibody; Includes:

[0032] In essence, the drug ABT-301 of the present invention exhibits preferential inhibitory activity against group I and IIb histone deacetylases (HDACs), including group I HDACs HDAC1, HDAC2, and HDAC3, and group IIb HDACs HDAC6 and HDAC10. In the treatment of colorectal cancer, ABT-301 not only induces apoptosis of tumor cells but also enhances the therapeutic effect of immunotherapy, particularly for the refractory MSS subtype of colorectal cancer.

[0033] Generally, the anti-PD-1 antibody suitable for use in the pharmaceutical kit of the present invention may be cemiplimab, pembrolizumab, nivolumab, or RMP1-14. Preferably, the anti-PD-1 antibody suitable for use in the pharmaceutical kit of the present invention is RMP1-14. Alternatively, the pharmaceutical kit of the present invention may comprise an anti-PD-L1 antibody, which may be atezolizumab, avelumab, or durvalumab. Preferably, the anti-PD-L1 antibody suitable for use in the pharmaceutical kit of the present invention is avelumab.

[0034] Without being bound by theory, other immune checkpoint inhibitor options may also be utilized in the pharmaceutical kits of the present invention, such as anti-CTLA4 antibodies, such as ipilimumab, tremelimumab, and zalifrelimab.

[0035] Optionally, or in addition, the pharmaceutical kits of the present invention can also include other immunotherapeutic agents, such as CC motif chemokine ligand 3 (CCL3), CC motif chemokine ligand 26 (CCL26), CXC motif chemokine ligand 7 (CXCL7), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-β (IL-6), interleukin-β (IL-6), interleukin-γ ... -7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15); apremilast, imiquimod, lenalidomide, pomalidomide, sipulcel-T, thalidomide; anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD11a antibody, anti-CD20 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-EGFR antibody, anti-HER2 antibody, anti-PCDP1 antibody, anti-SLAMF7 antibody, and anti-Trop-2 antibody.

[0036] For the purpose of treating colorectal cancer, the pharmaceutical kit of the present invention may further comprise a chemotherapeutic or targeted therapeutic agent. Exemplary chemotherapeutic agents include actinomycin D, aminoglutethimide, amsacrine, anastrozole, anthracyclines, bexarotene, bleomycin, buserelin, busulfan, camptothecin derivatives, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, exemestane, fludarabine, fluorouracil, formustane, gemcitabine, goserelin, and the like. These include, but are not limited to, cyclosporine, hycamtin, idarubicin, ifosfamide, imatinib, irinotecan, letrozole, leuprorelin, lomustine, melphalan, mercaptopurine, methotrexate, miltefosine, mitomycin, mitoxantrone, nimustine, oxaliplatin, paclitaxel, pentostatin, procarbazine, temozolomide, teniposide, testolactone, thiotepa, thioguanine, topotecan, treosulfan, tretinoin, triptorelin, trofosfamide, vinblastine, vincristine, vindesine, and vinorelbine.

[0037] According to embodiments of the present disclosure, the targeted therapeutic agent is selected from the group consisting of bortezomib, dasatinib, erlotinib, gefitinib, lapatinib, nilotinib, sorafenib, sunitinib, tofacitinib, crizotinib, venetoclax, obatoclax, navitoclax, gossypol, olaparib, rucaparib, niraparib, talazoparib, perifosine, apatinib, vemurafenib, dabrafenib, trametinib, vismodegib, sonidegib, salinomycin, and vintafoline. The therapeutic agent may be rituximab, temsirolimus, everolimus, rituximab, trastuzumab, alemtuzumab, cetuximab, panitumumab, bevacizumab, trichostatin A, phenylbutyrate, sodium butyrate, valproic acid, suberoylanilide hydroxamic acid, belinostat, citalinostat, entinostat, mocetinostat, nanatinostat, panobinostat, pracinostat, romidepsin, tucidinostat, and vorinostat.

[0038] Containers suitable for holding pharmaceutical agents can be manufactured from a variety of materials, such as glass or plastic. The first container can contain an effective amount of the pharmaceutical agent of the present invention, ABT-301, or a pharmaceutical formulation thereof, for treating colorectal cancer. The second container can contain an effective amount of an anti-PD-1 antibody or anti-PD-L1 antibody, or a pharmaceutical formulation thereof, for treating colorectal cancer. Alternatively, or additionally, the kit can further comprise a third container containing a pharmaceutically acceptable excipient (e.g., a pharmaceutically acceptable buffer), such as phosphate-buffered saline (PBS), Ringer's solution, or dextrose solution. Other materials desirable from a commercial or user standpoint may also be included, including other buffers, diluents, filters, needles, and syringes. The kit can further comprise a label or package insert on or associated with the container. The label or package insert will typically be written instructions on the label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions recorded on a magnetic or optical disk) are also acceptable. The label or package insert indicates that ABT-301 and the anti-PD-1 antibody or anti-PD-L1 antibody contained in the first and second containers, respectively, are used to treat colorectal cancer. The label or package insert may also include instructions for administering ABT-301 and the anti-PD-1 antibody or anti-PD-L1 antibody. The pharmaceutical kit of the present invention may be provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), boxes, etc.

[0039] 2. Treatment methods for colon cancer In another aspect of the present disclosure, the present invention relates to a method for treating a subject in need of treatment (e.g., a subject suffering from or suspected of having colorectal cancer) by use of the pharmaceutical kit of the present invention. The method of the present invention comprises the step of administering the pharmaceutical kit of the present invention to the subject, wherein: (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide is administered to the subject in an amount of about 10 μg / kg to 1 g / kg; The anti-PD-1 antibody or anti-PD-L1 antibody is administered to the subject in an amount of about 1 μg / kg to 100 mg / kg.

[0040] Essentially, the agent of the present invention, ABT-301, acts as an HDAC inhibitor, induces apoptosis in colon cancer cells, and can enhance the therapeutic effect of immunotherapy. According to some embodiments, the subject is a mouse, and ABT-301 is administered in an amount of 0.1 μg to 1 kg per kg of subject body weight per day (i.e., 0.1 μg to 1 kg / kg / day). Preferably, the agent of the present invention is administered in an amount of 1 μg to 100 g / kg / day. More preferably, the agent of the present invention is administered in an amount of 10 μg to 10 g / kg / day. Even more preferably, the agent of the present invention is administered in an amount of 0.1 mg to 10 g / kg / day. Even more preferably, the agent of the present invention is administered in an amount of 0.1 mg to 1 / kg / day. Even more preferably, the agent of the present invention is administered in an amount of 1 mg to 1 g / kg / day. According to some embodiments of the present disclosure, 25-100 mg / kg / day (e.g., 25, 50, or 100 mg / kg / day) of ABT-301 is sufficient to induce apoptosis of colon cancer cells and enhance the efficacy of immunotherapy.

[0041] Those skilled in the art can calculate the human equivalent dose (HED) of the drug ABT-301 based on the dose determined from the animal model. Thus, the drug ABT-301 can be administered to humans at a dose of 10 ng to 100 g (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, , 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 9 80, or 990 ng / kg / day; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 80, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, or 990 μg / kg / day;or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 9 90, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, or 990 mg / kg / day; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g / kg / day). Preferably, the drug ABT-301 is administered to a human in an amount of 100 ng to 10 g / kg / day. More preferably, the drug ABT-301 is administered to a human in an amount of 1 μg to 1 g / kg / day. Even more preferably, the drug ABT-301 is administered to a human in an amount of 10 μg to 1 g / kg / day. Even more preferably, the drug ABT-301 is administered to a human in an amount of 10 μg to 100 mg / kg / day. Even more preferably, the drug ABT-301 is administered to a human in an amount of 0.1 mg to 100 mg / kg / day. According to some embodiments of the present disclosure, the drug ABT-301 is administered to a human in an amount of 2.5 to 10 mg / kg / day (e.g., 2.5, 5, or 10 mg / kg / day).

[0042] Depending on the desired purpose, the drug ABT-301 can be administered by any suitable route, for example, enteral, oral, nasal, parenteral (such as intratumoral, intramuscular, intravenous, intraarterial, subcutaneous, intraperitoneal, intracerebral, intraventricular or intrathecal injection), or transmucosal administration. According to one embodiment of the present disclosure, the drug ABT-301 is administered orally.

[0043] For the purpose of inducing apoptosis of colon cancer cells and / or enhancing the effectiveness of immunotherapy, the drug ABT-301 can be administered to a subject once or multiple times. For example, the drug ABT-301 can be administered once for the entire course of treatment. Alternatively, the agent ABT-301 can be administered to a subject daily for at least 7 days; for example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days. In certain embodiments, when multiple doses are administered to a subject, the frequency of administration of the multiple doses to the subject is three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months, once seasonally (every three months), once every six months, or once a year. In certain embodiments, the frequency of administration of the multiple doses to the subject is once a day. In certain embodiments, the frequency of administration of the multiple doses to the subject is twice a week. In certain embodiments, when multiple doses are administered to a subject, the period from the first administration of the multiple doses to the last administration of the multiple doses is 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years, 15 years, 20 years, or the subject's lifetime. In certain embodiments, the period between the first and last administration of multiple doses is about 1 to 1.5 months.

[0044] According to embodiments disclosed herein, immunotherapies that may benefit from co-administration with the agent ABT-301 include an anti-PD-1 antibody or an anti-PD-L1 antibody, as described above. According to some embodiments, the subject is a mouse, and the anti-PD-1 antibody or anti-PD-L1 antibody is administered in an amount of 1 ng to 1 kg per kg of the subject's body weight per day (i.e., 1 ng to 1 kg / kg / day). Preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered in an amount of 10 ng to 100 g / kg / day. More preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered in an amount of 100 ng to 10 g / kg / day. Even more preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered in an amount of 1 μg to 10 g / kg / day. Even more preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered in an amount of 10 μg to 1 g / kg / day. Even more preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered in an amount of 100 μg to 100 mg / kg / day. According to some embodiments of the present disclosure, an amount of 1 mg to 10 mg / kg / day (e.g., 3.3 or 6.7 mg / kg / day) of the anti-PD-1 antibody or anti-PD-L1 antibody is sufficient to exhibit synergistic effects with the drug ABT-301 in the treatment of colorectal cancer.

[0045] If the subject is a human, the anti-PD-1 antibody or anti-PD-L1 antibody is administered at a dose of 0.1 ng to 100 g (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 8 20, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 90 0, 910, 920, 930, 940, 950, 960, 970, 980, or 990 ng / kg / day; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 8 40, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, or 990 μg / kg / day;or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 9 90, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, or 990 mg / kg / day; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 g / kg / day). Preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered to a human in an amount of 1 ng to 10 g / kg / day. More preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered to a human in an amount of 10 ng to 1 g / kg / day. Even more preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered to a human in an amount of 100 ng to 1 g / kg / day. Even more preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered to a human in an amount of 1 μg to 100 mg / kg / day. Even more preferably, the anti-PD-1 antibody or anti-PD-L1 antibody is administered to a human in an amount of 10 μg to 10 mg / kg / day. According to some embodiments of the present disclosure, the anti-PD-1 antibody or anti-PD-L1 antibody is administered to a human in an amount of 100 μg to 1 mg / kg / day (e.g., 330 or 670 μg / kg / day).

[0046] Alternatively, the actual dosage of the drugs ABT-301 and anti-PD-1 antibody or anti-PD-L1 antibody of the present invention can be determined by the attending physician based on the subject's physical and physiological factors, including, but not limited to, age, sex, weight, body surface area, the disease being treated, the severity of the condition, medical history, the presence of other medications, and the route of administration.

[0047] Non-limiting routes of administration include, but are not limited to, enteral, oral, nasal, parenteral, topical, or transmucosal administration, where parenteral administration may be intratumoral, intramuscular, intravenous, intraarterial, subcutaneous, intraperitoneal, intracerebral, intraventricular, or intrathecal injection. In one example, the anti-PD-1 antibody or anti-PD-L1 antibody is administered to a subject via the intraperitoneal route.

[0048] As will be appreciated, the drug of the present invention, ABT-301, can be administered to a subject before, simultaneously with, or after the administration of an anti-PD-1 antibody or anti-PD-L1 antibody, depending on medical considerations under the guidance of a medical professional. For the purpose of treating colorectal cancer, the anti-PD-1 antibody or anti-PD-L1 antibody can be administered to a subject once or multiple times. For example, the anti-PD-1 antibody or anti-PD-L1 antibody can be administered once for the entire course of treatment. Alternatively, the anti-PD-1 antibody or anti-PD-L1 antibody can be administered to a subject daily for at least 7 days; for example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days. In certain embodiments, when multiple doses are administered to a subject, the frequency of administration of the multiple doses to the subject is three times a day, twice a day, once a day, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months, once seasonally (every three months), once every six months, or once a year. In certain embodiments, the frequency of administration of the multiple doses to the subject is once a day. In certain embodiments, the frequency of administration of the multiple doses to the subject is twice a week. In certain embodiments, when multiple doses are administered to a subject, the period from the first administration of the multiple doses to the last administration of the multiple doses is 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 7 years, 10 years, 15 years, 20 years, or the subject's lifetime. In certain embodiments, the period between the first and last administration of multiple doses is about 1 to 1.5 months.

[0049] Colon cancer treatable by the pharmaceutical kit and / or method of the present invention may be MSS-type or MSI-type colon cancer. Furthermore, the colon cancer may be primary colon cancer or metastatic colon cancer. Optionally, the colon cancer may be resistant to chemotherapy (e.g., 5-fluorouracil (5-FU)), radiation therapy (e.g., ultraviolet (UV) irradiation), or immunotherapy (e.g., adoptive immune cell therapy (AIT)). Therefore, the pharmaceutical kit and / or method of the present invention provides a potential means for treating colon cancer patients who have developed resistance to cancer treatment.

[0050] Optionally or additionally, the method of the present invention includes administering an additional anti-cancer treatment to the subject before, simultaneously with, or after administering the pharmaceutical kit of the present invention to the subject. The additional anti-cancer treatment is at least one of surgery, radiation therapy, hyperthermia, immunotherapy, chemotherapy, or targeted therapy. Exemplary agents used in immunotherapy, chemotherapy, or targeted therapy are described above; for the sake of brevity, their listing will be omitted here.

[0051] Alternatively, the drug of the invention, ABT-301, may be administered alone to patients with colorectal cancer without combination treatment (e.g., concomitant or prior treatment) with an anti-PD-1 antibody or an anti-PD-L1 antibody and still achieve a discernible degree of efficacy in killing colorectal cancer, and vice versa.

[0052] Essentially, the subjects treatable by the pharmaceutical kits and / or methods of the present invention are mammals, and preferably the subjects are human.

[0053] The following examples are provided to clarify certain aspects of the present invention and to assist those skilled in the art in practicing the present invention. These examples are not to be considered as limiting the scope of the present invention in any way. Without further explanation, it is believed that those skilled in the art can make the most of the present invention based on the description provided herein. All publications cited herein are incorporated herein by reference in their entirety.

[0054] Example

[0055] Materials and Methods 1. HDAC Enzyme Activity Assay To evaluate the HDAC enzyme activity of ABT-301, an HDAC enzyme activity assay was performed using the biochemical HDAC assay service platform provided by Reaction Biology Corp. (Malvern, PA, USA). The HDAC enzyme activity assay was performed using a synthetic HDAC substrate, the structure of which is shown in Formula (I). The moiety required for signal generation was linked to the carboxyl of acetyllysine (K(Ac)), the target of deacetylation by HDAC. After deacetylation by HDAC, the fluorescent signal group, 7-amino-4-methylcoumarin (AMC), can be quantified.

[0056] [ka]

[0057] The fluorescent peptides used to measure HDAC enzymatic activity are shown in Table 1 below. [Table 1]

[0058] Histone deacetylase activity was detected by the fluorescence release of AMC from the aforementioned fluorescent peptide substrate, which accompanies deacetylase activity. The half-maximal inhibitory concentration (IC 50 ) is 10 doses IC 50 The IC50 dose was determined as the drug concentration that reduced by 50% the increase in HDAC activity observed in control wells during compound incubation in the same format. Controls: No inhibitor control (DMSO vehicle only), and for each target, one target-specific control compound was administered at 10 doses IC50. 50HDAC reference compounds were tested in the following format: (1) trichostatin A (TSA), a target-specific control compound for HDACs 1, 2, 3, 6, 8, and 11; (2) TMP269, a target-specific control compound for HDACs 4, 5, 7, and 9; and (3) quisinostat, a target-specific control compound for HDAC 10.

[0059] 2. Cell culture CT26.WT cells (ATCC No. CRL-2638™), murine colon carcinoma cells normally classified as microsatellite stable, were maintained in RPMI supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 mM HEPES, and 1 mM sodium pyruvate. MC-38 cells (purchased from Kerafast, Inc.), derived from C57BL / 6 murine colon adenocarcinoma cells normally classified as microsatellite unstable, were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 mM HEPES, and 1× non-essential amino acids. All cells were maintained at 37°C in a humidified atmosphere of 5% CO2.

[0060] 3. Animals Five- to seven-week-old female BALB / c mice (BALB / cByJNarl) (for the CT26 subcutaneous tumor model) and five- to seven-week-old female C57BL / 6 mice (C57BL / 6JNarl) (for the MC-38 subcutaneous tumor model) were housed in standard cages in the animal facility at the Industrial Technology Research Institute (ITRI). A one-week acclimation period was allowed prior to the start of the study, and the mice's health was monitored daily. Animals were housed in a room with a temperature of 22-26°C, humidity of 40-70%, positive pressure, 60% air recirculation, 15-20 air changes per hour, and a controlled light-dark cycle (12-12 h).

[0061] 4. CT26 Subcutaneous Tumor Model CT26 cells (2 x 10 5) was suspended in 100 μl of PBS and subcutaneously inoculated into the right flank of female BALB / c mice. Tumors were measured with a caliper, and tumor size was calculated using the following formula: tumor volume (V) = (L × S 2 ) / 2 (L is the longest diameter, mm; S is the shortest diameter, mm). Tumor size and mouse weight were monitored and recorded two to three times a week. The antitumor activity of the treatment was expressed as the tumor growth inhibition rate (%TGI), calculated using the following formula: [1 - (final tumor volume in the treatment group - initial tumor volume in the treatment group) / (final tumor volume in the vehicle group - initial tumor volume in the vehicle group)] × 100. The weight of each mouse was also compared with the day of treatment initiation (day 0) and expressed as a percentage of the value on day 0. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, USA) and approved by the Institutional Animal Care and Use Committee (IACUC) of ITRI (IACUC approval numbers: ITRI-IACUC-2022-031, ITRI-IACUC-2023-031, and ITRI-IACUC-2023-010).

[0062] 4-1. Single dose of ABT-301 (with or without avelumab) Treatment was initiated on day 5 after CT26 cell inoculation, and the day treatment began was designated day 0. The mean tumor volume was 50–100 mm. 3 At the time of tumor size, mice were divided into four groups according to S-type classification. The four groups included: (1) vehicle; (2) avelumab (100 μg, intraperitoneally (IP), twice weekly (BIW); (3) ABT-301 (100 mg / kg, orally (PO), once daily (QD)); and (4) ABT-301 (100 mg / kg, PO, QD) + avelumab (100 μg, IP, BIW). Eight mice were used in each group. Treatment was administered for 43 days. ABT-301 was prepared in 0.5% carboxymethylcellulose + 0.1% Tween 80 in 5% dextrose. Avelumab was prepared in PBS. The treatment regimens are summarized in Table 2.

[0063] [Table 2]

[0064] 4-2. Multiple administration of ABT-301 (with or without avelumab) The average tumor volume is approximately 80 to 100 mm 3 Treatment was initiated when the sigma-positive ... Treatment was administered for the specified duration (24 days) or until a humane endpoint was reached. Ten mice were used in each group. ABT-301 was prepared in 0.5% carboxymethylcellulose + 0.1% Tween 80 in 5% dextrose. Avelumab was prepared in PBS. The treatment schedule is summarized in Table 3.

[0065] [Table 3]

[0066] 4-3. Multiple administration of ABT-301 (with or without combination with RMP1-14) The average tumor volume is approximately 80 to 100 mm 3Treatment was initiated when the sigma-positive mice reached 100 mg / kg, and the day of treatment initiation was designated as day 0. Mice were divided into eight groups: (1) vehicle; (2) RMP1-14 (200 μg, IP, BIW); (3) ABT-301 (25 mg / kg, PO, QD); (4) ABT-301 (25 mg / kg, PO, QD) + RMP1-14 (200 μg, IP, BIW); (5) ABT-301 (50 mg / kg, PO, QD); (6) ABT-301 (50 mg / kg, PO, QD) + RMP1-14 (200 μg, IP, BIW); (7) ABT-301 (100 mg / kg, PO, QD); and (8) ABT-301 (100 mg / kg, PO, QD) + RMP1-14 (200 μg, IP, BIW). Treatments were administered for the specified duration (27 days) or until a humane endpoint was reached. Ten mice were used in each group. ABT-301 was prepared in 0.5% carboxymethylcellulose + 0.1% Tween 80 in 5% dextrose. RMP1-14 was prepared in PBS. The treatment schedule is summarized in Table 4.

[0067] [Table 4]

[0068] 4-4. HDAC inhibitors (with or without RMP1-14) In this study, HDAC inhibitors, including ABT-301, vorinostat, and citalinostat, were used. 3Treatment was initiated when the sigma-positive mice reached 100 mg / kg, and the day of treatment initiation was designated day 0. Mice were randomly assigned to eight groups: (1) vehicle; (2) RMP1-14 (200 μg, IP, BIW); (3) ABT-301 (100 mg / kg, PO, QD); (4) ABT-301 (100 mg / kg, PO, QD) + RMP1-14 (200 μg, IP, BIW); (5) vorinostat (100 mg / kg, PO, QD); (6) vorinostat (100 mg / kg, PO, QD) + RMP1-14 (200 μg, IP, BIW); (7) citalinostat (50 mg / kg, IP, QD); and (8) citalinostat (50 mg / kg, IP, QD) + RMP1-14 (200 μg, IP, BIW). Treatments were administered for the specified period (31 days) or until a humane endpoint was reached. Eight or nine mice were used in each group. ABT-301 and vorinostat were formulated in 0.5% carboxymethylcellulose + 0.1% Tween 80 in 5% dextrose. Citalinostat was formulated in 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. RMP1-14 was formulated in PBS. The treatment schedules are summarized in Table 5.

[0069] [Table 5]

[0070] 5. MC-38 Subcutaneous Tumor Model MC-38 cells (2 x 10 5 ) was suspended in 100 μl of PBS and inoculated subcutaneously into the right flank of female C57BL / 6 mice. Tumors were measured with a caliper, and tumor size was calculated using the following formula: tumor volume (V) = (L × S 2) / 2 (L is the longest diameter, mm; S is the shortest diameter, mm). Tumor size and mouse weight were monitored and recorded two to three times a week. The antitumor activity of the treatment was expressed as the tumor growth inhibition rate (%TGI), calculated using the following formula: [1 - (final tumor volume in the treatment group - initial tumor volume in the treatment group) / (final tumor volume in the vehicle group - initial tumor volume in the vehicle group)] × 100. The weight of each mouse was also compared with that on the day treatment began (day 0) and expressed as a percentage of the value on day 0. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NRC, USA) and approved by the ITRI IACUC (IACUC approval number: ITRI-IACUC-2023-003).

[0071] 5-1. Single administration of ABT-301 (with or without avelumab or RMP1-14) Treatment was initiated on day 6 after MC-38 cell inoculation, and the day of treatment initiation was designated as day 0. The mean tumor volume was 50-100 mm 3 At the time of tumor size, mice were divided into six groups according to S-type classification. The six groups included: (1) vehicle; (2) ABT-301 (100 mg / kg, PO, QD); (3) avelumab (200 μg, IP, BIW); (4) ABT-301 (100 mg / kg, PO, QD) + avelumab (100 μg, IP, BIW); (5) RMP1-14 (200 μg, IP, BIW); and (6) ABT-301 (100 mg / kg, PO, QD) + RMP1-14 (200 μg, IP, BIW). Treatment was administered for the designated period (29 or 36 days) or until a humane endpoint was reached. Each group contained eight mice. ABT-301 was formulated in 0.5% carboxymethylcellulose in 5% dextrose + 0.1% Tween 80. Avelumab and RMP1-14 were formulated in PBS. Treatment regimens are summarized in Table 6.

[0072] [Table 6]

[0073] 6. Statistical analysis Graphs and data analysis were performed using Excel and GraphPad Prism. Statistical analysis was performed using GraphPad Prism. Statistical significance was determined using Student's t-test or one-way ANOVA. A P value of less than 0.05 was considered statistically significant.

[0074] Example 1 Characterization of the HDAC inhibitory activity of ABT-301 In this example, the primary objective is to understand the HDAC enzyme activity profile exhibited by ABT-301. The procedures for assessing HDAC enzyme activity are described in the "Materials and Methods" section, and the results are shown in Table 7. Based on these results, ABT-301 exhibited clear HDAC enzyme activity against HDAC1, HDAC2, and HDAC3 (classified as HDAC group I), as well as HDAC6 and HDAC10 (classified as HDAC group IIb). However, ABT-301 did not exhibit activity against HDAC4, HDAC5, HDAC7, and HDAC9 (belonging to HDAC group IIa) or HDAC11 (corresponding to HDAC group IV). These results clearly suggest that ABT-301 functions as an inhibitor of HDAC groups I and IIb.

[0075] [Table 7]

[0076] In the HDAC enzymology study, vorinostat and tucidinostat were additionally included for comparative analysis alongside ABT-301. These two compounds belong to the HDAC class I and / or IIb inhibitors. Nevertheless, ABT-301 generally demonstrated superior inhibitory activity against these HDACs within HDAC class I and / or IIb.

[0077] Example 2 Effect of ABT-301 on Inhibition of MSS Subtype Colon Cancer

[0078] 2.1 Initial Evaluation of ABT-301 (in Combination with Avelumab) In this example, the primary objective is to evaluate the anticancer effect of ABT-301 against MSS subtype colorectal cancer and its ability to enhance the efficacy of immunotherapy against MSS subtype colorectal cancer, as this subtype accounts for the majority (approximately 90%) of colorectal cancer patients.

[0079] To this end, we conducted an initial study according to the procedures detailed in Section 4-1. Single Administration of ABT-301 (with or without Combination with Avelumab) in the "Materials and Methods" section. In this study, immunotherapy was achieved by administering avelumab to animals, and the results are shown in Figures 1A-1G. The results of tumor growth in CT26 tumor-bearing mice are detailed in Figures 1A-1D, focusing on demonstrating complete response (CR) of the designated treatment to tumor growth in mice. Compared to the vehicle group, administration of avelumab or ABT-301 alone to mice did not substantially increase the number of mice showing CR to tumors (i.e., the CR rates for the vehicle, avelumab, and ABT-301 groups were 0 / 8, 1 / 8, and 2 / 8, respectively). On the other hand, combined administration of avelumab and ABT-301 significantly improved the CR rate of treated mice (the CR rate for the combined treatment group was 7 / 8).

[0080] The tumor volumes recorded over 28 days and on day 28 are shown in Figures 1E-1F and Table 8. While ABT-301 and avelumab each exhibited different levels of antitumor activity, the combination therapy demonstrated the greatest inhibition of tumor growth. No significant differences (%) in body weight were observed between each treatment group and the vehicle group on day 28 (data not shown).

[0081] [Table 8]

[0082] Furthermore, the combination therapy significantly increased the survival rate of treated mice compared with mice treated with vehicle, avelumab, and ABT-301 (Figure 1G and Table 9). Taken together, these results demonstrate that although ABT-301 and avelumab alone can exhibit anticancer activity, the combination therapy of ABT-301 and avelumab can potently enhance the anti-tumor growth effect of avelumab against CT26 tumors, as reflected by tumor growth response and tumor volume suppression. Furthermore, the combination therapy of ABT-301 and avelumab extended the survival time of CT26 tumor-bearing mice compared with treatment with ABT-301 or avelumab alone.

[0083] [Table 9]

[0084] 2.2 Further Evaluation of ABT-301 (in Combination with Avelumab) Based on the findings in Example 2.1, studies were further expanded to multiple doses of ABT-301 (with or without avelumab) to investigate the cancer-inhibiting potential of ABT-301 in colorectal cancer and its ability to enhance the efficacy of avelumab in treating colorectal cancer. The procedures are detailed in Section "4-2. Multiple Administration of ABT-301 (with or without Avelumab)" in the "Materials and Methods" section, and the results are shown in Figures 2A-2J. According to Figures 2A-2H, compared with the vehicle group (Figure 2A), avelumab monotherapy could produce some anticancer activity, as indicated by the complete response (CR) rate (i.e., 7 out of 10 mice showed CR in the avelumab group; Figure 2B). However, increasing the ABT-301 dose from 25 mg / kg to 100 mg / kg only slightly enhanced the therapeutic effect (i.e., PD=9 and 10 in the ABT-301(25) group). From a PD and PR of 1 in the ABT-301(25) + avelumab group to a PD of 8 and SD of 2 in the ABT-301(100) group; Figures 2C, 2E, and 2G. Nevertheless, the combination of ABT-301 and avelumab significantly improved CR in treated mice, even at a minimum ABT-301 dose of 25 mg / kg (Figures 2C vs. 2D, 2E vs. 2F, and 2G vs. 2H). Furthermore, the combination therapy demonstrated a dose-dependent therapeutic effect with increasing ABT-301 dose (i.e., from a PD of 1 and CR of 9 in the ABT-301(25) + avelumab group to a CR of 10 in the ABT-301(100) + avelumab group; Figures 2D, 2F, and 2H).

[0085] The tumor volumes recorded for 24 days and on day 21 are shown in Figures 2I-2J and Table 10. Although ABT-301 and avelumab each exhibited different levels of antitumor activity, the combination therapy further suppressed tumor growth. Meanwhile, no significant difference (%) in body weight was observed between each treatment group and the vehicle group on day 24 (data not shown).

[0086] [Table 10]

[0087] Overall, ABT-301 and avelumab synergistically inhibited CT26 tumor growth. Combination treatment with ABT-301 and avelumab significantly enhanced the anti-tumor growth effect of avelumab on CT26 tumors, and ABT-301 strongly enhanced the anti-tumor growth effect of avelumab on CT26 tumors. This was supported by an increased CR rate and tumor volume suppression, even at a minimum dose of 25 mg / kg. These findings suggest a synergistic therapeutic effect, allowing for a low dose of ABT-301 to achieve potent anti-tumor effects.

[0088] 2.3 Evaluation of the Combination of ABT-301 and RMP1-14 In this example, we expanded our investigation into the potential of ABT-301 to enhance immunotherapy in the treatment of colorectal cancer to include various doses of ABT-301 in combination with another immune checkpoint inhibitor, specifically the anti-PD-1 antibody RMP1-14. The procedure is described in Section 4-3, "Multiple Administration of ABT-301 (with or without RMP1-14)," of the "Materials and Methods" section, and the results are shown in Figures 3A-3J. Similar to the results in Figures 2A-2H, administration of RMP1-14 alone can result in some anticancer activity, as indicated by the CR rate (i.e., 4 out of 10 mice showed CR in the RMP1-14 group; Figure 3B), compared with the vehicle group (Figure 3A). However, increasing the ABT-301 dose from 25 mg / kg to 100 mg / kg only slightly enhanced the therapeutic effect (i.e., from PD = 8, SD = 1, and PR = 1 in the ABT-301(25) group to PD = 7, PR = 2, and CR = 2 in the ABT-301(100) group; Figures 3C, 3E, and 3G). Nevertheless, the combination of ABT-301 and RMP1-14 significantly improved CR to tumors in treated mice, even at a minimum ABT-301 dose of 25 mg / kg (Figures 3C vs. 3D, 3E vs. 3F, and 3G vs. 3H). Furthermore, the combination therapy demonstrated a dose-dependent therapeutic effect with increasing ABT-301 dose (i.e., from PD = 2, PR = 1, and CR = 7 in the ABT-301(25) + RMP1-14 group to CR = 10 in the ABT-301(100) + RMP1-14 group; Figures 3D, 3F, and 3H).

[0089] Findings regarding tumor volume over 27 days (with particular focus on day 17) are shown in Figures 3I-3J and summarized in Table 11. ABT-301 and RMP1-14 each exhibited different levels of antitumor activity. However, the combination therapy demonstrated significant inhibition of tumor growth. Furthermore, no statistically significant differences in body weight (%) were observed between each treatment group and the vehicle group at day 27 (data not shown).

[0090] [Table 11]

[0091] Taken together, ABT-301 and RMP1-14 synergistically attenuated CT26 tumor growth. Combination treatment with ABT-301 and RMP1-14 significantly enhanced the anti-tumor growth effect of RMP1-14 against CT26 tumors, and ABT-301 subsequently potently enhanced the anti-tumor growth effect of RMP1-14 against CT26 tumors. This is supported by the increased CR rate and suppression of tumor volume, even at a minimum ABT-301 dose of 25 mg / kg. These findings suggest that a synergistic therapeutic effect exists when ABT-301 is administered in combination with RMP1-14, and that potent anti-cancer effects may be achieved with lower doses of ABT-301.

[0092] Example 3 Effect of ABT-301 on Inhibition of MSI-H Subtype Colon Cancer

[0093] 3.1 Evaluation of ABT-301 (in combination with avelumab or RMP1-14) To investigate the efficacy of ABT-301 in treating MSI-H subtype colon cancer and its potential to enhance the efficacy of immunotherapy against MSI-H subtype colon cancer, the following experiments were performed.

[0094] The study was performed according to the procedures detailed in Section "5-1. ABT-301 Single Administration (with or without Combination with Avelumab or RMP1-14)" in the "Materials and Methods" section. Immunotherapy was performed by administering avelumab or RMP1-14 to mice, and the results are shown in Figures 4A-4I. The results of tumor growth in MC-38 tumor-bearing mice are shown in Figures 4A-4F, focusing on demonstrating the CR of the designated treatment on tumor growth in treated mice. According to the results, compared with the vehicle group, administration of ABT-301 alone did not significantly increase the number of mice showing CR to tumors (i.e., the CR rates for both the vehicle and ABT-301 groups were 0 / 8; Figures 4A-4B). According to the literature, immune checkpoint inhibitors significantly improved the efficacy of MSI- compared with MSS subtype tumors. These drugs demonstrated high response rates and survival rates in the treatment of MSI-H subtype colon cancer, thereby resulting in higher survival rates. Similarly, avelumab or RMP1-14 monotherapy was also observed to achieve some anticancer efficacy (CR rates were 4 / 8 and 5 / 8 in the avelumab and RMP1-14 groups, respectively; Figures 4C and 4E). However, even under these circumstances, ABT-301, when combined with avelumab or RMP1-14, further enhanced the efficacy of avelumab or RMP1-14 in the treatment of MSI-H subtype colon cancer. The combined administration of ABT-301 with avelumab or RMP1-14 significantly increased the CR rate in treated mice (CR rates were 6 / 8 and 8 / 8 in the ABT-301 + avelumab and ABT-301 + RMP1-14 groups, respectively; Figures 4D and 4F).

[0095] Tumor volume data recorded over 25 days (with a focus on day 25) are shown in Figures 4G–4H and summarized in Table 12. ABT-301, avelumab, and RMP1-14 each exhibited different levels of antitumor activity. However, the combination therapy demonstrated more potent tumor growth inhibition. Mice in the vehicle group were euthanized on day 20 due to reaching the humane endpoint. Furthermore, no statistically significant differences in body weight (%) were observed between each treatment group and the vehicle group on day 25 (data not shown).

[0096] [Table 12]

[0097] Furthermore, although mice treated with avelumab or RMP1-14 showed a significant survival rate compared with mice treated with the vehicle, combination therapy (either ABT-301 + avelumab or ABT-301 + RMP1-14) substantially increased the survival rate of treated mice (Figure 4I and Table 13). Overall, although avelumab or RMP1-14 alone can exhibit anticancer activity, the combination of ABT-301 with avelumab or RMP1-14 can potently enhance the antitumor growth effect of avelumab or RMP1-14 against MC-38 tumors, as evidenced by the complete response to tumor growth and the suppression of tumor volume. Furthermore, combined treatment with ABT-301 and avelumab or RMP1-14 extended the survival time of MC-38 tumor-bearing mice compared with treatment with avelumab or RMP1-14 alone.

[0098] [Table 13]

[0099] Example 4 Effect of combined use of HDAC inhibitor and RMP1-14 on colon cancer growth This example aimed to evaluate the efficacy of combined treatment of RMP1-14 with different HDAC inhibitors against colorectal cancer. To this end, this study tested animals co-administered with RMP1-14 different HDAC inhibitors, including ABT-301 (a pan-HDAC inhibitor), vorinostat (a pan-HDAC inhibitor), and citalinostat (a type-specific HDAC inhibitor targeting HDAC6). The study procedures are detailed in Section "4-4. HDAC Inhibitors (with or without RMP1-14)" herein, and the results are shown in Figures 5A-5K. The results of tumor growth in CT26 tumor-bearing mice are shown in Figures 5A-5H. Compared to the vehicle group, administration of RMP1-14 alone or an HDAC inhibitor alone (i.e., ABT-301, vorinostat, or citalinostat alone) induced a visible response to tumor growth in mice. However, co-administration of HDAC inhibitors and RMP1-14 induced varying degrees of response in mice, particularly in terms of colon cancer volume. The results showed that co-administration of ABT-301 and RMP1-14 induced a better response against tumors than co-administration of vorinostat and RMP1-14 or co-administration of citalinostat and RMP1-14 (Figures 5D, 5F, and 5H).

[0100] In another batch of experiments, administration of RMP1-14 alone or other HDAC inhibitors alone (i.e., ABT-301, PCI-34051 (a type-specific HDAC inhibitor targeting HDAC8), or irebenostat (a type-specific HDAC inhibitor targeting HDAC11) alone) was found to induce no visible response to tumor growth in mice compared with the vehicle group. Conversely, co-administration of ABT-301 and RMP1-14 showed a better response to tumor growth compared with co-administration of PCI-34051 and RMP1-14 or co-administration of irebenostat and RMP1-14 (data not shown).

[0101] Changes in tumor volume were recorded over a 49-day period, with particular focus on days 22 and 31. See Figures 5I–5K and Table 14. Mice treated with the combination of ABT-301 and RMP1-14 exhibited the smallest tumor volume among all experimental groups (Figure 5I). On day 22, administration of ABT-301, vorinostat, citalinostat, or RMP1-14 alone demonstrated varying degrees of antitumor activity. However, combination therapy demonstrated more potent tumor growth inhibition, with the combination of ABT-301 and RMP1-14 demonstrating the most significant inhibitory activity (Figure 5J and Table 14). Mice in the vehicle, ABT-301, vorinostat, and citalinostat groups reached the humane endpoint and were subsequently euthanized. On day 31, even after the end of treatment, mice treated with the combination of ABT-301 and RMP1-14 exhibited the smallest tumor volume among all experimental groups. These results demonstrate that the combination of ABT-301 and RMP1-14 effectively and sustainably suppressed tumor growth, whereas other combination treatments resulted in tumor regrowth (Figure 5K). Furthermore, no statistically significant differences in body weight (%) were observed between each treatment group and the vehicle group throughout the entire experimental period (data not shown).

[0102] [Table 14]

[0103] Taken together, these data suggest that the combination of a pan-HDAC inhibitor and an anti-PD-1 antibody (e.g., RMP1-14) exhibits superior anti-cancer activity compared with the combination of a type-specific HDAC inhibitor (e.g., citalinostat, PCI-34051, or irebenostat) and an anti-PD-1 antibody. However, among the combinations of pan-HDAC inhibitors and anti-PD-1 antibodies, the combination of ABT-301 and RMP1-14 was able to enhance the anti-cancer activity compared with the combination of other pan-HDAC inhibitors (e.g., vorinostat) and RMP1-14.

[0104] In conclusion, the present invention provides an improved therapeutic approach for the treatment of colorectal cancer, including both MSS and MSI-H subtypes, by combining the drug ABT-301 with an anti-PD-1 antibody or an anti-PD-L1 antibody. This combination therapy shows improved therapeutic efficacy, thereby reducing the burden on patients affected by the disease.

[0105] It will be understood that the above description of the embodiments is merely illustrative, and that various modifications may be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. While various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more specific embodiments, those skilled in the art could make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. 1. A pharmaceutical kit for treating colon cancer in a subject, comprising: a first container containing (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide; and a second container containing an anti-PD-1 antibody or an anti-PD-L1 antibody; A pharmaceutical kit comprising:

2. The pharmaceutical kit of claim 1, wherein the anti-PD-1 antibody is selected from the group consisting of cemiplimab, pembrolizumab, nivolumab, and RMP1-14.

3. The pharmaceutical kit of claim 1, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

4. The pharmaceutical kit according to claim 1 , wherein the colon cancer is microsatellite-stable colon cancer or microsatellite-unstable colon cancer.

5. A method for treating colorectal cancer in a subject, comprising administering to the subject the pharmaceutical kit of claim 1, the (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide is administered to the subject in an amount of about 10 μg / kg to 1 g / kg; The method, wherein the anti-PD-1 antibody or anti-PD-L1 antibody is administered to the subject in an amount of about 1 μg / kg to 100 mg / kg.

6. 6. The method of claim 5, wherein the anti-PD-1 antibody is selected from the group consisting of cemiplimab, pembrolizumab, nivolumab, and RMP1-14.

7. 6. The method of claim 5, wherein the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

8. The method of claim 5, wherein the colon cancer is microsatellite-stable colon cancer or microsatellite-unstable colon cancer.

9. 6. The method of claim 5, wherein the (E)-N-hydroxy-3-(1-(phenylsulfonyl)-indolin-5-yl)-acrylamide is administered to the subject in an amount of about 2.5 to 10 mg / kg.

10. 6. The method of claim 5, wherein the anti-PD-1 antibody or anti-PD-L1 antibody is administered to the subject in an amount of about 100 μg / kg to 1 mg / kg.

11. The method of claim 5 , wherein the subject is a human.

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