Activation of Procaspase-3 and Immunotherapy for the Treatment of Cancer

Abstract

PAC-1, a procaspase-3 activator that crosses the blood-brain barrier, has been identified as an effective approach to induce immune-stimulated destruction of cancer cells. Studies have shown that PAC-1 induces MLH1 cleavage in cancer cells, and MLH1 inactivation leads to increased mutational burden and neoantigen presentation by major histocompatibility complex (MHC) products. Here, we describe a mechanism-based strategy to effectively harness the power of immunotherapy for cancer treatment.

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 854,823, filed May 30, 2019, and No. 62 / 944,404, filed December 6, 2019, which are incorporated herein by reference.

[0002] government support This invention was made with Government support under Grant No. R01 CA120439 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention Successful immunotherapy has revolutionized the treatment of melanoma, lung and bladder cancer, and shows great promise for several other tumor types. The dramatic results seen in some patients (e.g., durable responses even in late-stage disease) have raised hopes that such therapies may also be effective in other intractable cancers. There is a clear correlation between the mutational burden in tumors and the efficacy of immune checkpoint inhibitors. This notion is reflected in the approval of pembrolizumab (Keytruda, an antibody against PD-1) for patients with DNA mismatch repair deficiency and / or microsatellite instability (MSI). Unfortunately, for many cancers with low mutational burden, immunotherapy clinical trials have been largely disappointing, and the fact that less than 10% of cancers have an MSI phenotype illustrates the challenges of broadly scaling up immunotherapy success. The challenges of immunotherapy for glioblastoma (GBM) are even more significant given the low expression of neoantigens, the lack of T cell infiltration in tumors, and the difficulty of most agents crossing the blood-brain barrier (BBB).

[0004] A recent important development is the clinical approval of DNA microsatellite instability (MSI) as a biomarker of clinical benefit of PD-1 inhibition (with pembrolizumab) regardless of tumor origin. This approval is based on remarkable preclinical and clinical data showing that mismatch repair deficiency (dMMR) predicts response to PD-1 inhibition in solid tumors. Tumors with dMMR / MSI are known to harbor 100–1000 somatic mutations (10-fold higher than MMR-competent cancers, Figure 1A) and are presumed to have increased neoantigens. However, dMMR / MSI is present in a low proportion of cancers, probably less than 10%, including less than 5% of GBM. Sporadic MSI is caused by epigenetic silencing of the MLH1 promoter. Silencing of MLH1 is commonly used as a marker of MMR deficiency. The correlation between MLH1 silencing and the number of somatic mutations has been demonstrated in many studies and is shown in Figure 1.

[0005] Importantly, a recent report (Germano, G., et al., Nature 2017, 552, 116) validates that inactivation of MLH1 (via CRISPR / Cas9 knockout) leads to higher mutational burden and increased neoantigen profile. This MLH1 knockout-induced phenotype leads to cancer cells establishing minimally sympathetic tumors in mice, suggesting that dMMR is sufficient to mount an immune response. Furthermore, genomic knockout of MLH1 dramatically increases the response to immune checkpoint inhibitors (i.e., anti-PD-1 + anti-CTLA-4). These results suggest that loss of MLH1 function alters the phenotype with increased mutational burden, ultimately leading to increased neoantigen expression, immune recognition, and sensitivity to immune checkpoint inhibitors in vivo.

[0006] If we could selectively induce a decrease in MLH1 function in cancer cells, it could significantly improve patient response to immunotherapies such as checkpoint inhibitors and neoantigen peptide vaccines. Provocatively, multiple large-scale proteomics studies have revealed that MLH1 is the top substrate of caspase-3, with 0% protein remaining after 6 hours. Moreover, MLH1 is a substrate exclusively for active caspase-3, and no proteolysis by other active caspases (caspase-1, 2, 6, 7, 8) was observed.

[0007] Cleavage of procaspase-3 (PC-3) by caspase-3 is a key milestone in apoptosis, as this caspase catalyzes the hydrolysis of hundreds of protein substrates, leading to cell death. A hallmark of cancer is the ability of cancer cells to evade apoptosis through mutations and dysregulation of apoptotic proteins. Several anticancer drug discovery strategies focus on inhibiting these mutated proteins. A complementary approach is to activate proapoptotic proteins such as PC-3 with small molecules. PC-3 is downstream in the apoptotic cascade compared to frequently mutated proteins, and based on the low mutation frequency of PC-3 in cancer and the robust expression of procaspase-3 enzyme in many cancer types, including lymphoma, leukemia, multiple myeloma, glioblastoma (GBM), pancreatic cancer, liver cancer, non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, osteosarcoma, and meningioma, small molecule-mediated PC-3 activation is being actively explored as an anticancer drug strategy. Summary of the Invention [Problem to be solved by the invention]

[0008] Existing immunotherapy approaches to treat cancer are ineffective when neoantigens are expressed in low amounts, so there is a need for drugs that can selectively target cancer cells and increase the amount of neoantigens expressed, allowing for the eradication of cancer cells by immunotherapy.

[0009] Selective activation of procaspase-3 to caspase-3 in tumors leads to quantitative cleavage of MLH1, resulting in dMMR / MSI, thereby significantly enhancing the efficacy of immunotherapy. PAC-1 is used herein to selectively induce immune stimulation in cancer, including cleavage of MLH1, which converts MSS tumors into dMMR / MSI tumors, making them more susceptible to immunotherapy treatment. Results suggest that immune stimulation by PAC-1 promotes the induction of a stress response, thereby altering the tumor microenvironment and increasing the degree of immune inflammation. These results bring the power of immunotherapy to more cancer patients for dramatic and durable responses. [Means for solving the problem]

[0010] Thus, the present disclosure provides a composition comprising: (a) procaspase-3 activator; (b) at least one second active agent, where the second active agent is a checkpoint inhibitor, a cancer vaccine, a metabolic regulator, a macrophage inhibitor, or an immune stimulatory or modulatory agent; and (c) optionally, a pharma- ceutically acceptable diluent, excipient, or carrier. In various embodiments, the pro-caspase-3 activator is PAC-1. [ka]

[0011] The present disclosure also provides a method for treating cancer comprising simultaneously or sequentially administering to a subject in need thereof a therapeutically effective amount of a procaspase-3 activator and an effective amount of a second active agent, wherein the second active agent is an immunotherapeutic agent, and the effect of the second active agent is enhanced by administration of the procaspase-3 activator.

[0012] Certain embodiments of the method of treating cancer include administering to a subject PAC-1 and an anti-PD-1 antibody, where PAC-1 is administered daily for 21 or more consecutive days to provide a total daily dose of PAC-1 of about 100 mg / kg to about 125 mg / kg, and the anti-PD-1 antibody is administered two or four times for 21 or more consecutive days, where the dose of anti-PD-1 antibody is about 10 mg / kg, and where each dose of anti-PD-1 antibody is administered on a separate day.

[0013] The present disclosure also provides the use of the compositions described herein for use in medical therapy. The medical therapy can treat cancer, for example, breast cancer, triple negative breast cancer, ovarian cancer, lung cancer, endometrial cancer, pancreatic cancer, prostate cancer, lymphoma, melanoma, leukemia, multiple myeloma, glioblastoma, liver cancer, non-small cell lung cancer, osteosarcoma, meningioma, renal cancer, metastatic renal cell carcinoma, thyroid cancer, or colon cancer. Embodiments of the present disclosure also provide the use of the compositions described herein for the manufacture of a medicament for treating a mammalian disease, for example, a human cancer. The medicament can include a pharmaceutical acceptable diluent, excipient, or carrier. [Brief description of the drawings]

[0014] The following drawings form part of this specification and are included to further illustrate certain embodiments or various aspects of the present disclosure. In some instances, embodiments of the present disclosure can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may emphasize certain specific examples or aspects of the present disclosure. However, one of ordinary skill in the art will appreciate that some of the examples or aspects can be used in combination with other examples or aspects of the present disclosure.

[0015] [Figure 1]A) Microsatellite instability (MSI) and B) MLH1 silencing are strongly correlated with an increased number of somatic mutations, shown here in data from Vogelstein and co. for colon cancer (Proc Natl Acad Sci USA 2015,112,118). MSS, stable microsatellite.

[0016] [Diagram 2] Synergistic effects of PAC-1 and immunotherapy. Caspase-3 induced by PAC-1 cleaves specific proteins that sensitize cancer to various immunotherapeutic approaches.

[0017] [Diagram 3] PAC-1 treatment leads to cleavage of MLH1 in the absence of apoptotic death markers. Cell lines were incubated with the indicated concentrations of PAC-1 for 72 h and then analyzed by Western blot for MLH1 protein levels, PARP-1, cleaved PARP-1 (c-PARP-1 is an apoptosis maker), and β-actin (loading control). Cell line types are shown with special emphasis on HFF-1, a normal cell line, demonstrating cancer cell-specific MLH1 cleavage.

[0018] [Figure 4]Treatment of mice bearing coexisting tumors with PAC-1 increases the number of tumor-infiltrating lymphocytes. A) C57BL / 6 mice orthotopically implanted with GL261 neurospheres. After tumors were allowed to establish for 10 days, mice (n=3 / group) were either treated or not with PAC-1 (100mg / kg POx10 days) and then sacrificed. Tumors were stained with CD3 (brown) to identify T cell TILs. Data shown as mean CD3+ per 4HPF / mouse. Magnification 100x. B) C57BL / 6 mice implanted subcutaneously with B16F10 cells. After tumors were allowed to establish for 7 days, mice (n=8 / group) were either treated or not with PAC-1 (100mg / kg IPx2x14 days) and then sacrificed. Tumors were stained with CD3 (brown) to identify T cell TILs. Data shown as mean CD3+ per 10HPF / mouse. The magnification is l00x.

[0019] [Diagram 5] Validation for IHC studies of PD-L1 and MLH1. Positive PD-L1 expression in (A) human tonsil and (B) canine lymph node. Canine glioma (C) H&E and (D) PD-L1 IHC. E) Nuclear MLH1 IHC of human U87 and (F-H) three canine glioma cell lines.

[0020] [Figure 6] Graph showing the effect of combining PAC-1 with immunotherapy. PAC-1 was administered at 100 mg / kg once daily. 1=vehicle + isotope; 2=vehicle + anti-PD-1 + anti-CTLA-4; 3=PAC-1 + isotope; 4=PAC-1 + anti-PD-1 + anti-CTLA-4.

[0021] [Figure 7] Graph showing that the combination of PAC-1 and anti-PD-1 antibodies leads to improved survival in the late metastatic K7M2 model. MST = median survival. In the figure, aPD1 = anti-PD-1.

[0022] [Figure 8] Development of the CT-26_WT subcutaneous model in BALB / c mice.

[0023] [Figure 9] Growth after two (A) versus four (B) doses of CT-26_WT in BALB / c mice. Open circles = vehicle + anti-IgG2A antibody, squares = PAC-1 (100 mg / kg) + anti-IgG2a antibody, triangles = vehicle + anti-PD-1 mAb, inverted triangles = PAC-1 (100 mg / kg) + anti-PD-1 mAb.

[0024] [Figure 10] Analysis of treatment of BALB / c mice with PAC-1 and anti-PDL1 mAb. Open circles = vehicle + anti-IgG2A antibody, squares = PAC-1 (100 mg / kg) + anti-IgG2a antibody, triangles = vehicle + anti-PD-1 mAb, inverted triangles = PAC-1 (100 mg / kg) + anti-PD-1 mAb.

[0025] [Figure 11] Development of CT-26_TdTomato subcutaneous tumor model in BALB / C mice.

[0026] [Figure 12] A) Example of treatment protocol. B) Cytokine array of plasma from BALB / c mice treated with PAC-1.

[0027] [Figure 13] Analysis of neutrophil and macrophage populations in lungs, PBMCs, and spleens 14 days after treatment with PAC-1 - after tumor challenge. Open circles = vehicle + anti-IgG2A antibody, squares = PAC-1 + anti-IgG2a antibody, triangles = vehicle + anti-PD-1 mAb, inverted triangles = PAC-1 + anti-PD-1 mAb.

[0028] [Figure 14]Analysis of T cell, B cell, and NK cell populations in lungs, PBMCs, and spleens of BALB / c mice 26 days after combinatorial PAC-1 and anti-PD-1 treatment. Open circles = vehicle + anti-IgG2A antibody, squares = PAC-1 + anti-IgG2a antibody, triangles = vehicle + anti-PD-1 mAb, inverted triangles = PAC-1 + anti-PD-1 mAb.

[0029] [Figure 15] Expression of PD-L1 on the surface of dendritic cells and CD45- tumor cells 26 days after tumor challenge in lungs, PBMCs, and spleens of BALB / c mice. Open circles = vehicle + anti-IgG2A antibody, squares = PAC-1 + anti-IgG2a antibody, triangles = vehicle + anti-PD-1 mAb, inverted triangles = PAC-1 + anti-PD-1 mAb.

[0030] [Figure 16] Development of MC38 lung metastasis model in C57BL / 6 mice. 1=vehicle, 2=PAC-1, 3=anti-PD-1, 4=PAC-1+anti-PD-1. PAC-1 was administered by intraperitoneal injection at a dose of 100 mg / kg, and anti-PD-1 was administered by intraperitoneal injection at a dose of 10 mg / kg.

[0031] [Figure 17] Survival curves in the MC38 lung metastasis model. 1=vehicle, 2=PAC-1, 3=anti-PD-1, 4=PAC-1+anti-PD-1. PAC-1 was administered by intraperitoneal injection at a dose of 100 mg / kg, and anti-PD-1 was administered by intraperitoneal injection at a dose of 10 mg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Description Herein, we disclose the development of a novel mechanism-based strategy to selectively convert tumors with low mutational burden into tumors with high mutational burden, making them ideal candidates for immunotherapy. This strategy is premised on the targeted inactivation of the tumor suppressor MLH1. As disclosed herein, there is a strong correlation between MLH1 silencing and response to anti-PD-1 antibodies, a convincing association between MLH1 gene silencing and the number of somatic mutations in tumors is shown, and DNA damage due to loss of MLH1 function induces a highly immunogenic stress response. The goal was to bring the power and potential of immunotherapy to GBM through drug-mediated tumor-selective inactivation of MLH1. MLH1 is the major cellular substrate of caspase-3, and in the disclosed method, selective cleavage of MLH1 can be induced in cancer cells by selectively activating procaspase-3 to caspase-3 in tumor cells using a small molecule called PAC-1.

[0033] PAC-1 is an orally bioavailable, BBB-penetrant experimental therapeutic agent that has demonstrated safety in human cancer patients and is currently undergoing clinical evaluation (in combination with radiation and temozolomide) for GBM. The overall aim of this application is to achieve a mechanism-based synergy of drug-induced MLH1 cleavage and immunotherapy in a sophisticated model of GBM. The central hypothesis was that drug-mediated MLH1 cleavage induces tumor-selective DNA damage and MSI, increasing the amount (and immunogenicity) of potential neoantigens. Furthermore, the caspase-3-inducing activity of PAC-1 promotes an inflammatory environment within the tumor, thus transforming a “cold” GBM tumor into a “hot” tumor that is amenable to a variety of immunotherapeutic attacks (Figure 2).

[0034] definition The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the described terms have the following meanings. All other terms and phrases used herein have their ordinary meanings as would be understood by one of ordinary skill in the art. Such ordinary meanings are defined in Hawley's Condensed Chemical Dictionary 14 th These can be obtained by referring to technical dictionaries such as, for example, R. J. Lewis, John Wiley & Sons, New York, NY, 2001.

[0035] The phrases "one embodiment," "an embodiment," and the like in this specification indicate that the embodiment being described may include a particular aspect, feature, structure, moiety, or characteristic, but not all embodiments necessarily include that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment that is referenced elsewhere in this specification. Furthermore, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one of ordinary skill in the art to affect or connect such aspect, feature, structure, moiety, or characteristic to other embodiments, whether or not expressly described.

[0036] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" encompasses a plurality of such compounds, such that reference to compound X encompasses a plurality of compounds X. Additionally, it should be noted that the claims may be drafted to exclude any element. As such, this statement is intended as a predicate basis for using exclusive terminology, such as "solely," "solely," and the like, in connection with any element described herein, and / or for reciting claim elements or using "negative" limitations.

[0037] The term "and / or" means any one of the items, any combination of the items, or all of the items with which the term is associated. The phrases "one or more" ("one or more") and "at least one" are readily understood by those of ordinary skill in the art, particularly when read in the context of their usage. For example, the phrase can mean 1, 2, 3, 4, 5, 6, 10, 100, or any upper limit that is about 10-fold, 100-fold, 1000-fold higher than the stated lower limit.

[0038] As one of ordinary skill in the art would understand, all numerical values, including numerical values ​​expressing amounts of ingredients, properties such as molecular weight, reaction conditions, and the like, are approximate and are understood to be optionally modified in all instances by the term "about." These values ​​may vary depending on the desired properties one of ordinary skill in the art seeks to obtain using the teachings herein. It is also understood that these values ​​inherently contain variability necessarily resulting from the standard deviation found in their respective testing measurements. When values ​​are expressed as approximations by use of the preposition "about," it will be understood that the particular value without the modifier "about" also forms an additional aspect.

[0039] The terms "about" and "approximately" are used interchangeably. Both terms can refer to a variation of ±5%, ±10%, ±20%, or ±25% of the specified value. For example, "about 50" percent means, in some embodiments, a variation of 45 to 55 percent, or as otherwise defined in a particular claim. In integer ranges, the term "about" can include one or two integers greater than and / or less than the integers recited at each end of the range. Unless otherwise indicated herein, the terms "about" and "approximately" are intended to include values, e.g., weight percent, close to the recited range that are equivalent in terms of the functionality of the individual components, compositions, or embodiments. The terms "about" and "approximately" can also modify the endpoints of the recited ranges, as described above in this paragraph.

[0040] As will be understood by those skilled in the art, for all purposes, particularly in terms of providing a written description, all ranges described herein encompass all possible subranges and combinations of subranges, as well as the individual values, particularly integer values, that make up the range. Thus, it is understood that each unit between two particular units is also disclosed. For example, if 10 to 15 is disclosed, 11, 12, 13, and 14 are also disclosed individually and as part of a range. A range described (e.g., weight percent or carbon group) includes each particular value, integer, decimal, or identity within the range. Any range described is fully described and can be readily recognized as being capable of being broken down into at least equal halves, thirds, quarters, fifths, or tenths of the same range. As a non-limiting example, each range described herein can be readily broken down into a lower third, middle third, upper third, etc. Also, as will be appreciated by those of skill in the art, all terms such as "up to," "at least," "greater than," "less than," "over," "or greater than," and the like, are inclusive of the recited numbers, and such terms refer to ranges which can then be broken down into subranges as described above. Similarly, all ratios referenced herein also include all subratios falling within the broader ratio. Thus, the specific values ​​recited for radicals, substituents, and ranges are merely illustrative and do not exclude other defined or other values ​​within the defined ranges for radicals and substituents. Moreover, it will be understood that the endpoints of each range are significant both in relation to the other endpoint and independently of the other endpoint.

[0041] Also, one of ordinary skill in the art will readily recognize that where members are grouped in a common manner, such as in a Markush group, the disclosure encompasses not only the entire group as described as a whole, but also each member of the group individually, and all possible subgroups of the main group. Moreover, for all purposes, the disclosure encompasses not only the main group, but also the main group absent one or more of the group members. Thus, the disclosure contemplates the explicit exclusion of any one or more of the members of a described group. Thus, provisos may be applied to any of the disclosed categories or embodiments, whereby, for example, for use in an express negative limitation, any one or more of the described elements, species, or embodiments may be excluded from such category or embodiment.

[0042] "Contacting" refers to the act of touching, coming into contact, or immediate or close proximity, including at the cellular or molecular level, to effect a physiological response, chemical response, or physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

[0043] By "effective amount" is meant an amount effective to treat a disease, disorder, and / or condition or to produce a described effect. For example, an effective amount can mean an amount effective to reduce the progression or severity of the disease or condition being treated. Determining a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, that is, for example, an amount effective to treat or prevent a disease or disorder or treat a symptom of a disease or disorder in a host. Thus, an "effective amount" generally means an amount that produces a desired effect.

[0044] Also, as used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of an agent or composition or combination of compositions administered that is sufficient to alleviate to some extent one or more symptoms of the disease or condition being treated, resulting in reduction and / or alleviation of the signs, symptoms, or causes of the disease, or other desired changes in a biological system. For example, a therapeutic "effective amount" is the amount of a composition containing a compound described herein that is required to clinically significantly reduce a symptom of the disease. An appropriate "effective" amount in an individual case can be determined using techniques such as dose escalation studies. Dosages can be administered in single or multiple doses. However, the precise determination of what is considered an effective amount can be made based on factors specific to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of disease, route of administration of the composition, type or extent of adjunctive therapy used, ongoing disease process, and type of treatment desired (e.g., active vs. conventional treatment).

[0045] The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathological or medical condition from occurring (e.g., prophylaxis), (ii) inhibiting or arresting the occurrence of a disease, pathological or medical condition, (iii) alleviating a disease, pathological or medical condition, and / or (iv) reducing symptoms associated with a disease, pathological or medical condition. Thus, the terms "treat", "treatment" and "treating" can extend to prophylaxis and can include preventing, preventing, prophylactic, reducing, halting or reversing the progression or severity of the condition or symptom being treated. Thus, the term "treatment" can include medical, therapeutic and / or prophylactic administration, as appropriate.

[0046] As used herein, a "subject" or "patient" refers to an individual (individual) who is suffering from or at risk for a disease or other malignancy. A patient may be human or non-human, and may include animal strains or species used as "model systems" for research purposes, such as the mouse model described herein. Similarly, a patient may include either an adult or a juvenile (e.g., a child). Furthermore, a patient may refer to any organism, preferably a mammal (e.g., human or non-human), that can benefit from administration of the compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the mammalian class, such as humans, non-human primates such as chimpanzees, other apes and monkeys, farm animals such as cows, horses, sheep, goats, pigs, domestic animals such as rabbits, dogs, cats, and laboratory animals including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment of the methods provided herein, the mammal is a human.

[0047] As used herein, the terms "providing," "administering," and "introducing" are used interchangeably to mean placing a composition of the present disclosure in a subject by a method or route that at least partially localizes the composition to a desired site. The composition can be administered by any suitable route that results in delivery to the desired site in the subject.

[0048] The compositions described herein may be administered with additional compositions or used in combination with other therapeutic agents to prolong the stability and activity of the compositions.

[0049] The terms "inhibit," "inhibiting," and "inhibition" mean to slow, stop, or reverse the growth or progression of a disease, infection, condition, or population of cells. Inhibition can be, for example, by greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99% compared to the growth or progression that occurs in the absence of treatment or contact.

[0050] The term "substantially" as used herein is a broad term and is used in its ordinary sense. This includes, but is not limited to, the majority, but not necessarily all of what is specified. For example, the term may refer to a value that may not be 100% complete. The complete value may be less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, or about 20%.

[0051] The term "immunotherapy" refers to treating disease by activating or suppressing the immune system, for example with "immunotherapeutic agents." Immunotherapies designed to induce or amplify an immune response are classified as activating immunotherapies, while those designed to reduce or suppress an immune response are classified as suppressing immunotherapies. Immunotherapy is treating disease by activating or suppressing the immune system. Immunotherapies designed to induce or amplify an immune response are classified as activating immunotherapies, while those designed to reduce or suppress an immune response are classified as suppressing immunotherapies. Cancer immunotherapy aims to stimulate the immune system to destroy tumors.

[0052] The term "isotype" refers to a control that is a primary antibody that does not have specificity for the target, but that matches the class and type of the primary antibody used in this application. Isotype controls are used as negative controls to distinguish specific antibody signals from non-specific background signals.

[0053] Embodiments of the present disclosure The present disclosure provides a composition comprising: (a) procaspase-3 activator; (b) at least one second active agent, wherein the second active agent is a checkpoint inhibitor, a cancer vaccine, a metabolic regulator, a macrophage inhibitor, or an immune stimulatory or modulatory agent; and (c) optionally, a pharma- ceutically acceptable diluent, excipient, or carrier.

[0054] In various embodiments, the pro-caspase-3 activator is PAC-1. [ka]

[0055] In various additional embodiments, the pro-caspase-3 activator is a compound disclosed in U.S. Pat. Nos. 8,592,584; 8,778,945; 8,916,705; or 9,249,116, the formulas and compounds of which are incorporated herein by reference.

[0056] In additional embodiments, the second active agent has the effect of inducing apoptosis in cancer cells, and PAC-1 enhances the effect of the second active agent by a greater than additive effect, and PAC-1 primes the vulnerability of the cancer cells to the second active agent.

[0057] In various other embodiments, the composition (e.g., a pro-caspase-3 activator) inhibits mismatch repair (MMR) proteins. In additional embodiments, the composition is a mediator of caspase-3 cleavage of MutL homolog 1 (MLH1) protein. In further embodiments, the composition induces DNA microsatellite instability (MSI). In still other embodiments, the composition selectively targets cancer cells.

[0058] In some further embodiments, the MMR protein comprises MutL homolog 1 (MLH1) protein, and degradation of the MMR protein (e.g., MLH1 protein) mediated by caspase-3 activation via a procaspase-3 activator results in loss of the MMR protein (i.e., dMMR), which can further induce DNA microsatellite instability (MSI) and neoantigen expression, thereby enhancing the efficacy of immunotherapy, and the procaspase-3 activator increases tumor-infiltrating lymphocytes in cancer.

[0059] In other additional embodiments, the at least one second active agent is at least one checkpoint inhibitor that modulates an immune response via programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain containing 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), tumor necrosis factor receptor superfamily member 4 (TNFRSF4 or OX40), tumor necrosis factor receptor superfamily member 9 (TNFRSF9 or 4-1BB), glucocorticoid-inducible TNFR-related protein (GITR), inducible T-cell costimulator (ICOS), or a combination thereof.

[0060] In various additional embodiments, the second active agent is indoleamine-pyrrole 2,3-dioxygenase (IDO), adenosine A 2A receptor (A2AR), transforming growth factor beta (TGF-β), C-X-C chemokine receptor type 4 (CXCR-4), C-C chemokine receptor type 4 (CCR4), tumor necrosis factor receptor (CD27), interleukin 2 receptor subunit beta (CD122), death receptor 5 (DR5), inhibitor of apoptosis proteins (IAPs), glutaminase, colony stimulating factor 1 receptor (CSF1R), toll-like receptors (TLRs), dendritic cells (DCs), or combinations thereof.

[0061] In yet another embodiment, the second active agent is selected from the group consisting of ADXS11-001, ADXS31-142, AMP-224, AMP-514, atezolimumab, atezolizumab, avelumab, bevacizumab, cemiplimab, BLZ945, BMS-936559, BMS986016, BMS986156, BMS986205, CB839, CIMAvax, CMP001, CP 870893, CPI-444, CRS207, CV301, DC vaccine, DNX2401, DS-8273a, durvalumab, epacadostat, FAZ053, FPA008, GDC0919, GSK3174998, GVAX, GWN323, IMCgp100, IMP321, impr ime)PGG, indoximid, ipilimumab, JTX-2011, LAG525, LCL161, LK-301, LY2157299, LY2510924, LY3022855, MBG453, MEDI0562, MEDI0680, MEDI6469, MEDI9447, MGN1703, mogamulizumab, MOXR0916, neoantigen vaccine, NEO‐PV‐01, NIS793, nivolumab, NKTR‐214, PBF509, PDR001, pembrolizumab, peptide vaccine, pexidartinib (PLX3397), PF‐04518600, PF‐3512676, REGN2810, REGN3767, RO7009789, SD101, Talimogene talimogene laherparepvec, TPIV200 / huFR-1, tremelimumab, TroVax, TSR022, ulocuplumab, urelumab, utomilumab, varlilumab, viagenpumatucel-L (HS-110), or combinations thereof.

[0062] In other embodiments, the checkpoint inhibitor is anti-PD-1, anti-CTLA-4, or a combination thereof; the anti-PD-1 is nivolumab or pembrolizumab and the anti-CTLA-4 is ipilimumab or tremelimumab, or a combination thereof.

[0063] In some embodiments, the disclosed compositions include a pharma- ceutically acceptable diluent, excipient, carrier, or combination thereof. In other embodiments of the disclosed compositions, a) the carrier includes water, a buffer, a sugar, a cellulose, a cyclodextrin, dimethylsulfoxide, polyethylene glycol, a tocopherol, a liposome, a micelle, or a combination thereof, or b) the excipient includes a binder, a lubricant, an adsorbent, a vehicle, a disintegrant, a preservative, or a combination thereof.

[0064] In various other embodiments, the concentration of PAC-1 is from about 0.1 μM to about 50 μM. In other embodiments, the concentration of PAC-1 is from about 0.1 μM to about 1 μM, from about 1 μM to about 10 μM, from about 2 μM to about 15 μM, from about 3 μM to about 20 μM, from about 4 μM to about 25 μM, from about 5 μM to about 30 μM, from about 10 μM to about 40 μM, from about 15 μM to about 50 μM, or from about 0.01 μM to about 100 μM.

[0065] In additional embodiments, the concentration of the second active agent is from about 1 nM to about 100 μM. In other embodiments, the concentration of the second active agent is from about 1 nM to about 100 nM, from about 10 nM to about 1 μM, from about 100 nM to about 1 μM, from about 1 μM to about 5 μM, from about 1 μM to about 10 μM, from about 5 μM to about 15 μM, from about 10 μM to about 20 μM, from about 10 μM to about 30 μM, from about 15 μM to about 40 μM, from about 20 μM to about 50 μM, or from about 50 μM to about 100 μM.

[0066] In further embodiments, the compositions disclosed herein selectively target cancer cells, wherein the cancer cells are bladder cancer, breast cancer, colon cancer, endometrial cancer, glioblastoma, leukemia, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple myeloma, ovarian cancer, osteosarcoma, pancreatic cancer, prostate cancer, renal cancer, or thyroid cancer cells, wherein the breast cancer is optionally triple-negative breast cancer, the lung cancer is optionally non-small cell lung cancer, and the renal cancer is optionally metastatic renal cell carcinoma.

[0067] The present disclosure also provides a method of inhibiting the growth or proliferation of cancer cells, comprising contacting the cancer cells with an effective amount of the disclosed composition, thereby inhibiting the growth or proliferation of the cancer cells. In other embodiments, the growth or proliferation of cancer cells is inhibited by inhibiting mismatch repair (MMR) proteins. In further embodiments, the growth or proliferation of cancer cells is inhibited by caspase 3 activation-mediated degradation of MutL homolog 1 (MLH1) protein. In yet other embodiments, DNA microsatellite instability (MSI) is induced.

[0068] The present disclosure further provides a method of inducing apoptosis in cancer cells comprising contacting the cancer cells with an effective amount of a composition disclosed herein, thereby inducing apoptosis by suppressing mismatch repair (MMR) proteins in the cancer cells. In other embodiments, degradation of MutL homolog 1 (MLH1) protein is mediated by procaspase-3 activator-mediated caspase-3 activation, thereby inducing apoptosis in the cancer cells.

[0069] Furthermore, the present disclosure provides a method of treating cancer comprising simultaneously or sequentially administering to a subject in need thereof a therapeutically effective amount of a procaspase-3 activator and an effective amount of a second active agent, where the second active agent is an immunotherapeutic agent, and the effect of the second active agent is enhanced by administration of the procaspase-3 activator.

[0070] In yet other additional embodiments, the procaspase-3 activator is PAC-1 or has a molecular weight of about 200 to about 800, about 250 to about 550, about 300 to about 600, about 350 to about 550, or about 350 to about 450, and the procaspase-3 activator directly activates procaspase-3 to caspase-3.

[0071] In further embodiments, the second active agent comprises a checkpoint inhibitor, a cancer vaccine, a metabolic regulator, a macrophage inhibitor, an immune stimulator, or a regulator; or a combination thereof.

[0072] In various embodiments, degradation of MutL homolog 1 (MLH1) protein by caspase 3 induces DNA microsatellite instability (MSI) and neoantigen expression, thereby enhancing the efficacy of cancer treatment. In other embodiments, mismatch repair (MMR) proteins are inhibited by procaspase-3 activators. In further embodiments, procaspase-3 activators, such as PAC-1, increase tumor infiltrating lymphocytes (TILs) in cancer (or cancer cells).

[0073] In various other embodiments, the immunotherapeutic agent is a checkpoint inhibitor, which modulates the immune response via programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain containing 3 (TIM-3), lymphocyte activation gene 3 (LAG-3), tumor necrosis factor receptor superfamily member 4 (TNFRSF4 or OX40), tumor necrosis factor receptor superfamily member 9 (TNFRSF9 or 4-1BB), glucocorticoid-inducible TNFR-related protein (GITR), inducible T-cell costimulator (ICOS), or a combination thereof.

[0074] In still other various embodiments, the second active agent is atezolimumab, avelumab, bevacizumab, BMS986016, BMS986156, CP870893, durvalumab, FAZ053, GSK3174998, GWN323, IMP321, ipilimumab, JTX-2011, LAG525, MBG453, MEDI0562, MEDI0680, MEDI6469 , MOXR0916, nivolumab, PDR001, pembrolizumab, PF-04518600, REGN2810, REGN3767, RO7009789, tremelimumab, TSR022, urelumab, utomilumab, or a combination thereof.

[0075] In various additional embodiments, the concentration of PAC-1 is from about 0.1 μM to about 50 μM and the concentration of the second active agent is from about 1 nM to about 100 μM. In further embodiments, the concentration of PAC-1 is from about 1 μM to about 10 μM. In other embodiments, the concentration of the second active agent is from about 1 nM to about 1 μM.

[0076] In various embodiments, as one of ordinary skill in the art would readily recognize, the concentrations of PAC-1 and the second active agent described throughout this disclosure can also be described and interpreted as ratios of PAC-1 to the second active agent, e.g., by converting the concentrations described herein to the corresponding molar ratios of PAC-1 and the second active agent.

[0077] In various other embodiments, the cancer is bladder cancer, breast cancer, colon cancer, endometrial cancer, glioblastoma, leukemia, liver cancer, lung cancer, lymphoma, melanoma, meningioma, multiple myeloma, ovarian cancer, osteosarcoma, pancreatic cancer, prostate cancer, renal cancer, or thyroid cancer, where the breast cancer is optionally triple-negative breast cancer, the lung cancer is optionally non-small cell lung cancer, and the renal cancer is optionally metastatic renal cell carcinoma.

[0078] In some various embodiments, the compound PAC-1 and the second active agent are administered to the subject simultaneously. In still other embodiments, the compound PAC-1 and the second active agent are administered to the subject sequentially. In additional embodiments, the compound PAC-1 is administered to the subject before the second active agent. In further embodiments, the compound PAC-1 is administered to the subject after the second active agent.

[0079] Additionally, the present disclosure provides a composition for preparing a medicament for use in treating cancer, the composition comprising: (a) Compound PAC-1; (b) at least one second active agent, wherein the second active agent is a checkpoint inhibitor, a cancer vaccine, a metabolic regulator, a macrophage inhibitor, or an immune stimulatory or modulatory agent; and (c) optionally a pharma- ceutically acceptable diluent, excipient, carrier, or combination thereof; where cancer is treated therewith.

[0080] In additional embodiments, the concentration of PAC-1 is about 0.1 μM to about 500 μM and the concentration of the second active agent is about 1 nM to about 1000 μM. In yet other additional embodiments, the second active agent is atezolimumab, avelumab, bevacizumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, tremelimumab, urelumab, utomilumab, or combinations thereof. In yet other embodiments, the cancer is lymphoma, melanoma, leukemia, multiple myeloma, glioblastoma, pancreatic cancer, liver cancer, non-small cell lung cancer, breast cancer, ovarian cancer, colon cancer, osteosarcoma, or meningioma.

[0081] In various embodiments, the compounds PAC-1 and the second active agent are administered to a subject once daily (qd), twice daily (bid), three times daily (tid), or four times daily (qid), with a total daily dose of PAC-1 ranging from about 1 mg / kg to about 150 mg / kg, from about 10 mg / kg to about 125 mg / kg, or from about 5 mg / kg to about 250 mg / kg. In other embodiments, each dose of PAC-1 (or the second active agent) is about 10 mg, about 25 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 175 mg, about 250 mg, about 375 mg, about 450 mg, about 500 mg, about 625 mg, about 750 mg, about 1000 mg, or from about 10 mg to about 2000 mg. In a further embodiment, each dose of PAC-1 (or a second active agent) is about 50 mg / m 2 ~about 250mg / m 2 , or about 10 mg / m 2 ~about 500mg / m 2 In some other embodiments, the total daily dose of the second active agent is from about 1 mg / kg to about 100 mg / kg, or from about 5 mg / kg to about 150 mg / kg.

[0082] In some embodiments, a composition administered to a patient in need of treatment for cancer comprises PAC-1 and alpha-PD-1, wherein the dosage of PAC-1 is about 100 mg / kg to about 150 mg / kg (or about 125 mg / kg) and the dosage of alpha-PD-1 is about 150 micrograms to about 250 micrograms (or about 200 micrograms); in various embodiments, survival of the patient is extended as compared to a control.

[0083] In the present disclosure, ranges, limits, and deviations are provided for variables such as volume, mass, percentages, ratios, etc. One of ordinary skill in the art will appreciate that ranges such as "number 1" to "number 2" refer to a continuous range of numbers, including whole numbers and fractions. For example, 1 to 10 refers to 1, 2, 3, 4, 5, ... 9, 10. This also refers to 1.0, 1.1, 1.2, 1.3, ... 9.8, 9.9, 10.0, and also 1.01, 1.02, 1.03, etc. Also, when a disclosed variable is a number less than "number 10," it refers to a continuous range including whole numbers and fractions less than number 10, as described above. Similarly, when a disclosed variable is a number greater than "number 10," it refers to a continuous range including whole numbers and fractions greater than number 10. These ranges may be modified by the term "about," the meaning of which is described above.

[0084] Results and Discussion Immunotherapy with checkpoint inhibitors has become an effective treatment for certain cancers (e.g., melanoma, NSCLC, urothelium) and can induce durable responses in some cancer patients. Currently, dozens of clinical trials combining immune checkpoint inhibitors with small molecule drugs are ongoing. As described here, a mechanistic hypothesis is explored that direct activation of procaspase-3 promotes cleavage of the key protein MLH1 in DNA mismatch repair, thereby dramatically enhancing the efficacy of immune checkpoint inhibitors by increasing potential neoantigens targeted by T cells.

[0085] Background of the initiativeImmunotherapy using immune checkpoint inhibitors has shown great promise, but many clinical trials have been plagued by low response rates and failures. A recent important development has been the clinical approval of DNA microsatellite instability (MSI) as a biomarker for clinical efficacy of PD-1 blockade (with pembrolizumab) regardless of tumor origin. This approval is based on remarkable preclinical and clinical data showing that mismatch repair deficiency (dMMR) predicts response to PD-1 blockade in solid tumors. This is because tumors with dMMR / MSI are known to harbor 100s-1000s of somatic mutations (10-fold higher than MMR-positive cancers, Figure 1A), possibly leading to elevated levels of neoantigens and enhanced T-cell infiltration. However, dMMR / MSI only exists in a small proportion of all cancers, probably less than 10%. Sporadic MSI is caused by epigenetic silencing of the MLH1 promoter, and MLH1 silencing is commonly used as a marker of MMR deficiency. The correlation between MLH1 silencing and the number of somatic mutations has been demonstrated in many studies and is powerfully illustrated in Figure 1.

[0086] Selective induction of MSI in cancer cells significantly increases patient responses to immune checkpoint inhibitors (e.g., those targeting PD-1 or CTLA-4). Multiple large-scale proteomic studies have revealed that MLH1 is the top substrate of caspase-3, with 0% protein remaining after 6 hours (compared to 70% for MEK1 / 2 at the same time point). Furthermore, MLH1 is a substrate exclusively for active caspase-3, and proteolysis by other active caspases (caspases-1, 2, 6, 7, and 8) was not observed. These data suggest that selective activation of PC-3 in tumors can quantitatively cleave MLH1 and result in dMMR / MSI, which in turn significantly enhances the efficacy of immune checkpoint inhibitors, as shown diagrammatically in Figure 2.

[0087] Mechanism of action.In this disclosure, we show that selective induction of MLH1 cleavage in cancers using PAC-1 can render them more susceptible to treatment with immune checkpoint inhibitors (Figure 2). Furthermore, treatment with PAC-1 induces a stress response, thereby altering the tumor microenvironment and increasing the degree of immune inflammation. These results bring the power of immunotherapy, i.e., dramatic and durable responses, to even more cancer patients. In summary, cleavage and inactivation of MLH1 by caspase 3 stimulates the innate immune system, leading to both point mutations and indels (neoantigens derived from new open reading frames), which are immunogenic. Thus, this chemically induced degradation of MLH1 promotes anticancer drug-induced immune responses.

[0088] result The MSS / MSI status of colon cancer cell lines has been reported (Ahmed, D., et al., Oncogenesis 2013, 2, e71), allowing the selection of an MSS colon cancer cell line, HT-29. To date, studies focusing on MLH1 cleavage have utilized strategies that induce widespread and high levels of apoptotic cell death (e.g., with staurosporine). HT-29 cells were treated with sublethal doses of PAC-1. As shown in Figure 3, treatment of HT-29 cells with PAC-1 induced PC-3 activation and MLH1 cleavage, but little PARP-1 cleavage at these times and concentrations. This result further validates that MLH1 is an excellent substrate for caspase-3. Importantly, the concentration of PAC-1 used in these experiments is sustainable for several weeks in human cancer patients (450 mg at C min = 3.2 μM, C max =7.8μM).

[0089] In addition, PAC-1 alone inhibited TIL (CD3 + Experiments have also been performed in syngeneic GL261 and B16F10 mouse models showing that IL-16 expression increased the number of IL-16 cells (Figure 4).

[0090] The bulk of data on PAC-1 suggests that it does not induce cancer in vivo. This is confirmed by a) treatment of pet dogs with cancer, some of which have been treated with PAC-1 for >6 months and have remained free of secondary malignancies >12 months after the end of treatment, b) extensive IND-enabled toxicity studies in rats and laboratory dogs, including an 84-day continuous treatment dog study, and c) data from human clinical trials. Several patients have taken PAC-1 beyond the 2-month clinical trial period, two of which have taken it for >10 months (one taking 450 mg daily) without adverse effects. It should be noted that anticancer drugs can induce secondary cancers, e.g., nearly one-third of patients treated with vemurafenib alone have developed secondary malignancies. However, this has not been observed so far with PAC-1. Just as PAC-1 selectively induces cleavage of PC-3 in cancer cells, the resulting caspase-3 activity should selectively induce cleavage of MLH1 in cancer cells. Notably, Turcot syndrome, a cancer predisposition syndrome termed constitutional mismatch repair deficiency (CMMRD), is correlated with biallelic germline mutations in MMR genes, resulting in early onset of GBM. In Turcot syndrome and other CMMRD syndromes (e.g., Lynch syndrome), the importance of maintaining MMR protein function has been noted, suggesting that non-targeted, pan-biological induction of MLH1 cleavage or loss is not a viable therapeutic strategy. However, the PAC-1 strategy exploits the well-known overexpression of PC-3 in cancer cells (including GBM) to induce targeted cleavage of MLH1 in tumors, leaving MMR proteins in normal cells unaffected.

[0091] Pharmaceutical Formulations The compounds and compositions described herein can be used to prepare pharmaceutical compositions for treatment, for example, by combining the compounds with a pharma- ceutically acceptable diluent, excipient, or carrier. The compounds may be added to the carrier in the form of a salt or solvate. For example, if the compounds are sufficiently basic or acidic to form stable non-toxic acid or base salts, it may be appropriate to administer the compounds as salts. Examples of pharma-ceutically acceptable salts are organic acid addition salts formed with acids that form physiologically acceptable anions, such as tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α-ketoglutarate, and β-glycerophosphate. Suitable inorganic salts can also be formed, such as hydrochloride, halide, sulfate, nitrate, bicarbonate, carbonate, and the like.

[0092] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound, such as an amine, with a suitable acid to provide a physiologically acceptable ionic compound, and alkali metal (sodium, potassium, lithium, etc.) or alkaline earth metal (calcium, etc.) salts of carboxylic acids can also be obtained in a similar manner.

[0093] The compounds of the formulae described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms that can be specifically adapted for a selected route of administration, for example, oral or parenteral administration by intravenous, intramuscular, topical, or subcutaneous routes.

[0094] The compounds described herein can be administered systemically in combination with a pharma- ceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, the compounds can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the patient's diet. The compounds can also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 0.1% of the active compound. The percentage of the compositions and preparations may vary and conveniently be about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10% by weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions can be such that an effective dosage level will be obtained.

[0095] Tablets, troches, pills, capsules, etc. may contain one or more of the following: binders, such as tragacanth gum, acacia, corn starch, gelatin, excipients, such as dicalcium phosphate, disintegrants, such as corn starch, potato starch, alginic acid, and lubricants, such as magnesium stearate. Sweeteners, such as sucrose, fructose, lactose, and aspartame, and flavorings, such as peppermint, wintergreen oil, and cherry flavoring, may also be added. When the unit dosage form is a capsule, in addition to the above types of materials, it may contain liquid carriers, such as vegetable oils and polyethylene glycols. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar. Syrups and elixirs may contain the active compound, sucrose or fructose as a sweetener, methylparaben or propylparaben as a preservative, a dye, and a flavoring, such as cherry or orange flavoring. Materials used in preparing unit dosage forms should be pharma- ceutically acceptable and substantially non-toxic in the amounts employed.Furthermore, the active compounds can be incorporated into sustained-release preparations and devices.

[0096] The active compound can be administered intravenously or intraperitoneally by infusion or injection. A solution of the active compound or its salts can be prepared in water, optionally mixed with a non-toxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycol, triacetin, or mixtures thereof, or in pharma-ceutically acceptable oils. Under normal storage and use conditions, the preparations can contain a preservative to prevent the growth of microorganisms.

[0097] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient, optionally encapsulated in liposomes, suitable for the extemporaneous preparation of a sterile injectable or infusible solution or dispersion. The final dosage form must be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be, for example, a solvent or liquid dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Prevention of the action of microorganisms can also be achieved by various antibacterial and / or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers, sodium chloride, and the like. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, such as, for example, aluminum monostearate and / or gelatin.

[0098] Sterile injections can be prepared by placing the required amount of active compound in a suitable solvent, adding various ingredients as described above as necessary, and then optionally sterilizing by filtration. In the case of aseptic powder for preparing sterile injection solutions, the preparation method includes vacuum drying and freeze-drying technology, which gives a powder of active ingredient plus any additional desired ingredient present in the solution.

[0099] For topical administration, the compounds can be applied in pure form, e.g., when they are liquids, but it will generally be desirable to administer the active agent to the skin as a composition or formulation, e.g., in combination with a dermatologically acceptable carrier, which can be a solid, liquid, gel, etc.

[0100] Effective solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, etc. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol / glycol mixtures in which the compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can also be added to optimize properties for specific applications. The resulting liquid compositions can be applied from absorbent pads, impregnated into bandages or other dressings, or sprayed onto the affected area using pump or aerosol sprayers.

[0101] Additionally, thickening agents such as synthetic polymers, fatty acids, salts or esters of fatty acids, fatty alcohols, modified celluloses, modified mineral materials, and the like can be used in conjunction with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for application directly to the skin of the user.

[0102] Examples of dermatological compositions for delivering active agents to the skin are known in the art, see, for example, U.S. Patent Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combination with the compounds described herein, which can optionally be substituted for components of such compositions or which can be added to the compositions.

[0103] Useful dosages of the compositions described herein can be determined by comparing in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art, see, for example, U.S. Patent No. 4,938,949 (Borch et al.). The amount of compound or its active salt or derivative required for therapeutic use will vary depending on the particular compound or salt selected, as well as the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be left to the discretion of the attending physician or clinician.

[0104] In general, however, a suitable dosage will be in the range of about 0.5 to about 100 mg / kg of body weight per day, for example, about 10 to about 75 mg / kg, for example, 3 to about 50 mg per kg of recipient body weight per day, preferably in the range of 6 to 90 mg / kg / day, and most preferably in the range of 15 to 60 mg / kg / day.

[0105] The compounds are conveniently formulated in unit dosage form, for example containing 5-1000 mg, conveniently 10-750 mg, and most conveniently 50-500 mg of active ingredient per unit dosage form, hi one embodiment, the present disclosure provides a composition comprising a compound of the present disclosure formulated in such a unit dosage form.

[0106] The compound may be administered in a dosage form of, for example, 5 to 1000 mg / m 2 , advantageously 10 to 750 mg / m 2 , most conveniently 50-500 mg / 2 It is convenient to administer the compound in a unit dosage containing m of the active ingredient. The desired dosage may conveniently be administered as a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, for example, into a number of discrete loosely spaced administrations.

[0107] The desired dose may conveniently be administered as a single dose or as divided doses administered at appropriate intervals, for example as multiple inhalations from an inhaler or as multiple loosely spaced doses applied to the eye, for example as multiple eye drops.

[0108] The compounds described herein are effective anti-tumor agents, with increased efficacy and / or reduced toxicity compared to immunotherapy alone or other cancer treatments.

[0109] The present disclosure provides a method of treating cancer in a mammal, comprising administering to a mammal having cancer an effective amount of a compound or composition described herein. Mammals include primates, humans, rodents, dogs, cats, cows, sheep, horses, pigs, goats, cattle, vertebrates, etc. Cancer refers to various types of malignant neoplasms, such as colon cancer, breast cancer, ovarian cancer, osteosarcoma, melanoma, leukemia, etc., and is generally characterized by unwanted cell proliferation, such as unregulated growth, lack of differentiation, invasion of local tissues, metastasis, etc.

[0110] The ability of the compounds of the present disclosure to treat cancer can be determined using assays well known in the art. For example, the design of treatment protocols, evaluation of toxicity, data analysis, quantification of tumor cell killing, and biological significance of using transplantable tumor screens are known. In addition, the ability of compounds to treat cancer can be determined using tests such as those described below.

[0111] The following examples are intended to illustrate the above disclosure and should not be construed to narrow its scope. Those skilled in the art will readily recognize that the examples suggest many other ways in which the present disclosure can be practiced. It should also be understood that numerous variations and modifications can be made while remaining within the scope of the present disclosure. EXAMPLES

[0112] Example 1 Experimental procedure for the 4T1 efficacy model Reagents. The following antibodies were purchased from Bio X Cell: anti-mouse CTLA-4 monoclonal antibody (9H10), anti-mouse PD-1 monoclonal antibody (RMP1-14), rat IgG2a isotype control (2A3), and polyclonal Syrian hamster IgG.

[0113] Cell lines. The 4T1 murine breast cancer cell line was obtained from ATCC and cultured in complete RPMI 1640 containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C for 24 h at 4 °C for 1 h at 25 °C. 2 The cells were cultured in an incubator at 37°C.

[0114] 4T1 orthotopic tumor model. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana-Champaign. 6-8 week old female BALB / c mice were purchased from Charles River and acclimated for 7 days. Mice were lightly sedated with i.p. xylazine (16 mg / kg) and ketamine (100 mg / kg). After sedation, 100 μL (10 million cells / mL) of 4T1 cells in chilled HBSS were injected into the right second mammary gland of the mice. Orthotopic growth tumors were established 1 week later. Twelve days after inoculation of 4T1 cells, tumor-bearing mice were randomly assigned to one of four treatment groups (n=6): vehicle + isotype, vehicle + anti-PD-1 / anti-CTLA-4, PAC-1 + isotype, and PAC-1 + anti-PD-1 / anti-CTLA-4. PAC-1 was formulated in HPβCD (10 mg / mL in 200 mg / mL HPβCD, pH 5.5).

[0115] All antibodies were diluted to appropriate concentrations in sterile PBS (pH 7.0). Vehicle or 100 mg / kg PAC-1 was administered intraperitoneally for 5 consecutive days for 3 weeks. Isotype or 10 mg / kg anti-PD-1 + 10 mg / kg anti-CTLA-4 antibodies were administered intraperitoneally 4 hours after PAC-1 on days 13, 16, 20, and 23 after tumor implantation. Tumors were measured every 2 or 3 days using calipers, and tumor volumes were (0.5 × l × w 2) formula. Thirty days after inoculation of 4T1 cells, the mice were sacrificed. The tumors were then excised and their masses were measured. All statistical analyses were performed using unpaired two-tailed Student's test, with a p value of <0.05 considered statistically significant (see Figure 6).

[0116] Example 2 Tumor research. In GL261 tumors treated with PAC-1, immune cell infiltration is increased. In addition to the data showing that PAC-1 induces caspase-3-mediated cleavage of MLH1 in cancer cells, there are many other lines of evidence supporting the synergistic combination of PAC-1 with immunotherapy strategies (including checkpoint inhibitors and neoantigen vaccines): 1) the transcriptional profile of cancer cells treated with PAC-1 showed an increase in key genes such as TNFα, agonists of the innate immune system IL-1β, and IL-8, without an increase in markers associated with resistance to anti-PD-1 therapy (IPRES: e.g. CCL2, CCL7, CCL8, CCL13, etc.); 2) a study from another group showed that PAC-1 can promote extrinsic cell death in culture when tested in combination with the immune cytokine TRAIL. 3) PAC-1 is effective in in vivo settings where the immune system is intact, e.g., in syngeneic mouse (EL4, K7M2, GL261) (Figure 7) and rat (9L) models, and in canine cancer patients. 4) The Gandhi group at MD Anderson (Blood 2015,125,1126) has shown that PAC-1 and derivatives have minimal toxicity to PMBC. 5) PAC-1 has not been observed to induce myelosuppression (in mice, rats, dogs, or humans), even when used at very high doses in IND-enabling rat and dog studies. 6) PAC-1 selectively inflicts DNA damage in cancer cells, further validating findings that activation of caspase-3 can result in significant genomic instability. As a first step in exploring the potential of PAC-1 to stimulate immune responses, we have demonstrated that single-agent PAC-1 inhibits TILs (CD3 + Experiments were performed using a syngeneic GL261 mouse model that showed that IL-1 increased the number of IL-1 cells (Figure 4).

[0117] Relevance of immune checkpoints in canine gliomas. Recent studies have confirmed the expression of PD-L1 in various canine tumor types. However, there are no published studies on PD-L1 in canine gliomas. We tested the cross-reactivity of a commercially available mouse monoclonal anti-human PD-L1 antibody (Abcam, clone ABM4E54) in archived canine gliomas (Figure 5), revealing that 75% of tumors expressed PD-L1. This frequency is comparable to human GBM, where PD-L1 was identified in 88% and 72% of primary and recurrent samples, respectively. In addition to PD-L1, the antibody was confirmed to cross-react with the nuclear target MLH1 in human and canine glioma cell lines, allowing quantitative assessment of MLH1 cleavage following PAC-1 treatment.

[0118] Example 3 Immunotherapy in a PAC-1 and syngeneic colon cancer model (CT-26 cells). FIG. 8 shows the development of the CT-26 WT subcutaneous disease model in BALB / c mice. On day 0, BALB / c mice were inoculated with 1×10 6 CT-26_WT cells were injected subcutaneously. At various daily intervals, selected mice were injected (ip) with empty vehicle, PAC-1 (100 mg / kg), anti-PD-1 antibody (10 mg / kg; 2 doses), anti-PD-1 antibody (10 mg / kg; 4 doses), or a combination of PAC-1 (100 mg / kg) and anti-PD-1 antibody (10 mg / kg), as shown in Table 1.

[0119] [Table 1]

[0120] Figure 9 shows the large variation in the single agent PAC-1 in controlling the growth of sc CT-26_WT in BALB / c mice. Furthermore, the combination of PAC-1 and anti-PD-1 mAb significantly reduced the growth of CT-26_WT in BALB / c mice compared to vehicle + anti-IgG control. Figure 10 shows that the combination of PAC-1 and anti-PD-1 monoclonal antibody (mAb) reduced the growth of CT-26_WT cells in BALB / c mice compared to control mice (injected with empty vehicle + anti-IgG antibody). The relative contribution of PAC-1 in this combination therapy becomes more prominent when the dose of anti-PD-1 mAb is reduced from four to two doses. These experiments also show that days 14 and 21 are suitable times to perform TIL analysis.

[0121] FIG. 11 shows the development of the CT-26_TdTomato subcutaneous tumor model in BALB / c mice. CT-26_TdTomato cells 1x10 6 The mice were inoculated subcutaneously into the hind flank. Ten days after inoculation (tumor volume approximately 150 mm 3 ), and mice were subjected to the following treatments: Group 1 : 3 mice - vehicle + rat IgG isotype mAb (10 mg / kg) Group 2 : 3 mice - PAC-1 (125 mg / kg) + rat IgG isotype mAb (10 mg / kg) Group 3 : 3 mice - vehicle + anti-PD1 mAb (10 mg / kg) Group 4 : 3 mice - PAC-1 (125 mg / kg) + anti-PD1 mAb (10 mg / kg)

[0122] BALB / C mice in group 4 (PAC-1 + anti-PD1 mAb) were able to reject CT-26-TdT after 5 days of continuous PAC-1 and 2 injections of anti-PD1. At day 47, the mice still appear tumor-free. The anti-PD1 group was able to eliminate tumors after 3 injections of anti-PD1 mAb. At day 47, the mice still appear tumor-free. At day 47, the mice still appear tumor-free. 6 CT-26_TdTomato cells were rechallenged. After rechallenge, no significant increase in tumor volume was observed.

[0123] Figure 12A shows a cytokine array demonstrating that PAC-1 is immunogenic and results in increased cytokines that promote macrophage differentiation, B-cell and T-cell proliferation. As shown in Figure 12B, approximately 100ul of blood was collected by retro-orbital bleeding into a heparinized vial. White blood cells were centrifuged at 8000g for 10 minutes and plasma / supt was transferred to a new tube. Cytokine arrays were performed in four groups using pooled samples from 2-3 mice. Non-tumor bearing + vehicle Non-tumor bearing + PAC-1 (5 doses) Tumor-bearing + vehicle Tumor-bearing + PAC-1 (5 doses)

[0124] Signals were quantified using ImageJ. Data were normalized by calculating the ratio of PAC-1 / vehicle mean pixel density as shown in the plots.

[0125] Figure 13 shows that neutrophils and macrophages appear to be increased in the lung tumor microenvironment following PAC-1 treatment at day 14 after tumor challenge. At day 26, the population of macrophages and dendritic cells in the tumor microenvironment is decreased.

[0126] Figure 14 shows CD4+ expression in the lung tumor microenvironment 26 days after combinatorial treatment with PAC-1 and anti-PD-1. + T hThe increase in cells in the lungs shows that FoxP3 + T regs The rate was lowest in the combination therapy group.

[0127] Figure 15 shows that dendritic cells and CD45 - This suggests that increased PD-L1 expression on tumor cells may contribute to T cell exhaustion.

[0128] Example 4 Immunotherapy with PAC-1 in a syngeneic colon cancer model (MC-38 cells). Figure 16 shows the development of an MC-38 metastasis model in C57BL / 6 mice and treatment with a combination of PAC-1 and anti-PD-1 antibodies. MC-38 cells were inoculated at 1x10 6 Cells / mouse were injected via the tail vein. PAC-1 was injected at 100 mg / kg (ip) and anti-PD-1 was injected at 10 mg / kg over a period of 23 days following MC38 injection. Mice injected with the PAC-1 / anti-PD-1 combination gained weight starting approximately at day 24 and continuing through day 32, demonstrating significant weight regain.

[0129] Figure 17 shows the survival curves of mice injected with empty vehicle control, PAC-1, anti-PD-1 antibody, or a combination of PAC-1 and anti-PD-1 antibody after challenge with MC-38 cells. These results show that mice injected with both PAC-1 and anti-PD-1 antibody have a stable survival probability after about 32 days.

[0130] Example 5. Pharmaceutical dosage forms. The following formulations illustrate representative pharmaceutical dosage forms that can be used for the therapeutic or prophylactic administration of a composition of the formulas described herein, a composition specifically disclosed herein, or a pharma- ceutically acceptable salt thereof (hereinafter referred to as "Composition X").

[0131] (i) 1 tablet mg / tablet "Composition X" 100.0 Lactose 77.5 Povidone 15.0 Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesium stearate 3.0 300.0

[0132] (ii) Tablet 2 mg / tablet "Composition X" 20.0 Microcrystalline Cellulose 410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0 500.0

[0133] (iii) Capsules mg / capsule "Composition X" 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5 Pregelled Starch 120.0 Magnesium stearate 3.0 600.0

[0134] (iv) Injection 1 (1 mg / mL) mg / mL "Composition X" (free acid form) 1.0 Sodium phosphate dibasic 12.0 Sodium phosphate monobasic 0.7 Sodium chloride 4.5 1.0N Sodium Hydroxide Solution qs (Adjust pH to 7.0-7.5) Water for injection qs ad 1mL

[0135] (v) Injection 2 (10mg / mL) mg / mL "Composition X" (free acid form) 10.0 Sodium phosphate monobasic 0.3 Sodium phosphate dibasic 1.1 Polyethylene glycol 400 200.0 0.1N sodium hydroxide solution qs (Adjust pH to 7.0-7.5) Water for injection qs ad 1mL

[0136] (vi) Aerosols mg / can "Composition X" 20 Oleic Acid 10 Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000 Dichlorotetrafluoroethane 5,000

[0137] (vii) Topical gel 1 wt.% "Composition X" 5% Carbomer 934 1.25% Triethanolamine qs (Adjust pH to 5-7) Methylparaben 0.2% Purified water qs up to 100g

[0138] (viii) Topical gel 2 wt.% "Composition X" 5% Methylcellulose 2% Methylparaben 0.2% Propylparaben 0.02% Purified water qs up to 100g

[0139] (ix) Topical ointment wt.% "Composition X" 5% Propylene glycol 1% Anhydrous ointment base 40% Polysorbate 80 2% Methylparaben 0.2% Purified water qs up to 100g

[0140] (x) Topical cream 1 wt.% "Composition X" 5% White Beeswax 10% Liquid Paraffin 30% Benzyl alcohol 5% Purified water qs up to 100g

[0141] (xi) Topical cream 2 wt.% "Composition X" 5% Stearic acid 10% Glyceryl monostearate 3% Polyoxyethylene stearyl ether 3% Sorbitol 5% Isopropyl palmitate 2% Methylparaben 0.2% Purified water qs up to 100g

[0142] These formulations can be prepared by conventional procedures well known in the art of pharmacy. It will be understood that the above pharmaceutical compositions can be varied in accordance with well known pharmaceutical techniques to vary the amount and type of active ingredient "Composition X". The aerosol formulation (vi) can be used in combination with a standard metered dose aerosol dispenser. Furthermore, the specific ingredients and ratios are for illustrative purposes. The ingredients may be substituted with suitable equivalents or in different proportions depending on the desired characteristics of the targeted dosage form.

[0143] Although specific embodiments have been described above with reference to disclosed embodiments and examples, such embodiments are merely illustrative and are not intended to limit the scope of the present disclosure. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the disclosure in its broader aspects as defined in the following claims.

[0144] All publications, patents, and patent documents are incorporated herein as if individually incorporated by reference. No limitations inconsistent with the present disclosure should be understood therefrom. The present disclosure has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made while remaining within the spirit and scope of the present disclosure.

Claims

1. (a) PAC-1; 【Chemistry 1】 (b) at least one second active agent, wherein the second active agent is an anti-PD-1 antibody selected from the group consisting of RMP1-14, atezolizumab, avelumab, cemiplimab, durvalumab, and pembrolizumab; and (c) a pharma- ceutically acceptable diluent, excipient, or carrier; 23. A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, wherein the anti-PD-1 antibody is atezolizumab, avelumab, cemiplimab, durvalumab, or pembrolizumab.

3. 3. The pharmaceutical composition of claim 1, wherein the concentration of PAC-1 is 0.1 μM to 50 μM and the concentration of the anti-PD-1 antibody is 1 nM to 100 μM.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, comprising a pharma- ceutically acceptable diluent, excipient, or carrier, wherein a) the carrier comprises water, a buffer, a sugar, a cellulose, a cyclodextrin, dimethylsulfoxide, polyethylene glycol, tocopherol, a liposome, a micelle, or a combination thereof; or b) the excipient comprises a binder, a lubricant, an adsorbent, a vehicle, a disintegrant, a preservative, or a combination thereof.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition selectively targets cancer cells, and the cancer cells are colon cancer or osteosarcoma.

6. A combination of pharmaceutical compositions for treating colon cancer or osteosarcoma, comprising a composition comprising a therapeutically effective amount of PAC-1 and a composition comprising a therapeutically effective amount of an anti-PD-1 antibody, wherein the composition comprising a therapeutically effective amount of PAC-1 and the composition comprising a therapeutically effective amount of an anti-PD-1 antibody are administered simultaneously or sequentially to a subject in need thereof, and the effect of the composition comprising the anti-PD-1 antibody is enhanced by administration of the composition comprising PAC-1.

7. The combination of claim 6, wherein the concentration of PAC-1 in the composition comprising a therapeutically effective amount of PAC-1 is between 0.1 μM and 50 μM, and the concentration of the anti-PD-1 antibody in the composition comprising a therapeutically effective amount of the anti-PD-1 antibody is between 1 nM and 100 μM.

8. The composition comprising a therapeutically effective amount of PAC-1, wherein the concentration of PAC-1 is between 1 μM and 10 μM, and the composition comprising a therapeutically effective amount of an anti-PD-1 antibody, wherein the concentration of the anti-PD-1 antibody is between 1 nM and 1 μM; or 8. The pharmaceutical composition combination of claim 6 or 7, wherein the total daily dose of PAC-1 is 10 mg / kg to 125 mg / kg and the total daily dose of the anti-PD-1 antibody is 1 mg / kg to 100 mg / kg.

9. The combination of any one of claims 6 to 8, wherein the anti-PD-1 antibody is atezolizumab, avelumab, durvalumab, or pembrolizumab.

10. The combination of pharmaceutical compositions according to any one of claims 6 to 9, wherein the cancer is osteosarcoma.

11. A composition comprising a therapeutically effective amount of PAC-1 and a composition comprising a therapeutically effective amount of an anti-PD-1 antibody are administered simultaneously to a subject; or 11. The combination of pharmaceutical compositions according to any one of claims 6 to 10, wherein a composition comprising a therapeutically effective amount of PAC-1 and a composition comprising a therapeutically effective amount of an anti-PD-1 antibody are administered sequentially to a subject, wherein the composition comprising a therapeutically effective amount of PAC-1 is administered to the subject before the composition comprising a therapeutically effective amount of an anti-PD-1 antibody, or wherein the composition comprising a therapeutically effective amount of PAC-1 is administered to the subject after the composition comprising a therapeutically effective amount of an anti-PD-1 antibody.

12. A composition comprising a therapeutically effective amount of PAC-1 and a composition comprising a therapeutically effective amount of an anti-PD-1 antibody, wherein the composition is administered to a subject once per day (q.d.), twice per day (b.i.d.), three times per day (t.i.d.), or four times per day (q.i.d.), and the total daily dose of PAC-1 is between 1 mg / kg and 150 mg / kg; or Each dose of PAC-1 is 70 mg, 175 mg, 250 mg, 375 mg, 450 mg, 500 mg, 625 mg, 750 mg, or 1000 mg; or each dose of PAC-1 was 50 mg / m ２ ~250mg / m ２ The pharmaceutical composition according to any one of claims 6 to 11, 13. The combination of any one of claims 6 to 12, wherein a composition comprising a therapeutically effective amount of PAC-1 is administered every day for 21 or more consecutive days to provide a total daily dose of PAC-1 of 100 mg / kg to 125 mg / kg, and a composition comprising a therapeutically effective amount of an anti-PD-1 antibody is administered two or four times for 21 or more consecutive days, wherein the dose of the anti-PD-1 antibody is 10 mg / kg, and each dose of the anti-PD-1 antibody is administered on a separate day.

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