Combination immunotherapies for treating pancreatic cancer

Abstract

Provided herein are methods of treating a cancer in a subject that include (a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor, and (b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL1-alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor, thereby treating the cancer in the subject.

Description

[0001] COMBINATION IMMUNOTHERAPIES FOR TREATING PANCREATIC CANCER

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority- to U.S. Provisional Patent Application No. 63 / 654,407, filed on May 31, 2024. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated herein by reference in its entirety-.

[0004] BACKGROUND

[0005] Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies worldwide, characterized by- early metastasis, resistance to conventional therapies, and a dismal prognosis. Despite notable progress in understanding the genetics of pancreatic ductal adenocarcinoma (PDAC), discerning transcriptional subty pes, and an increased understanding of its tumor microenvironment, efficacious treatment modalities remain elusive.

[0006] MICAL2 is a member of the MICAL (molecules interacting with CasL) protein family, evolutionarily conserved flavin monooxygenases whose canonical function is the oxidation and resultant depolymerization of actin. MICAL2 was first linked to malignant disease when its splice variants were found to be overexpressed in prostate cancer, and more recently, studies have revealed that MICAL2 may promote epithelial to mesenchymal transition (EMT), migration and invasion in non-small cell lung cancer (NSCLC), gastric cancer, and breast cancer. However, MICAL2 has not been implicated in pancreatic cancer biology-, nor have MICAL2 regulated pathways and the specific roles of MRTF-A versus B in oncogenic phenotypes been comprehensively characterized.

[0007] SUMMARY

[0008] Provided herein are methods of treating a cancer in a subject that include (a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; and (b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL 1 -alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor, thereby treating the cancer in the subject.

[0009] Also provided herein are methods of reducing tumor size in a subject that include (a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; and (b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL 1 -alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor, thereby reducing tumor size in the subject.

[0010] In some embodiments, the MICAL2 inhibitor comprises an inhibitor}' nucleic acid. In some embodiments, the inhibitory nucleic acid comprises a small interfering RNA (siRNA) or a small hairpin RNA (shRNA). In some embodiments, the MICAL2 inhibitor represses MICAL2 gene expression.

[0011] In some embodiments, the MICAL2 inhibitor and the one or more checkpoint inhibitors are administered at the same time. In some embodiments, the MICAL2 inhibitor and the one or more checkpoint inhibitors are administered sequentially. In some embodiments, the MICAL2 inhibitor is administered before the one or more checkpoint inhibitors are administered.

[0012] In some embodiments, the ILl-alpha inhibitor comprises an anti-ILl -alpha antibody. In some embodiments, the PD-1 inhibitor comprises an anti-PD-1 antibody . In some embodiments, the LAG3 inhibitor comprises an anti-LAG3 antibody. In some embodiments, the TIGIT inhibitor comprises an anti-TIGIT antibody.

[0013] In some embodiments, the administering comprises intravenous administration, subcutaneous administration, intraperitoneal administration, rectal administration, oral administration, or combinations thereof. In some embodiments, the cancer is a pancreatic cancer, glioblastoma, gastric cancer, or neo-angiogenic capillary endothelia. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is a pancreatic ductal adenocarcinoma (PDAC).

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0015] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

[0016] FIGs. 1A-1D show that MICAL2 silencing by shRNA reduces the grow th of pancreatic cancers in human and mouse models whether established in a subcutaneous or orthotopic site. Loss of MICAL2 expression impairs orthotopic and subcutaneous tumor growth of murine (KPC46, KPC 1199) (FIGs. 1A and 1C) and human (AsPc-1) pancreatic cancer (FIG. IB). FIGs. 2A-2B show that established syngeneic orthotopic pancreatic cancers expressing MICAL2 regress after doxycycline mediated induction of MICAL2 silencing. FIG. 2A shows tumor growth kinetics demonstrating tumor burden change after flank injection of KPC46 cells with doxycycline (dox)-inducible short hairpin RNA targeting MICAL2 (shMICAL2) or short hairpin RNA, control (shCNT). Tumors were allowed to reach 3 mm in diameter prior to doxycycline (dox) chow7administration. FIG. 2B shows average tumor burden at the end of the study showing specific reduction in the inducible shMICAL2 group on the doxycycline (dox) diet.

[0017] FIG. 3 shows that depletion of MICAL2 in syngeneic orthotopic pancreatic cancer results in changes in the tumor immunophenotype, as assayed by single cell RNA sequencing. These are marked by an increase in cycling CD4 / 8+ T cells, cytotoxic T cells and the expression of granzymes and CCL5, all associated with cytotoxic T cell phenotypes. Results also showdown regulation of IL1 -alpha and other immunosuppressive signals such as ApoE expression. FIG. 4 show s flow cytometry analysis of PD1, TIGIT, and LAG3 checkpoint expression in KPC46 orthotopic tumors, from MICAL2 expressing and silenced tumors demonstrating the upregulation of checkpoint protein expression on T cells, including PD-1, TIGIT and LAG-3. FIGs. 5A-5C show that in syngeneic orthotopic tumors established from 2 distinct KPC cell lines (KPC46 and 1199), the growth control seen after MICAL2 silencing is relieved by administration of a CD8+ T cell depleting antibody. This demonstrates that the growth control observed in the setting of MIC AL2 knockdown is CD8+ T cell dependent. FIG. 5A shows CD8 T cell depletion in KPC 1199 orthotopic tumor models. FIG. 5B shows quantification of FIG. 5A. FIG. 5C shows adoptive CD8 T-Cell transfer extracted from shMICAL2 tumors and injected into KPC46 shCNT, wherein results show that adoptive transfer of T cells from MIC AL2 knockdown tumors can control the grow th of tumors expressing MICAL2, again demonstrating that loss of MICAL2 expression engenders a specific and effective anti-tumor T cell immune response.

[0018] FIGs. 6A-6B show' that adoptive transfer of CD8+ T cells from MICAL2 knockdown tumors can control the growth of MIC AL2 expressing pancreatic cancer lung metastases established by tail vein injection. FIG. 6A shows images of pancreatic cancer lung metastases. FIG. 6B shows tumor weight quantification of the pancreatic cancer lung metastases.

[0019] FIGs. 7A-7B show that the addition of antibodies to either PD1 or IL- 1 -alpha alone can enhance growth control after MIC AL2 knockdow n and that the combination of both antibodies with MICAL2 knockdown can result in complete clinical responses in orthotopic tumor models. FIG. 7A shows images of orthotopic tumor growth control with shRNA knockdown of MICAL2 and the addition of anti ILl-a and / or anti PD-1 antibody therapy. FIG. 7B shows tumor w eight quantification of the orthotopic tumors.

[0020] DETAILED DESCRIPTION

[0021] Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies worldwide, characterized by early metastasis, resistance to conventional therapies, and a dismal prognosis. Despite notable progress in comprehending the genetics of PDAC, discerning transcriptional subtypes, and an increased understanding of its tumor microenvironment, efficacious treatment modalities remain elusive. It is discouraging that the most effective therapeutic approaches still predominantly comprise traditional cytotoxic therapies, which often entail significant adverse effects on patients’ quality of life.

[0022] In recent years, research has shed light on large, highly active chromatin regions known as “super-enhancers” (SE), which play a critical role in defining cell identity and cell state in both normal and malignant cells. Previous studies have indicated that histone 3 lysine 27 acetylation (H3K27ac) marks serve as a reliable indicator for demarcating SE regions. These chromatin regions can regulate key genes that govern the cell phenotype. In tumor cells, this regulatory mechanism may encompass both oncogene and non-oncogene drivers of the transformed state. It was hypothesized that PDAC is driven and sustained by SE- associated genes and that delineating these genes could reveal novel promising therapeutic targets for drug development. Using chromatin immunoprecipitation and sequencing (ChlP- seq), MICAL2 (microtubule associated monooxygenase, calponin and LIM domain containing 2) w as identified as a putative SE-associated gene in human PDAC samples and its overexpression (OE) was confirmed at the RNA and protein levels in both human tissues and cell lines as well as in murine models.

[0023] MICAL2 is a member of the MICAL (molecules interacting with CasL) protein family, consisting of evolutionarily conserved flavin monooxygenases, the canonical function of which is the oxidation and resultant depolymerization of actin. Unique to its other family members MICAL 1 and MICAL3, MICAL2 has no autoinhibitory domain and is thus constitutively active. MICAL2, which is present in both the cytoplasm and the nucleus, was previously shown to indirectly regulate serum response factor (SRF)-mediated transcription through its modulation of nuclear G actin levels. G actin acts to sequester myocardin-related transcription factors A and B (MRTF-A and MRTF-B), coactivators of SRF. Nuclear accumulation of MRTFs is associated with the upregulation of genes associated with cell migration, fibrosis, and epithelial-mesenchymal transition (EMT), although MRTF-A has been the subject of most cancer-related studies. Interestingly, the role of MRTF-B in oncogenesis was previously much more uncertain, with some studies linking it to mesenchymal and hepatocellular tumor progression, whereas a recent study concluded that it acts as a tumor suppressor in human and murine colorectal cancers. Furthermore, recent work demonstrated its important role in sustaining SRF signaling in pancreatic cancer cells. Garg et al., Cancer Res. 2025 Mar 14;85(6): 1049-1063. MICAL2 was first linked to malignant disease when its splice variants were found to be overexpressed in prostate cancer, and more recently, studies have revealed that MICAL2 may promote EMT, migration, and invasion in lung, gastric, and breast cancers.

[0024] MICAL2 is also a potent dnver of pancreatic cancer growth and metastasis, wherein high MIC AL2 expression is associated with a worse prognosis in pancreatic cancer patients. MICAL2 expression increases pancreatic cancer cell proliferation, migration and colony formation and it promotes tumor growth and metastasis. As MICAL2 has been identified as a highly ranked super-enhancer-associated gene in human PDAC. in some embodiments, MICAL2 expression can be correlated with poor prognosis in patients who had undergone surgical resection. In some embodiments, MICAL2 promotes PDAC growth and metastasis, regulates patterns of gene expression associated with KRAS. Thus, the present disclosure describes methods of using MICAL2 as a potential therapeutic target for PDAC, and specifically targeting MICAL2 in combination with other immunotherapies can form an effective anticancer therapeutic strategy.

[0025] Described herein are methods of treating a cancer in a subject that include (a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; and (b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL 1 -alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TI GIT inhibitor. Also described herein are methods of reducing tumor size in a subject that include (a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; and (b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL 1 -alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor.

[0026] Various non-limiting aspects of these methods are described herein and can be used in any combination without limitation. Additional aspects of various components of the methods described herein are known in the art.

[0027] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and ‘"the” include plural referents unless the context clearly dictates otherwise.

[0028] As used herein, a '‘cell” can refer to a eukaryotic cell, optionally obtained from a subj ect or a commercially available source.

[0029] As used herein, the term “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. In some embodiments, if the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The expression level of a gene may be determined by measuring the amount of mRNA or protein in a cell or tissue sample; further, the expression level of multiple genes can be determined to establish an expression profile for a particular sample.

[0030] As used herein, “nucleic acid” or “nucleic acid molecule” is used to include any compound and / or substance that comprise a polymer of nucleotides. In some embodiments, a polymer of nucleotides is referred to as polynucleotides. Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2‘-amino- LNA having a 2’-amino functionalization, and 2’-amino-a-LNA having a 2’-amino functionalization) or hybrids thereof. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).

[0031] A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A deoxyribonucleic acid (DNA) can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid (RNA) can have one or more bases selected from the group consisting of uracil (U). adenine (A), cytosine (C), or guanine (G). In some embodiments, the term “nucleic acid’' or “nucleic acid molecule"’ refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing know n analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is DNA. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is RNA.

[0032] As used herein, the term “subject” refers to an organism, typically a mammal (e.g., a human). In some embodiments, a subject is suffering from a relevant disease, disorder, or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered. In some embodiments, the subject can be an animal, human or non-human. Non-limiting examples of non-human subjects can include mice, rats, hamsters, rabbits, cats, dogs, horses, pigs, donkeys, monkeys, and / or other non-human primates such as apes and lemurs. In some embodiments, the subject is a human.

[0033] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 10% or ± 5%, are within the intended meaning of the recited value.

[0034] Methods of Treating Cancer

[0035] Provided herein are methods of treating a cancer in a subject that include (a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; and (b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL1 -alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor, thereby treating the cancer in the subject.

[0036] As used herein, the term '‘treating” means a reduction in the number, frequency, severity, or duration of one or more (e.g., two, three, four, five, or six) symptoms of a disease or disorder in a subject (e.g., any of the subjects described herein), and / or results in a decrease in the development and / or worsening of one or more symptoms of a disease or disorder in a subject.

[0037] As used herein, the term '‘administration” typically refers to the administration of a composition to a subject or system to achieve delivery' of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be oral, enteral, parenteral, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, enteral, intra-arterial, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intracistemal, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), by patch, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, administration may involve methods of delivery' that include, but are not limited to, use of external and / or implanted infusion pumps, liquid formulation, capsulated formulation, or slow-release encapsulation. In some embodiments, the administering comprises intravenous administration, subcutaneous administration, intraperitoneal administration, rectal administration, oral administration, or combinations thereof.

[0038] As used herein, the term “therapeutically' effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, stabilizes one or more characteristics of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. A therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment.

[0039] In the context of cancer treatment, a “therapeutically effective amount” is an amount which, when administered to an individual diagnosed with a cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual. For example, “therapeutically effective amount” of a composition described herein reverses (in a therapeutic treatment) the development of a malignancy or helps achieve or prolong remission of a malignancy. A pharmaceutically effective amount administered to an individual to treat a cancer in that individual may be the same or different from a pharmaceutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the therapeutic methods described herein are not to be interpreted as, restricted to, or otherwise limited to a “cure” for cancer; rather the methods of treatment are directed to the use of the described compositions to “treat” a cancer, i.e., to affect a desirable or beneficial change in the health of an individual who has cancer. Such benefits are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, a stabilization of patient condition, a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further metastasis, a decrease in opportunistic infections, an increased survivability, a decrease in pain, improved motor function, improved cognitive function, improved feeling of energy (vitality, decreased malaise), improved feeling of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e.g., as the result of treatments described herein) may also be assessed by taking samples of cancer cells from the site of a tumor (e.g., over the course of treatment) and testing the cancer cells for the level of metabolic and signaling markers to monitor the status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenofype. In some embodiments, a pharmaceutically effective amount may be formulated and / or administered in a single dose. In some embodiments, a pharmaceutically effective amount may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen. Inhibitor of MICAL2

[0040] In some embodiments, an inhibitor of MICAL2 inhibits expression of &MICAL2 gene. In some embodiments, an inhibitor of MICAL2 comprises an inhibitory' nucleic acid. Inhibitory nucleic acids in any of the methods and compositions described herein can include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases / locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of a target RNA (e.g.. MICAL2 gene) and modulate its function. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA- induced gene activation (RNAa); small activating RNAs (saRNAs), or any combinations thereof. See, e.g., WO 2010040112, which is herein incorporated by reference in its entirety. In some embodiments, the inhibitory nucleic acid inhibits MICAL2 by knockdown of the MICAL2 gene expression. In some embodiments, an inhibitory nucleic acid comprises a small interfering RNA (siRNA), a small hairpin RNA (shRNA), an antisense oligonucleotide (ASO), or a small nuclear RNA (snRNA) targeting a MICAL2 nucleic acid.

[0041] In some embodiments, an inhibitor of MICL A2 inhibits a MIC AL2 protein. In some embodiments, an inhibitor of MICLA2 comprises a small molecule inhibitor. siRNA / shRNA

[0042] In some embodiments, an inhibitory nucleic acid can comprise an interfering RNA, including but not limited to a small interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”). Methods for constructing interfering RNAs are yvell known in the art. For example, the interfering RNA can be assembled from tw o separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e.. an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, interfering RNA is assembled from a single oligonucleotide, where the self- complementary sense and antisense regions are linked by means of nucleic acid based or non- nucleic acid-based linker(s). The interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary7structure, having self- complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The interfering RNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.

[0043] In some embodiments, the interfering RNA coding region encodes a self- complementary7RNA molecule having a sense region, an antisense region, and a loop region. Such an RNA molecule when expressed desirably forms a "hairpin" structure and is referred to herein as an “shRNA.” The loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length. In some embodiments, the sense region and the antisense region are between about 15 and about 20 nucleotides in length. Following post-transcriptional processing, the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. The siRNA is then capable of inhibiting the expression of a gene with which it shares homology. For details, see Brummelkamp et al., Science 296:550-553, (2002); Lee et al, Nature Biotechnol., 20, 500- 505, (2002); Miyagishi and Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes & Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002); Sui, Proc. Natl. Acad. Sd. USA, 99(6), 5515-5520, (2002); Yu et al. Proc NatlAcadSci USA 99:6047- 6052, (2002).

[0044] In some embodiments, an inhibitory nucleic acid comprises a small hairpin RNA (shRNA). In some embodiments, the shRNA comprises a sequence selected from SEQ ID NOs: 1-3. In some embodiments, the inhibitory nucleic acid has at least 95% sequence identity (e.g.. at least 96%, at least 97%, at least 98%, at least 99%) to a sequence selected from shRNA SEQ ID NOs: 1-3.

[0045] [Table 1] Human (h) and Mouse (m) shRNA sequences

[0046] In some embodiments, an inhibitory nucleic acid comprises a small interfering RNA (siRNA). In some embodiments, the siRNA comprises a sequence selected from SEQ ID NOs: 4-11. In some embodiments, the inhibitory nucleic acid has at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99%) to a sequence selected from siRNA SEQ ID NOs: 4-11.

[0047] [Table 2] Human (h) and Mouse (m) siRNA sequences In some embodiments, an inhibitory nucleic acid can be 10 to 50 (e.g., 10 to 40, 10 to 35, 10 to 30, 10 to 20, 20 to 50, 20 to 40, 20 to 30, 30 to 50. 30 to 40. or 40 to 50) nucleotides in length. In some embodiments, an inhibitory nucleic acid can have a complementary portion of 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, or 50 nucleotides in length, or any range therewithin.

[0048] In some embodiments, an inhibitory nucleic acid is sufficiently complementary to the target RNA (e.g., MICAL2 gene), i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. As used herein, “complementary” refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non- naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a target RNA, then the bases are considered to be complementary to each other at that position. In some embodiments, 100% complementarity is not required. In some embodiments, an inhibitory nucleic acid described herein can have at least 80% sequence complementarity to a target region within the target RNA, e.g.. 90%. 95%. or 100% sequence complementarity to the target region within the target RNA. The inhibitory nucleic acid can be directed to hybridize sufficiently well (least 80% sequence complementarity) and with sufficient specificity to one or more of the human MICAL2 sequences corresponding to NM 001393937.1; NM 00I346293.2; NM 001346297.2; NM 014632.4;

[0049] NM_001346295.2; NM_001282663.2; NM_001346299.2; NM_001346296.2; NM_001346292.2; NM_001346298.2; NM_001346294.2; NM_001282668.2; NM_001282664.1 ; NM_001282665. 1 ; NM_001282666.1 ; or NM_001282667.1.

[0050] For further disclosure regarding inhibitory nucleic acids, see, e.g.. US2010 / 0317718 (antisense oligos); US2010 / 0249052 (double-stranded ribonucleic acid (dsRNA)); US2009 / 0181914 and US2010 / 0234451 (LNAs); US2007 / 0191294 (siRNA analogues); US2008 / 0249039 (modified siRNA); and WO2010 / 129746 and W02010 / 040112 (inhibitory nucleic acids), which are herein incorporated by reference in their entireties.

[0051] The methods described herein include administering one or more checkpoint inhibitors with an inhibitor of MICAL2. Specifically, the methods described herein include administering one or more MICAL2 inhibitors with one or more checkpoint inhibitors selected from the group consisting of an ILl-alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor. Anti-ILl-alpha antibody

[0052] IL- la is pro-inflammatory cytokine that plays a role in a number of different activities including inflammation, immune responses, tumor metastasis, and hematopoiesis. In some embodiments, the methods described herein comprise administering an anti-IL-1 -alpha antibody. In some embodiments, the anti-IL-1 antibody comprises an IL- la binding site and specifically binds to IL- la. In some embodiments, an anti-IL-1 -alpha antibody can interfere with IL- 1 alpha binding to an IL-1 alpha receptor. In some embodiments, an anti-IL-1 -alpha antibody specifically binds IL-lalpha or an IL- 1 alpha receptor. In some embodiments, an example of an anti-IL-1 -alpha antibody can include, but is not limited to. anakinra (KINERET).

[0053] In some embodiments, a method described herein includes (a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; (b) administering to the subject a therapeutically effective amount of an anti-ILl -alpha antibody; and optionally (c) administering to the subject a therapeutically effective amount of an anti-PD-1 antibody, thereby treating the cancer in the subject.

[0054] Anti-PD-1 antibody

[0055] PD-1 refers to the programmed death- 1 protein, a T-cell co-inhibitor, also known as CD279. PD-1 is a member of the CD28 / CTLA-4 / ICOS family of T-cell co-inhibitors. PD-1 is a 288-amino acid protein with an extracellular N-terminal domain which is IgV-like, a transmembrane domain and an intracellular domain containing an immunoreceptor tyrosinebased inhibitory (ITIM) motif and an immunoreceptor tyrosine-based switch (ITSM) motif. The PD-1 receptor has two ligands, PD-ligand-1 (PD-L1) and PD-L2.

[0056] In some embodiments, the methods described herein comprise administering an anti- PD-1 antibody. In some embodiments, the anti-PD-1 antibody is an antibody which binds specifically to the antigen PD-1. In some embodiments, the anti-PD-1 antibody selectively binds human PD-1. In some embodiments, the anti-PD-1 antibody selectively binds to the extracellular domain of human PD-1. In some embodiments, the anti-PD-1 antibody selectively binds to one or more of full-length human PD-1, PD-1 Aex2, PD-1 Aex3, PD- lAex2,3, and PD-l ex2,3,4. In some embodiments, the anti-PD-1 antibody binds to homologs of human PD-1. In some embodiments, the anti-PD-1 antibody binds to a homolog of human PD-1 from a species selected from monkeys, mice, dogs, cats, rats, cows, horses, goats, and sheep. In some embodiments, the anti-PD-1 antibody inhibits the binding of PD-1 to its ligands. In some embodiments, the anti-PD-1 antibody inhibits the binding of PD-1 to PD-L1. In some embodiments, the anti-PD-1 antibody inhibits the binding of PD-1 to PD-L2. In some embodiments, the anti-PD-1 antibody inhibits the binding of PD-1 to PD-L1 and PD- L2. In some embodiments, the example of an anti-PD-1 antibody can include, but is not limited to, nivolumab (ONO-4538 / BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), durvalumab (IMFINZI). atezolizumab (TECENTRIQ), avelumab (BAVENCIO), cemiplimab (LIBTAYO), sintilimab (IBI308), toripalimab (JS 001), camrelizumab (SHR1210), dostarlimab (JEMPERLI), JTX-4014 (Jounce Therapeutics), spartalizumab (PDR001), tislelizumab (BGB-A317), INCMGA00012 (MGA012), AMP-224, AMP-514 (MEDI0680). KN035, CK-301, AUNP12, CA-170, BMS- 986189 (BRISTOL MYERS SQUIBB), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), and MPDL328OA (ROCHE)).

[0057] Anti-Lag3 antibody

[0058] LAG3 refers to Lymphocy te Activation Gene-3, wherein LAG3 is an immune checkpoint molecule expressed on activated T and NK, and some B and DC cells. Interaction of LAG3 with its ligand, the major histocompatibility complex (MHC) class II (MHCII) expressed on antigen presenting cells like macrophages and DC, inhibits the activation of T and NK cells and therefore, suppress the ability of the cells to recognize and kill cancer cells.

[0059] In some embodiments, the methods described herein comprise administering an anti- Lag3 antibody. In some embodiments, the anti-Lag3 antibody is an antibody which binds specifically to the antigen Lag3. In some embodiments, the anti-Lag3 antibody selectively binds human Lag3. In some embodiments, the anti-Lag3 antibody binds to homologs of human Lag3. In some embodiments, the anti-Lag3 antibody can cross-react with a LAG3 protein from a species other than human. In some embodiments, an example of anti-Lag3 antibody can include, but is not limited to, relatlimab (BMS-986016).

[0060] Anti-TIGIT antibody

[0061] T cell Ig and ITIM domain (TIGIT) is a surface protein expressed on regulatory, memory, and activated T cells that exerts immunosuppressive effects by binding to poliovirus receptor and modulating cytokine production by dendritic cells. TIGIT is also referred to as Vsig9, Vstm3, and WUCAM and has two ligands: CD155 (PVR or Necl-5) and CD112 (nectin-2, also known as PRR2 or PVRL2). TIGIT contains an Ig variable domain, a transmembrane domain, and an immunoreceptor tyrosine-based inhibitory motif, and it can bind CD155 and inhibit the cytotoxicity function of NK cells. Furthermore, TIGIT can suppress cytokine production (IFN-y) via the NF-KB pathway.

[0062] In some embodiments, the methods described herein comprise administering an anti- TIGIT antibody. In some embodiments, the anti -TIGIT antibody is an antibody which binds specifically to CD155 or CD112. In some embodiments, the anti-TIGIT antibody selectively binds human TIGIT. In some embodiments, the anti-TIGIT antibody binds to homologs of human TIGIT. In some embodiments, the anti-TIGIT antibody can cross-react with a TIGIT protein from a species other than human. In some embodiments, an example of anti-TIGIT antibody can include, but is not limited to. AB 154 (Arcus Biosciences), BGB-A1217 (BeiGene), BMS-986207 (BMS), COM902 (Compugen), IBI939 (Innovent Biologies), MK- 7684 (MSD), MTIG7192A (Genentech), and tiragolumab (Roche).

[0063] In some embodiments, the MICAL2 inhibitor and one or more checkpoint inhibitors are administered at the same time. In some embodiments, the MICAL2 inhibitor and the one or more checkpoint inhibitors are administered sequentially. In some embodiments, the MICAL2 inhibitor is administered prior to the administration of checkpoint inhibitor(s).

[0064] In some embodiments, a method described herein is used to treating a cancer in a subject, wherein the cancer is a pancreatic cancer, glioblastoma, gastric cancer, breast cancer, stomach cancer, kidney cancer, cervical cancer, ovarian cancer, hepatocellular carcinoma, head and neck squamous cancers, colorectal cancer, endometrial cancer, bladder cancer, or prostate cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is a pancreatic ductal adenocarcinoma (PDAC).

[0065] Methods of Reducing Tumor Size

[0066] Also provided herein are methods of reducing tumor size in a subject that include (a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; and (b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL 1 -alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor, thereby reducing tumor size in the subj ect. In some embodiments, the methods described herein can be used to reduce tumor size (tumor regression) in the subject having a cancer. In some embodiments, the methods provided herein can be used to reduce or eliminate the number of cancer cells present within a subject having a cancer. For example, any one of the methods described herein can be used to reduce the number of cancer cells present within a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, any one of the methods described herein can be used to reduce the size (e.g., volume) of one or more tumors present within a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some embodiments, the number of cancer cells present within a subject being treated can be monitored. Any appropriate method can be used to determine whether or not the number of cancer cells present within a subject is reduced. For example, imaging techniques can be used to assess the number of cancer cells present within a subject.

[0067] In some embodiments, any one of the methods provided herein can be used to improve survival of a subject having a cancer. For example, any one of the methods described herein can be used to improve the survival of a subject having cancer by, for example. 10. 20. 30. 40, 50, 60, 70, 80, 90, 95, or more percent. For example, any one of the methods described herein can be used to improve the survival of a subject having cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).

[0068] Pharmaceutical Compositions

[0069] The methods described herein can include the administration of pharmaceutical compositions and formulations comprising an inhibitor of MICAL2. In some embodiments, an inhibitor of MICAL2 comprises an inhibitory nucleic acid. In some embodiments, a composition comprising an inhibitor of MICAL2 is used in a method of treating cancer, e.g.. pancreatic cancer, in a subject, and the composition is administered with one or more checkpoint inhibitors.

[0070] In some embodiments, the pharmaceutical compositions are formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see. e.g., Remington: The Science and Practice of Pharmacy, 21st ed.. 2005.

[0071] In some embodiments, any one of the inhibitory nucleic acids described herein can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0072] Formulations of the pharmaceutical compositions described herein include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

[0073] Pharmaceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be an admixture with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

[0074] Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g.. sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose. or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the crosslinked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

[0075] Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences as described herein) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g.. lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.

[0076] In some embodiments, oil-based pharmaceuticals are used for administration of nucleic acid sequences. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Patent No. 5.716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

[0077] Pharmaceutical formulations can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally -occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, these injectable oil-in-water emulsions comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and / or an ethoxylated sorbitan trioleate.

[0078] The pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107-111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0079] In some embodiments, the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0080] In some embodiments, the pharmaceutical compounds can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

[0081] In some embodiments, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary' widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).

[0082] In some embodiments, the pharmaceutical compounds and formulations can be lyophilized. Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical as described herein and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg / mL protein, about 15 mg / mL sucrose, about 19 mg / mL NaCl. and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.

[0083] The compositions and formulations can be delivered by' the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery’ of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0084] Liposomes can also include "‘sterically stabilized” liposomes, i.e.. liposomes comprising one or more specialized hpids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.

[0085] The formulations can be administered for prophylactic and / or therapeutic treatments. In some embodiments, for therapeutic applications, compositions are administered to a subject who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount.

[0086] The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

[0087] The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability', metabolism, clearance, and the like (see, e.g., Hidalgo- Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59- 69; Johnson (1995) J. Pharm. Sci. 84: 1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods as described herein are correct and appropriate.

[0088] Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration, and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.

[0089] EXAMPLES

[0090] The disclosure is further described in the following examples, which do not limit the scope of the disclosure.

[0091] Example 1 - MICAL2 promotes heterotopic and orthotopic growth in vivo

[0092] When MICAL2 was silenced in vitro, reduced cell proliferation, migration, invasion, and a reversal of the EMT phenoty pe was observed. Therefore, it was next sought to determine how the loss of MICAL2 impacts tumorigenesis initially using orthotopic and subcutaneous mouse transplant models. FIG. 1A depicts the results after transplantation of KPC46 cells into the pancreas of syngeneic mice. Reduction in MICAL2 expression resulted in marked reductions in tumor growth. These results were recapitulated with human PDAC cells, when AsPCl cells were transplanted with constitutive KD of MICAL2 into immunodeficient NSG mice (FIG IB). A similar experiment was repeated in another murine pancreatic cancer cell line 1199 and again, MICAL2 silencing reduced tumor growth (FIG 1C). Next to determine whether the reduced growth of MICAL2-KD cells was due to failure of engraftment, and to determine if loss of MICAL2 would reduce the growth of an established tumor, dox-inducible KPC46 cells w ere established with shRNA targeting MICAL2 (ish-MICAL2) or control (ishcontrol). The cells were implanted into the flank of syngeneic animals and tumors w ere allowed to form as documented by both palpation and ultrasound over 10 days prior to dox induction. Tumor growth over 44 days was significantly abrogated in the ish-MICAL2-implanted mice compared with ish-control mice when treated with dox but not when kept on a regular dox-free diet (FIGs. 2A-2B).

[0093] In summary, reduction in the expression of MIC AL2 in PDAC cells inhibits the growth of pancreatic cancer in multiple model systems, including in both heterotopic and orthotopic locations, and in the setting of an established tumor. Example 2 - Depletion of MICAL2 results in changes in tumor immunophenotype

[0094] To understand the mechanisms inhibiting tumor growth after MICAL2 silencing, single cell RNA sequencing was performed on syngeneic tumors established form MICAL2 expressing and MICAL2 knockdown cells. This experiment demonstrated that depletion of MICAL2 in syngeneic orthotopic pancreatic cancer results in marked changes in the tumor immunophenotype. These changes are marked by an increase (teal) in cycling CD4 / 8+ T cells, cytotoxic T cells and the expression of granzymes and CCL5, all associated with cytotoxic T cell phenotypes. Furthermore, down regulation (orange) of ILl-alpha and other immunosuppressive signals such as ApoE expression was also observed demonstrating that MICAL2 promotes an immunosuppressive microenvironment that can be reversed by inhibiting MICAL2 expression (FIG. 3).

[0095] Example 3 - Flow cytometry analysis of PD1, TIGIT, and LAG3 checkpoint expression in tumors

[0096] The same syngeneic tumor models were evaluated by flow cytometry to understand the impact of MICAL2 expression on the expression of immune checkpoint proteins in CD8+ T cells. The percentages of CD8+ T cells expressing PD-1, TIGIT, and LAG3 were evaluated, and all demonstrated increased expression after loss of MICAL2 expression, strongly suggesting the potential for benefit from combining MICAL2 inhibition with the use of an antibody to these checkpoint proteins (FIG. 4).

[0097] Example 4 - CD8+ T cell depletion in tumor models

[0098] Next, to determine if the observed tumor growth control that occurs in the setting of MICAL2 knockdown was in fact related to an augmented anti-tumor immune response driven by CD8+ T cells, we conducted CD8+ T cell depletion in KPC1199 and KPC 46 orthotopic tumor models. It was observed that the growth control seen after MICAL2 silencing is relieved by administration of a CD8+ T cell depleting antibody (data for 1199 is depicted in FIG. 5A and 5B). These results clearly demonstrate that the growth control observed in the setting of MICAL2 knockdown is CD8+ T cell dependent. To further explore this idea, adoptive transfer of T cells w as performed from MICAL2 knockdown tumors into tumors expressing MICAL2. This experiment demonstrated that these T cells can control the growth of tumors expressing MICAL2, again showing that loss of MICAL2 expression engenders a specific and effective anti-tumor T cell immune response (FIG. 5C). Furthermore, adoptive transfer of CD8+ T cells from MICAL2 knockdown tumors were shown to control the growth of MICAL2 expressing pancreatic cancer lung metastases established by tail vein injection (FIGs. 6A-6B).

[0099] Example 5 - MICAL2 knockdown with addition of checkpoint inhibitors result in clinical responses in tumor models

[0100] To understand if the growth control observed after inhibition of MICAL2 expression could be augmented by other immunotherapy strategies, orthotopic tumor growth control was investigated with shRNA knockdown of MICAL2 and the addition of anti ILl-a and / or anti PD-1 antibody therapy (FIG. 7A). Results show that the addition of anti ILl-a and / or anti PD-1 antibody therapy can enhance growth control after MICAL2 knockdown and that the combination of both antibodies with MICAL2 knockdown can result in complete clinical responses in orthotopic tumor models (FIG. 7B).

Claims

WHAT IS CLAIMED IS:

1. A method of treating a cancer in a subject, the method comprising:(a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; and(b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL 1 -alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor, thereby treating the cancer in the subject.

2. A method of reducing tumor size in a subject, the method comprising:(a) administering to the subject a therapeutically effective amount of a composition comprising a MICAL2 inhibitor; and(b) administering to the subject a therapeutically effective amount of one or more checkpoint inhibitors selected from the group consisting of an IL 1 -alpha inhibitor, a PD-1 inhibitor, a LAG3 inhibitor, and a TIGIT inhibitor, thereby reducing tumor size in the subject.

3. The method of claim 1 or 2, wherein the MICAL2 inhibitor comprises an inhibitory nucleic acid.

4. The method of claim 3, wherein the inhibitory nucleic acid comprises a small interfering RNA (siRNA) or a small hairpin RNA (shRNA).

5. The method of any one of claims 1-4, wherein the MICAL2 inhibitor represses MICAL2 gene expression.

6. The method of any one of claims 1-5, wherein the MICAL2 inhibitor and the one or more checkpoint inhibitors are administered at the same time.

7. The method of any one of claims 1-5, wherein the MICAL2 inhibitor and the one or more checkpoint inhibitors are administered sequentially.

8. The method of claim 7, wherein the MICAL2 inhibitor is administered before the one more checkpoint inhibitors are administered.

9. The method of any one of claims 1-8, wherein the IL 1 -alpha inhibitor comprises an anti-ILl -alpha antibody.

10. The method of any one of claims 1-9, wherein the PD-1 inhibitor comprises an anti- PD-1 antibody.

11. The method of any one of claims 1-10, wherein the LAG3 inhibitor comprises an anti- LAG3 antibody.

12. The method of any one of claims 1-11, wherein the TIGIT inhibitor comprises an anti-TIGIT antibody.

13. The method of any one of claims 1-12, wherein the administering comprises intravenous administration, subcutaneous administration, intraperitoneal administration, rectal administration, oral administration, or combinations thereof.

14. The method of any one of claims 1-13, wherein the cancer is a pancreatic cancer, glioblastoma, gastric cancer, or neo-angiogenic capillary endothelia.

15. The method of claim 14, wherein the cancer is a pancreatic cancer.

16. The method of claim 15, wherein the cancer is a pancreatic ductal adenocarcinoma (PDAC).

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