Methods for treating cancer with compositions comprising amlexanox and immune modulators

Abstract

To provide compositions and methods for treating cancer in a subject.SOLUTION: In some embodiments, methods comprise generating an immune response in an individual by inducing expression of neoantigens on the surface of abnormal (such as proliferative) cells. In one embodiment, a method of treating cancer in a subject comprises administering amlexanox in combination with immune modulators such as checkpoint inhibitors, immune co-stimulatory molecules, TLR agonists, and TNFR superfamily agonists. In one embodiment, checkpoint inhibitors are selected from antibodies against PD-1, PD-L1, and CTLA-4.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 62 / 523,537, filed Jun. 22, 2017, entitled “Methods of Treating Cancer with Compositions Comprising Anlequinox and Immunomodulators,” the reference of which is incorporated herein by reference.

Summary of the Invention

[0002] Disclosed herein are compositions and methods for treating cancer in a subject. In some embodiments, the method includes generating an immune response in an individual by inducing the expression of neoantigens on the surface of abnormal (such as proliferative) cells. This can be achieved by promoting the readthrough of premature termination codons (PTCs) of mRNA and / or inhibiting nonsense-mediated decay (NMD) of mRNA.

[0003] In one embodiment, a method of treating a subject having cancer can include administering a therapeutically effective amount of a compound that promotes readthrough of premature termination codons (PTCs) in mRNA and inhibits nonsense-mediated decay (NMD) of mRNA. In some embodiments, the compound is anlequinox. In some embodiments, the method further includes administering a molecule that inhibits an immune checkpoint protein.

[0004] In one embodiment, a method of treating cancer in a subject includes administering anlequinox in combination with an immunomodulator such as a checkpoint inhibitor, an immunostimulatory molecule, a TLR agonist, a TNFR superfamily agonist, a cyclic dinucleotide, a T cell agonist, a cytokine, a chemokine, and an oncolytic virus.

[0005] In one embodiment, a method of treating a target cancer includes administering anlequinox in combination with a molecule that inhibits at least one immune checkpoint protein disclosed herein. In one embodiment, the checkpoint protein is selected from PD-1, PD-L1, and CTLA-4. In some embodiments, the method includes administering anlequinox in combination with an anti-PD-1 antibody. In some embodiments, the method includes administering anlequinox in combination with an anti-PD-L1 antibody. In some embodiments, the method includes administering anlequinox in combination with an anti-CTLA-4 antibody. In some embodiments, the method includes administering anlequinox in combination with anti-PD-1 and anti-CTLA-4 antibodies. In some embodiments, the method includes administering anlequinox in combination with anti-PD-L1 and anti-CTLA-4 antibodies.

[0006] In another embodiment, a method of killing cancer cells can include contacting the cancer cells with a composition comprising anlequinox in combination with a molecule that inhibits at least one immune checkpoint protein. In some embodiments, the method can be in vitro or in vivo.

[0007] In a further aspect, a method of killing cancer stem cells can include contacting the cancer stem cells with a composition comprising anlequinox in combination with a molecule that inhibits at least one immune checkpoint protein. In some embodiments, the method can be in vitro or in vivo.

[0008] In a further aspect, a method of inducing the expression of one or more neoantigens on the surface of abnormal cells includes contacting the abnormal cells with anlequinox in combination with a molecule that inhibits at least one immune checkpoint protein. In some embodiments, the method can be in vitro or in vivo.

[0009] In another embodiment, a method of generating an immune response in an individual in need of treatment comprises administering to the individual a therapeutically effective amount of a compound that promotes premature termination codon (PTC) readthrough in the mRNA and inhibits nonsense-mediated decay (NMD) of the mRNA. In some embodiments, the compound is amlexanox. In some embodiments, the method further comprises administering a molecule that inhibits an immune checkpoint protein. In some embodiments, the checkpoint proteins are PD-1, PD-L1, and CTLA-4.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0011] A major obstacle to the effectiveness of checkpoint blockade against cancer immunotherapy relates to the paucity of potent tumor neoantigens expressed by late-stage cancers that have undergone extensive immune editing. This process occurs early in the tumor life cycle, where populations of tumor cells that express immunogenic or potent tumor-specific antigens and thus become targets for cytotoxic T cells are eliminated. Thus, mature tumors are primarily composed of tumor cells that have evolved multiple immune evasion strategies, such as the expression of only weak tumor antigens, and are thus less likely to be effectively targeted by cytotoxic T cells. Despite the recent success of checkpoint blockade as a cancer immunotherapy, the effectiveness of these drugs is highly correlated with the availability of potent tumor neoantigens. In particular, the tumors in which these drugs are most effective are those with the highest mutational burdens, such as melanoma and non-small cell lung cancer (NSCLC), both of which are characterized by strong environmental mutagenesis from UV damage and smoking.

[0012] Accordingly, disclosed herein are methods and compositions for generating an immune response in an individual having cells that express one or more messenger RNA (mRNA) molecules having one or more premature termination codons (PTCs) by inducing the expression of one or more neoantigens on the surface of those cells. This method is based in part on the fact that inhibition of the molecular pathways associated with the regulation of nonsense-mediated decay (NMD) results in “readthrough” and subsequent translation of the PTC-bearing mRNA into polypeptides having amino acid sequences that often differ from the wild-type protein. Without being bound by theory, proteolysis and presentation of these peptides on the cell surface via major histocompatibility complex (MHC) molecules results in highly antigenic targets for attack by components of the immune system, such as T cells. As further described below, combinations of compounds that promote PTC readthrough and inhibit NMD not only effectively inhibit tumor cell replication in in vivo models, but also compounds specific for one or more immune checkpoint molecules synergistically enhance the anti-proliferative effect in treatment regimens. Accordingly, the methods disclosed herein are particularly useful for the treatment of diseases characterized by hyperproliferative cells, such as cancer, which results from the hypermutability of rapidly dividing cells. Cancer cells partially evade detection by the immune system by displaying only weakly antigenic or non-antigenic peptides on their surface. Accordingly, the compositions and methods disclosed herein provide an effective way to induce the expression of neoantigens on the surface of cancer cells, thereby rendering them vulnerable to attack by the immune system.

[0013] Parallel to the progress of immunotherapy, efforts in tumor vaccine development have led to the selection of RNAseq / exome sequencing performed on tumor samples for their ability to identify mutant transcripts that function as robust neoantigens and are subsequently used as the basis for vaccine development. Due to the nature of these mutation detection methods, the overwhelming majority of the detected mRNA species contain missense mutations in the coding sequences generated by nucleotide transitions and transversions, leading to either silent or single amino acid substitutions. These proteins have the ability to function as neoantigens, but identifying mutant mRNA species with differences in multiple amino acids has the potential to function as much more potent neoantigens.

[0014] A more desirable pool of tumor mRNAs that induce robust tumor neoantigens are those that contain premature termination codons (PTCs). These mRNA species contain much more deleterious mutations such as insertions, deletions, nonsense mutations, non-stop (delayed termination) mutations, etc. Paradoxically, however, these same PTC-containing species are very unstable and are rapidly degraded by the nonsense-mediated decay (NMD) pathway, and thus they are generally undetectable by RNA sequencing and rarely translated into proteins due to their very low abundance or complete absence. PTC-containing mRNAs can potentially encode many different amino acids from the wild-type sequence through frameshift of the reading frame and / or use of alternative stop codons. When proteins are transcribed from mRNA species containing PTCs, they can encode proteins with very diverse sequences, thus serving as a very potent source of tumor neoantigens. Therapeutic approaches aimed at blocking the NMD pathway and promoting readthrough of PTCs within tumors can translate transcripts containing PTCs and express potent neoantigens in vivo.

[0015] Early termination codon (PTC) mutations are those in which a sense codon is changed to one of the three stop codons (UAA, UAG, or UGA) by a base substitution or frameshift mutation. Studies on yeast, human genetic disorders, and immunoglobulin family gene expression have identified an RNA surveillance mechanism that minimizes the translation of nonsense RNAs containing such chain termination mutations and regulates RNA stability. This surveillance mechanism is called "nonsense-mediated mRNA decay" ("NMD"). NMD is a post-transcriptional mechanism that functions in both normal cells (e.g., B and T cells) and cells with gene mutations (e.g., cells with mutations in genes that control cell proliferation).

[0016] Many of the proteins involved in NMD are not conserved across species, but in Saccharomyces cerevisiae (yeast), there are three main factors in NMD: UPF1, UPF2, and UPF3 (UPF3A and UPF3B in humans), which constitute the conserved core of the NMD pathway. All three of these factors are trans-acting elements called up-frameshift (UPF) proteins. In mammals, UPF2 and UPF3 are part of the "exon-exon junction complex" (EJC) that binds to mRNA after splicing, along with other proteins that also function in NMD. The phosphorylation of UPF1 is controlled by the proteins SMG-1, SMG-5, SMG-6, and SMG-7.

[0017] The process of detecting abnormal transcripts occurs during the translation of mRNA. A common model for the detection of mammalian abnormal transcripts suggests that during the first round of translation, ribosomes remove exon-exon junction complexes bound to mRNA after splicing has occurred. After this first translation, if any of these proteins remain bound to the mRNA, NMD is activated. Exon-exon junction complexes located downstream of the PTC are not removed from the transcript because they are released before the ribosome arrives. Termination of translation results in the assembly of a complex composed of UPF1, SMG1 and the release factors eRF1 and eRF2 on the mRNA. If the EJC remains on the mRNA because the transcript contains a PTC, UPF1 contacts UPF2 and UPF3, causing phosphorylation of UPF1.

[0018] In vertebrates, typically, the position of the last exon junction complex relative to the stop codon determines whether a transcript undergoes NMD. If the stop codon is downstream of or within about 50 nucleotides of the final exon junction complex, the transcript is translated normally. However, if the stop codon is located about 50 nucleotides upstream of the exon junction complex, the transcript is downregulated by NMD. Phosphorylated UPF1 interacts with SMF-5, SMG-6 and SMG-7, promoting dephosphorylation of UPF1. SMG-7 is thought to be the terminal effector of NMD because it accumulates in P bodies, the cytoplasmic sites of mRNA decay. In both yeast and human cells, the major pathway of mRNA decay is initiated by removal of the 5' cap, followed by degradation by the exoribonuclease enzyme XRN1. Another pathway by which mRNA is degraded is deadenylation from 3'-5'.

[0019] Therefore, there are at least two ways to avoid the NMD pathway in order to induce translation of mRNA with a PTC into polypeptide without being bound by theory. 1) Providing a compound that promotes readthrough of the PTC, thus ensuring removal of all EJCs associated with the mRNA during the first round of translation by ribosomes, and / or 2) inhibiting one or more proteins associated with the NMD degradation complex (such as UPF1, UPF2, UPF3, eIF4AIII, MLN51, Y14 / MAGOH heterodimer, SMG-1, SMG-5, etc.) such as SMG-6 and / or SMG-7.

[0020] The present invention is not limited to the specific processes, compositions, or methodologies described, as these may vary. The terms used in the description are for the purpose of describing particular versions or embodiments only and are not intended to limit the scope of the invention. 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. Nothing in this specification should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0021] As used herein, "premature termination codon" (PTC) or "premature stop codon" refers to the introduction of a stop codon (prior to an endogenous stop codon) into an mRNA as a result of a mutation.

[0022] As used herein, "nonsense mutation" is a point mutation in a DNA sequence that results in a PTC or nonsense codon, leading to a transcribed mRNA and a truncated, incomplete, and usually non-functional protein product. Nonsense mutations are genetic mutations underlying various diseases, particularly those inherited genetically. For example, in cancer, nonsense mutations are generally acquired or are somatic mutations in tumors. In some embodiments, the nonsense mutation is a somatic mutation. In another embodiment, the nonsense mutation is not a germline mutation.

[0023] A "non-stop mutation" is a point mutation of an endogenous stop codon that causes continuous and inappropriate translation into the 3' untranslated region of mRNA. Non-stop mutations result in the incorporation of abnormal amino acid sequences and the utilization of downstream stop codons. In some embodiments, the non-stop mutation is a somatic mutation. In another embodiment, the non-stop mutation is not a germline mutation.

[0024] A "frameshift mutation" refers to a deletion or insertion of one or more nucleotides within an open reading frame such that the reading frame of the coding region is shifted by one or two nucleotides, for example, a deletion or insertion of a single nucleotide or dinucleotide. Thus, the amino acid sequence of a polypeptide translated from an mRNA having a frameshift mutation is very different from the corresponding wild-type sequence. In some embodiments, the frameshift mutation generates a PTC. In some embodiments, the frameshift mutation is a deletion of a nucleotide or dinucleotide that results in a +1 or +2 frameshift mutation. However, any number of nucleotide deletions can occur as long as the result is a frameshift mutation. Alternatively, an insertion of one or more nucleotides can cause a frameshift, and such mutations also form part of the present invention. Other genetic modifications that cause a frameshift also form part of the present invention. For example, exon skipping or retention of intron sequences or splice site mutations or mutations outside the coding region such as within introns or 5' or 3' untranslated regions that result in a change in the nucleotide sequence leading to translation initiation from a different position. This mutation can result in translation errors and the production of mutant proteins. In this type of gene mutation, the mutant protein has a completely mutant amino acid sequence and does not contain the wild-type sequence. In some embodiments, the frameshift mutation can result in an early stop codon (when occurring early in the mRNA) or a delayed stop codon (when occurring near an endogenous stop codon). In another embodiment, the frameshift mutation is not a germline mutation.

[0025] As used herein, a "non-functional" polypeptide refers to a polypeptide that, due to one or more mutations, is unable to perform its function in a cellular context as compared to the corresponding non-mutated (wild-type) polypeptide. A "functional" polypeptide is a polypeptide that can perform at least some cellular functions even if it has one or more mutated amino acids as compared to the corresponding non-mutated (wild-type) polypeptide.

[0026] As used herein, the term "readthrough" means skipping a premature stop codon in ribosomal translation, substituting an amino acid, or suppressing the degradation of mRNA containing a premature stop codon.

[0027] As used herein, the term "polypeptide" includes proteins, peptides, polypeptide fragments, and fusion polypeptides.

[0028] As used herein, the terms "animal", "patient", or "subject" include, but are not limited to, humans and non-human vertebrates such as wild animals, domestic animals, and farm animals. Preferably, the term refers to a human. The terms "subject", "patient", or "individual" are used interchangeably herein and refer to a mammalian subject to be treated. In some embodiments, the patient is human. In some cases, these methods can be used in the development of animal models of diseases including, but not limited to, experimental animals, veterinary applications, and rodents such as mice, rats, hamsters, and primates. In some embodiments, the patient is a patient in need thereof.

[0029] The transitional term "comprising", which is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes elements, steps, or components not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to specific materials or steps that "do not materially affect the basic and novel characteristics" of the claimed invention. In embodiments or claims where the term "comprising" is used as a transitional phrase, it is contemplated that such embodiments can also be replaced with the terms "consisting of" or "consisting essentially of".

[0030] Unless otherwise defined herein, 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.

[0031] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antioxidant" includes reference to one or more antioxidants known to those of ordinary skill in the art and their equivalents.

[0032] "Administration" when used in connection with a treatment means administering a therapeutic agent to a patient, thereby having a positive effect on the tissue targeted by the therapeutic agent. The compounds described herein can be administered alone or in combination with other pharmaceuticals (simultaneously or sequentially). For example, the compounds can be administered in combination with other anti-cancer or anti-tumor agents, or in combination with other cancer therapies other than chemotherapy, such as surgery or radiation therapy. In some embodiments, the compounds described herein can also be administered in combination with other therapeutic agents (i.e., as a combined formulation or as separate formulations).

[0033] A "therapeutically effective amount" or "effective amount" of a composition is a predetermined amount calculated to achieve a desired effect, i.e., to ameliorate, prevent, or improve an undesirable condition, disease, or symptom in a patient. The activities contemplated by this method can, if desired, include both therapeutic and / or prophylactic treatment. The specific dosage of a compound administered in accordance with the present invention to obtain a therapeutic and / or prophylactic effect will, of course, be determined by the particular circumstances of the case, including, for example, the compound administered, the route of administration, and the condition being treated. The effective amount to be administered can be determined by a physician in light of the relevant circumstances, including the condition being treated, the selection of the compound to be administered, and the route of administration selected. A therapeutically effective amount of a compound of the present invention is typically an amount sufficient to achieve an effective systemic or local concentration in the target tissue when administered in a physiologically acceptable excipient composition.

[0034] As used herein, the term "therapeutic agent" means an agent that is utilized to prevent, combat, ameliorate, prevent, or improve an undesirable condition, disease, or symptom in a patient.

[0035] "Pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not be harmful to its recipient.

[0036] The term "modulate" means that any of the recited activities are, for example, increased, enhanced, augmented, agonized (act as an agonist), or promoted. Modulation can increase the activity by 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc. over the baseline value. Modulation can also decrease the activity below the baseline value and act as an antagonist or inhibitor. Modulation can also normalize the activity to the baseline value.

[0037] The term "immunomodulatory agent" includes, but is not limited to, checkpoint inhibitors, costimulatory molecules, TLR agonists, TNFR superfamily agonists, cyclic dinucleotides, T cell agonists, cytokines, chemokines, oncolytic viruses, and other agents that stimulate or inhibit an immune response.

[0038] As used herein, the phrase "in need thereof" means that a patient has been identified as in need of a particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, an animal or mammal may be in need thereof. In some embodiments, the animal or mammal is in or will be moving into an environment where a particular disease, disorder, or condition is prevalent.

[0039] "Treatment" is an intervention that is intended to prevent the onset of a disorder or to change the course or symptoms of a disease. Thus, "treatment" can refer to therapeutic treatment or prophylactic or preventive means. In some embodiments, the treatment is for therapeutic treatment. In some embodiments, the treatment is for prophylactic or preventive treatment. Persons in need of treatment include those who already suffer from a disorder and those who prevent a disorder.

[0040] As used herein, the phrase "treatment of cancer" refers to inhibition of cancer cell replication, apoptosis, inhibition of cancer spread (metastasis), inhibition of tumor growth, reduction in the number of cancer cells or tumor growth, reduction in the grade of cancer malignancy (e.g., increased differentiation) or improvement of cancer-related symptoms.

[0041] As used herein, the term "alkyl" means a saturated hydrocarbon group that is straight-chain or branched-chain. The alkyl may be heteroalkyl.

[0042] As used herein, the term "substituted alkyl" refers to an alkyl in which one or more hydrogen atoms bonded to the carbon of the alkyl are replaced by another group.

[0043] As used herein, the term "heteroalkyl" refers to an alkyl group in which one or more C atoms are replaced by oxygen, nitrogen, sulfur or combinations thereof.

[0044] As used herein, the term "alkenyl" means a straight-chain or branched alkyl group having one or more double carbon-carbon bonds. The alkenyl may be heteroalkenyl.

[0045] As used herein, the term "substituted alkenyl" refers to an alkenyl in which one or more hydrogen atoms bonded to the carbon of the alkenyl are replaced by another group.

[0046] As used herein, the term "heteroalkenyl" refers to an alkenyl group in which one or more C atoms are replaced by oxygen, nitrogen, sulfur, or combinations thereof.

[0047] As used herein, the term "alkynyl" means a straight-chain or branched-chain alkyl group having one or more triple carbon-carbon bonds. Alkynyl can be heteroalkynyl.

[0048] As used herein, the term "substituted alkynyl" refers to an alkynyl in which one or more hydrogen atoms bonded to the carbon of the alkynyl are replaced by another group.

[0049] As used herein, the term "heteroalkynyl" refers to an alkynyl group in which one or more C atoms are replaced by oxygen, nitrogen, sulfur, or combinations thereof.

[0050] As used herein, the term "aryl" means a monocyclic, bicyclic, or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbon. In some embodiments, the aryl group has 6 to 20 carbon atoms or 6 to 10 carbon atoms. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, benzyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, tetrahydronaphthyl, etc. Aryl can be heteroaryl.

[0051] As used herein, the term "substituted aryl" refers to an aryl in which one or more hydrogen atoms bonded to any carbon atom are replaced by one or more functional groups.

[0052] As used herein, the term "heteroaryl" means an aromatic heterocyclic ring having up to 20 ring-forming atoms (e.g., C) and having at least one heteroatom ring member (ring-forming atom) such as sulfur, oxygen, or nitrogen. In some embodiments, the heteroaryl group has at least one or more heteroatom ring-forming atoms, each of which is independently sulfur, oxygen, or nitrogen.

[0053] As used herein, the term "arylalkyl" means a C1-6 alkyl substituted by aryl.

[0054] As used herein, the term "heterocyclic ring" means a 5- to 7-membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic ring system consisting of carbon atoms, any of the rings may be saturated or unsaturated, 1 to 3 heteroatoms selected from N, O and S, and the N and S heteroatoms may be optionally oxidized, the N heteroatoms may be optionally quaternized, and the heterocyclic ring containing any of the bicyclic groups defined above is fused to a benzene ring.

[0055] Disclosed herein are methods of treating a subject having cancer. In some embodiments, a method of treating a subject having cancer may include administering a therapeutically effective amount of a compound that promotes readthrough of premature termination codons (PTCs) of mRNA and inhibits nonsense-mediated decay (NMD) of mRNA. In some embodiments, a compound having both of these properties is amlexanox.

[0056] In some aspects, a method of treating a subject having cancer may include administering a therapeutically effective amount of an amlexanox compound.

[0057] In some aspects, amlexanox ((2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid) is represented by the following formula I,

Chemical formula

[0058] In some embodiments, anlexanox or a homolog thereof is represented by Formula II,

Chem.

[0059] In some aspects, anlexanox may be deuterated anlexanox represented by the following Formula III:

Chem.

[0060] In some embodiments, anlequinox is administered to a subject having a cancer selected from the group consisting of colon carcinoma, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, Merkel cell carcinoma, craniopharyngioma, ependymoma, pineal body, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, for example, acute lymphocytic leukemia, acute myelogenous leukemia, chronic leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease. In some embodiments, administration of anlequinox will induce the expression of neoantigens in these cancers, thereby inducing an immune response in the subject.

[0061] While not wishing to be bound by theory, cancers with a high frequency of nonsense mutations are thought to be more sensitive to anlexanox, inhibitors of the NMD complex, and other agents disclosed herein. Such cancers include, but are not limited to, uterine corpus endometrial carcinoma (UCEC), bladder urothelial carcinoma (BLCA), gastric cancer, head and neck cancer, kidney renal cell carcinoma, colon adenocarcinoma, esophageal cancer, lung squamous cell carcinoma, rectal adenocarcinoma, pancreatic adenocarcinoma, lung adenocarcinoma, cutaneous melanoma, hepatocellular carcinoma of the liver, etc. Such cancers may have a high remission rate when treated with anlexanox or other NMD complex inhibitors and when compared to standard chemotherapy regimens. In some embodiments, these nonsense mutations are not present in tumor suppressor genes. In some embodiments, administration of anlexanox will induce the expression of neoantigens in these cancers, thereby inducing an immune response in the subject.

[0062] In some aspects, a method of treating a subject's cancer comprises administering anlexanox in combination with a compound that promotes PTC readthrough disclosed herein. In some embodiments, a method of treating a subject's cancer comprises administering anlexanox in combination with a compound that inhibits the NMD complex disclosed herein. In some embodiments, the method comprises administering anlexanox in combination with a compound that promotes PTC readthrough and a compound that inhibits the NMD complex. In some embodiments, the methods disclosed herein comprise generating an immune response in an individual by inducing the expression of neoantigens on the surface of abnormal (e.g., proliferative) cells or cancer cells.

[0063] In some aspects, a method of treating a subject's cancer comprises administering anlexanox in combination with a molecule that inhibits at least one immune checkpoint protein disclosed herein. In some embodiments, the method comprises administering a therapeutically effective amount of anlexanox and a therapeutically effective amount of at least one checkpoint inhibitor.

[0064] Immune checkpoints are proteins within the immune system that either raise or lower signals (co-stimulatory molecules). Checkpoint modulators are designed to overcome one of the main ways cancer cells avoid detection by the immune system. T lymphocytes regularly monitor cells for signs of disease. When antigens on the surface of a cell suggest abnormality, T cells initiate an immune response that increases the expression of additional molecules, preventing the immune response from damaging normal tissues in the body. These proteins are known as immune checkpoints.

[0065] Cancer cells often use immune checkpoint proteins to avoid or suppress attacks by the immune system. Thus, the expression of immune checkpoint proteins on the surface of cancer cells prevents immune cells such as T cells from recognizing them as "foreign" or "abnormal". As a result, checkpoint inhibitors are the immune system by T cell recognition.

[0066] Any molecule that can inhibit one or more immune checkpoint proteins can be used in the methods disclosed herein. Such molecules are referred to as checkpoint inhibitors. These include, but are not limited to, antibodies or functional fragments thereof, inhibitory polypeptides, small molecule compounds, and / or inhibitory nucleic acids (antisense oligonucleotides, small interfering RNA (siRNA), small hairpin RNA (shRNA, etc.), and / or catalytic nucleic acids such as ribozymes).Immune checkpoint proteins suitable for targeting with checkpoint inhibitors for use in any of the methods disclosed herein include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276; e.g., MGA271), cytotoxic T-lymphocyte-associated protein 4 (CTLA4; also known as CD152; examples: ipilimumab, AGEN-1884 (Agenus)), programmed cell death ligand 1 (PD-L1; also known as CD274; examples: MDX-1105 (Bristol Myers Squibb), WBP-3155 (C-stone), LY3300054 (Eli Lilly)), programmed cell death protein 1 (PD-1; also known as CD279; e.g., pembrolizumab, SHR-1210 (Incyte), STI-A1110 (Sorrento), REGN2810 (Regeneron), CT-011 (pidilizumab; Curetech), PDR-001 (Novartis), BGB-A317 (BeiGene), TSR-042 (Tesaro), ENUMC-8 (Enumeral), MGD-013 (Macrogenics, bispecific antibody of PD1 and Lag3), B7-H4 (also known as VTCN1), T cell immunoglobulin and mucin domain-containing 3 (TIM3; also known as HAVCR2), B and T lymphocyte attenuator (BTLA; also known as CD272), indoleamine-pyrrole 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR; e.g., lirilumab), lymphocyte activation gene 3 (LAG-3; e.g., BMS-986016), T cell immunoreceptor with Ig and ITIM domains (TIGIT; also known as WUCAM and Vstm3), ILT-3, ILT-4, and / or one or more of the V-domain Ig suppressor of T cell activation (VISTA), including but not limited to these.

[0067] In some embodiments, the checkpoint inhibitor is one or more antagonistic antibodies such as, but not limited to, ipilimumab (Bristol-Myers Squibb), nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), durvalumab (Medimmune), atezolizumab (Genentech / Roche), tremelimumab (Medimmune), and / or avelumab (Pfizer).

[0068] In some embodiments, the immunostimulatory molecule can be used in combination with anlequinox and a checkpoint inhibitor. Immunostimulatory molecules are immunomodulatory factors and belong to members of the tumor necrosis factor (TNF) receptor superfamily and the B7-CD28 superfamily. Non-limiting examples include CD27, GITR, B7-H3, CD28, CD40, interleukin-2 receptor subunit beta (ILR2P; also known as CD122; e.g., NKTR-214), CD137 (also known as TNFRSF9, 4-1BB), and activators induced by lymphocyte activation (ILA), ICOS, and / or OX40 (also known as CD134 and TNFRSF4), but are not limited thereto. Many of these activators are agonist antibodies such as CDX-1127, TGN1412, MEDI0562, MEDI6469, MEDI6383, etc.

[0069] In some embodiments, a method of treating cancer in a subject comprises administering anlequinox in combination with one checkpoint inhibitor disclosed herein. In some embodiments, the method comprises administering anlequinox in combination with two checkpoint inhibitors disclosed herein. In some embodiments, the method comprises administering anlequinox in combination with one checkpoint inhibitor and one immunostimulatory molecule disclosed herein. In some embodiments, the method comprises administering anlequinox in combination with one of the checkpoint inhibitors listed in Table 1 and one of the immunostimulatory molecules listed in Table 1.

Table 1

[0070] In some embodiments, a method of treating a subject's cancer comprises administering anlequinox in combination with one immune co-stimulatory molecule disclosed herein. In some embodiments, the method comprises administering anlequinox in combination with two immune co-stimulatory molecules disclosed herein.

[0071] This method also provides a method of reducing the risk of postoperative complications, which includes administering an effective amount of anlequinox in combination with a molecule that inhibits at least one checkpoint protein before, during, or after surgery, and in certain non-limiting embodiments, in cancer treatment surgery.

[0072] The present disclosure also provides a method for preventing the occurrence of cancer, preventing or delaying recurrence, or reducing the recurrence rate of cancer, which includes directly administering an effective amount of anlequinox described herein in combination with a molecule that inhibits at least one checkpoint protein to a patient in need thereof. In some embodiments, the combination of anlequinox and a checkpoint inhibitor can be used as adjuvant therapy.

[0073] The present disclosure also provides a method of sensitizing a tumor or cancer to one or more other anti-cancer agents, comprising administering anlecanox together with a molecule that inhibits at least one checkpoint protein. The anti-cancer agent can be administered before, overlapping, simultaneously, and / or after the administration of anlecanox and the checkpoint inhibitor. In some embodiments, anlecanox and the checkpoint inhibitor are administered to a subject before cancer treatment, simultaneously with cancer treatment, after treatment, or during remission of cancer. When administered simultaneously, the anlecanox / checkpoint inhibitor and the other anti-cancer agent may be administered in a single formulation or in separate formulations, and in the separate case, optionally, by different modes of administration. Thus, the combination of an anlecanox / checkpoint inhibitor and one or more other anti-cancer agents may act synergistically to fight the tumor or cancer.

[0074] Indeed, administering an effective amount of anlecanox and / or a checkpoint inhibitor to a patient in need of such treatment may reduce the dosage of another anti-cancer agent having clinically significant efficacy. In the absence of administration of anlecanox and / or a checkpoint inhibitor, such efficacy of dose reduction of other anti-cancer agents may not be observed. Thus, the present invention provides a method for treating a tumor or cancer, comprising administering a reduced dosage of one or more other anti-cancer agents.

[0075] In some embodiments, the anti-cancer agent may be tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, megestrol acetate, anastrozole, letrozole, vorazole, exemestane, flutamide, nilutamide, bicalutamide, cyproterone acetate, goserelin acetate, leuprolide, finasteride, Herceptin, methotrexate, 5-fluorouracil, cytosine arabinoside, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mitramycin, cisplatin, carboplatin, melphalan, chlorambucil, busulfan, cyclophosphamide, ifosfamide, nitrosourea, thiotepa, vincristine, taxol, taxotere, etoposide, teniposide, amsacrine, irinotecan, topotecan, epothilone, gefitinib, erlotinib, sorafenib, an angiogenesis inhibitor, an EGF inhibitor, a VEGF inhibitor, a CDK inhibitor, a cytokine, a Her1 and Her2 inhibitor, or a monoclonal antibody.

[0076] In another embodiment, anlequinox and the checkpoint inhibitor are administered in combination with a radiotherapy regimen. The treatment may also include surgery and / or chemotherapy. For example, anlequinox and the checkpoint inhibitor can be administered in combination with radiotherapy and cisplatin (Platinol), fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin), and / or paclitaxel (Taxol). Treatment with anlequinox / checkpoint inhibitor enables the use of low-dose radiotherapy and / or less frequent radiotherapy, for example, reducing the incidence of severe sore throat that can interfere with swallowing function and cause undesirable weight loss or dehydration.

[0077] In some embodiments disclosed herein, there is a method of inhibiting tumor growth in an individual by administering anlecanox and one or more molecules (antibodies, such as monoclonal antibodies, etc.) that inhibit immune checkpoint proteins. The combination of anlecanox added to an immune checkpoint inhibitor is effective or more effective in suppressing tumor growth compared to a combination of two or more antibody-based immune checkpoint inhibition therapies administered without the combination of anlecanox. In addition, administration of a combination of anlecanox and a molecule that inhibits an immune checkpoint protein according to the methods described herein results in a reduction in side effects and adverse events (e.g., any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% is only for two or more antibody-based immune checkpoint inhibition therapies (e.g., a combination of an anti-PD-1 antibody and an anti-CTLA-4 antibody)).

[0078] In some aspects, the method comprises administering anlecanox in combination with a molecule that inhibits one or more of PD-1, PD-L1, and CTLA-4. In some embodiments, anlecanox of formula I is administered in combination with a molecule that inhibits one or more of PD-1, PD-L1, and CTLA-4. In some embodiments, anlecanox of formula II is administered in combination with a molecule that inhibits one or more of PD-1 and CTLA-4. In some embodiments, anlecanox of formula III is administered in combination with a molecule that inhibits one or more of PD-1, PD-L1, and CTLA-4. In some embodiments, the molecule that inhibits PD-1, PD-L1, and CTLA-4 is an antibody.

[0079] In some embodiments, the method comprises administering anlexanox in combination with an anti-PD-1 antibody. In some embodiments, the method comprises administering anlexanox in combination with an anti-PD-L1 antibody. In some embodiments, the method comprises administering anlexanox in combination with an anti-CTLA-4 antibody. In some embodiments, the method comprises administering anlexanox in combination with anti-PD-1 and anti-CTLA-4 antibodies. In some embodiments, the method comprises administering anlexanox in combination with anti-PD-L1 and anti-CTLA-4 antibodies.

[0080] In some embodiments, anlexanox is administered to an individual at any of the following ranges: about 0.5 to about 1 mg / kg, about 0.5 to about 2 mg / kg, about 0.5 to about 3 mg / kg, about 0.5 to about 4 mg / kg, about 0.5 to about 5 mg / kg, about 0.5 to about 10 mg / kg, about 0.5 to about 20 mg / kg, about 0.5 to about 50 mg / kg, about 1 to about 10 mg / kg, about 1 to about 50 mg / kg, about 10 to about 100 mg / kg, about 10 to about 150 mg / kg, about 50 to about 175 mg / kg, about 175 to about 200 mg / kg, about 200 to about 225 mg / kg, about 225 to about 250 mg / kg, about 250 to about 300 mg / kg, about 300 to about 350 mg / kg, about 350 to about 400 mg / kg, about 400 to about 450 mg / kg, or about 450 to about 500 mg / kg. The dosage may be daily, every two days, every three days, every four days, every five days, weekly, every two weeks, every three weeks, or every four weeks until remission.

[0081] In some embodiments, the checkpoint inhibitor is administered at a dosage of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.3 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 33.3 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg.

[0082] In preferred embodiments, the antibodies against PD-1, PD-L1 and CTLA-4 are administered at a dosage of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, or 30 mg / kg. The dosage may be daily, every two days, every three days, every four days, every five days, weekly, every two weeks, every three weeks, every four weeks, etc. until remission.

[0083] In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered every three days at a dose of about 1 mg / kg to about 15 mg / kg.

[0084] In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered weekly at a dose of about 1 mg / kg to about 15 mg / kg.

[0085] In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered every ten days at a dose of about 1 mg / kg to about 15 mg / kg.

[0086] In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered every two weeks at a dose from about 1 mg / kg to about 15 mg / kg.

[0087] In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered every three weeks at a dose of about 1 mg / kg to about 15 mg / kg.

[0088] In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered every four weeks at a dose of about 1 mg / kg to about 15 mg / kg.

[0089] In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered every eight weeks at a dose of about 1 mg / kg to about 15 mg / kg.

[0090] In one embodiment, anlequinox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every 12 weeks.

[0091] In one embodiment, anlequinox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every 3 days.

[0092] In one embodiment, anlequinox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every week.

[0093] In one embodiment, anlequinox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every 10 days.

[0094] In one embodiment, anlequinox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every 2 weeks.

[0095] In one embodiment, anlequinox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every 3 weeks.

[0096] In one embodiment, anlequinox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every 4 weeks.

[0097] In one embodiment, anrulexanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every 8 weeks.

[0098] In one aspect, anrulexanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 1 mg / kg to about 15 mg / kg every 12 weeks.

[0099] In certain aspects, anrulexanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 mg every 3 days. In one embodiment, anrulexanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 100 mg to about 1500 mg every 3 days.

[0100] In one aspect, anrulexanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 mg per week. In one embodiment, anrulexanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 100 mg to about 1500 mg per week.

[0101] In one aspect, anrulexanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 mg every 2 weeks. In one embodiment, anrulexanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 100 mg to about 1500 mg every 2 weeks.

[0102] In one aspect, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg daily, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 mg every 4 weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg daily, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 100 mg to about 1500 mg every 4 weeks.

[0103] In one aspect, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg daily, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 mg every 8 weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg daily, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 100 mg to about 1500 mg every 8 weeks.

[0104] In one aspect, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg daily, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 mg every 12 weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg daily, and the anti-PD-1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 100 mg to about 1500 mg every 12 weeks.

[0105] In one aspect, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg daily, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 mg every 3 days. In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 100 mg to about 1500 mg every 3 days.

[0106] In certain embodiments, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 mg per week. In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered weekly at a dose of about 100 mg to about 1500 mg.

[0107] In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 mg every two weeks. In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered every two weeks at a dose of about 100 mg to about 1500 mg.

[0108] In certain embodiments, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 mg every four weeks. In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered every four weeks at a dose of about 100 mg to about 1500 mg.

[0109] In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 mg every eight weeks. In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered every eight weeks at a dose of about 100 mg to about 1500 mg.

[0110] In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 every 12 weeks. In one embodiment, anleraxanox is administered daily at a dose of about 1 mg / kg to 50 mg / kg, and the anti-PD-L1 antibody and the anti-CTLA-4 antibody are each administered at a dose of about 100 mg to about 1500 mg every 12 weeks.

[0111] In certain embodiments, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-CTLA-4 antibody is administered at a dose of about 100 mg to about 1500 every three days. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 every three days. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 every three days.

[0112] In one aspect, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-CTLA-4 antibody is administered at a dose of about 100 mg to about 1500 per week. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 per week. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 per week.

[0113] In one aspect, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-CTLA-4 antibody is administered at a dose of about 100 mg to about 1500 every two weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 every two weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 every two weeks.

[0114] In one aspect, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-CTLA-4 antibody is administered at a dose of about 100 mg to about 1500 every four weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 every four weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 every four weeks.

[0115] In one aspect, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-CTLA-4 antibody is administered at a dose of about 100 mg to about 1500 every eight weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 every eight weeks. In one embodiment, anleraxanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 every eight weeks.

[0116] In one aspect, anlequinox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-CTLA-4 antibody is administered at a dose of about 100 mg to about 1500 every 12 weeks. In one embodiment, anlequinox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-1 antibody is administered at a dose of about 100 mg to about 1500 every 12 weeks. In one embodiment, anlequinox is administered at a dose of about 1 mg / kg to 50 mg / kg per day, and the anti-PD-L1 antibody is administered at a dose of about 100 mg to about 1500 every 12 weeks.

[0117] In another embodiment, anlequinox in combination with a molecule that inhibits one or more immune checkpoint proteins administered according to any of the methods disclosed herein provides at least about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 33.3%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% tumor suppression effect against tumors not treated with anlequinox and checkpoint inhibitors.

[0118] The compounds and molecules disclosed herein can be administered by conventional methods via any route by which they are active. Administration can be systemic, parenteral, topical, or oral. For example, administration can be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or intraocular routes, or by intravaginal, inhalation, depot injection, or implant, but is not limited thereto. Thus, the mode of administration of the molecules of the present disclosure (either alone or in combination with other pharmaceuticals) can be sublingual, injectable (including short-acting, depot, implant, and pellet forms injected subcutaneously or intramuscularly), but is not limited thereto), or by the use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal agents such as patches and creams. In some embodiments, anlequinox and checkpoint inhibitors can be administered directly to the tumor site.

[0119] In some aspects, anlequinox and checkpoint inhibitors can be administered directly to the tumor site. In some embodiments, anlequinox is administered to the tumor site and the checkpoint inhibitor is administered intravenously. In some embodiments, anlequinox is administered to the tumor site, followed by oral administration of anlequinox and intravenous administration of the checkpoint inhibitor.

[0120] In some embodiments, the method comprises orally administering anlequinox together with intratumoral injection of an immunomodulatory agent selected from oncolytic adenoviruses, CDNs, TLR agonists, TNFR superfamily agonists, and epigenetic regulatory compounds. In some embodiments, the method comprises orally administering anlequinox in combination with intratumoral injection of an immunomodulatory agent (oncolytic adenovirus, CDN, TLR agonist, TNFR superfamily agonist, and epigenetic regulatory compound), and further intravenous administration of a checkpoint inhibitor. In some embodiments, the method comprises orally administering anlequinox in combination with intratumoral injection of an immunomodulatory agent (oncolytic adenovirus, CDN, TLR agonist, TNFR superfamily agonist, and epigenetic regulatory compound), and further intravenous administration of an immunostimulatory molecule. In some embodiments, the method comprises orally administering anlequinox in combination with intratumoral injection of chemotherapy and an immunomodulatory agent. In some embodiments, the method comprises orally administering anlequinox in combination with intratumoral injection of radiotherapy and an immunomodulatory agent.

[0121] In some embodiments, anlequinox and a checkpoint inhibitor are administered intravenously. In some embodiments, anlequinox is administered orally and a checkpoint inhibitor is administered intravenously. In some embodiments, anlequinox and a checkpoint inhibitor are administered orally. In some embodiments, anlequinox and a checkpoint inhibitor are administered simultaneously or sequentially. For example, anlequinox can be administered first, followed by administration of antibodies against PD-1, PD-L1, CTLA-4. In some embodiments, antibodies against PD-1, PD-L1, and CTLA-4 can be administered first, followed by administration of anlequinox.

[0122] The specific mode of administration depends on the indication. The selection of the specific route of administration and dosing schedule is adjusted or titrated by the clinician according to methods known to the clinician in order to obtain an optimal clinical response. The amount of the compound administered is a therapeutically effective amount. The dosage administered depends on the characteristics of the subject being treated, such as the specific animal or human being treated, age, weight, health, the type of co-therapy if any, and the frequency of treatment, and can be readily determined by one of ordinary skill in the art (e.g., by a clinician).

[0123] In another embodiment, a method of killing cancer cells can include contacting the cancer cells with a composition comprising anlexanox in combination with a molecule that inhibits at least one immune checkpoint protein. In some embodiments, the method can be in vitro or in vivo. In some embodiments, the molecule that inhibits the checkpoint protein can be an antibody against PD-1, PD-L1, and CTLA-4. In some embodiments, the cancer cells are selected from colon carcinoma, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical carcinoma, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, Merkel cell carcinoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, e.g., acute lymphocytic leukemia, acute myelogenous leukemia, chronic leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease.

[0124] In a further aspect, a method of killing cancer stem cells can include contacting the cancer stem cells with a composition comprising anlexanox in combination with a molecule that inhibits at least one immune checkpoint protein. In some embodiments, the method can be in vitro or in vivo. In some embodiments, the molecule that inhibits the checkpoint protein can be an antibody against PD-1, PD-L1, and CTLA-4. In some embodiments, the method includes contacting the cancer stem cells with anlexanox and a molecular adjuvant such as a TNF receptor superfamily (TNFRSF) agonist, GM-CSF, a Toll-like receptor (TLR) ligand, and an intracellular DNA sensor agonist such as cyclic dinucleotides (CDNs) and CpG motifs.

[0125] In a further aspect, a method of inducing the expression of one or more neoantigens on the surface of abnormal cells includes contacting the abnormal cells with anlexanox in combination with a molecule that inhibits at least one immune checkpoint protein. In some embodiments, the method can be in vitro or in vivo. In some embodiments, an anlexanox of formula I is contacted in combination with a molecule that inhibits one or more of PD-1, PD-L1, and CTLA-4. In some embodiments, an anlexanox of formula II is contacted in combination with a molecule that inhibits one or more of PD-1 and CTLA-4 below. In some embodiments, an anlexanox of formula III is contacted in combination with a molecule that inhibits one or more of: PD-1, PD-L1, and CTLA-4 below. In some embodiments, the molecules that inhibit PD-1, PD-L1, and CTLA-4 are antibodies.

[0126] In another embodiment, a method of generating an immune response in an individual in need thereof promotes read-through of premature termination codons (PTCs) in mRNA and inhibits nonsense-mediated decay (NMD) of the mRNA. In some embodiments, the compound is anlexanox. In some embodiments, the method further comprises administering a molecule that inhibits an immune checkpoint protein. In some embodiments, the checkpoint proteins are PD-1 and CTLA-4. In some embodiments, the method comprises administering anlexanox in combination with a molecule that inhibits one or more of the following: PD-1, PD-L1, and CTLA-4. In some embodiments, an anlexanox of formula I is administered in combination with a molecule that inhibits one or more of PD-1, PD-L1, and CTLA-4. In some embodiments, an anlexanox of formula II is administered in combination with a molecule that inhibits one or more of PD-1 and CTLA-4. In some embodiments, an anlexanox of formula III is administered in combination with a molecule that inhibits one or more of the following: PD-1, PD-L1, and CTLA-4. In some embodiments, the molecule that inhibits PD-1, PD-L1, and CTLA-4 is an antibody.

[0127] In some aspects, a method of generating an immune response in an individual in need thereof comprises administering to the individual a therapeutically effective amount of a compound that promotes PTC read-through. In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a compound that promotes PTC read-through.

[0128] Any compound that can promote the read-through of mRNAs having a PTC is suitable for use in the present method. To date, most of the reported PTC read-through compounds that are active in mammalian cells have belonged to the aminoglycoside class of antibiotics. Certain types of aminoglycosides can lead through PTC mutations to ribosomes via the random insertion of amino acids by nearly the same type of transfer RNA (tRNA). The therapeutic potential of aminoglycosides is evaluated in the laboratory for various genetic models such as cystic fibrosis. In some embodiments, the PTC read-through compound is ataluren (previously known as PTC124).

[0129] In some embodiments, the compound that promotes PTC read-through is a 1,2,4-oxadiazole benzoic acid compound of formula IV:

Chemical formula

[0130] In a further aspect, the compound that promotes PTC lead-through is 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid.

[0131] In certain aspects, the compounds for promoting PTC lead-through for use in the methods disclosed herein are:

Chemical formula

[0132] In some embodiments, compounds that facilitate PTC readthrough for use in any of the methods disclosed herein include, but are not limited to, aminoglycosides such as amikacin, G418 (geneticin), gentamicin, or paromomycin. In other embodiments, the PTC readthrough compounds are aminoglycoside derivatives such as, but not limited to, NB54, NB74, NB84, or TC007. In further embodiments, the compounds for promoting PTC readthrough are non-aminoglycosides such as, but not limited to, negamycin or tylosin.

[0133] In a further aspect, compounds for promoting PTC readthrough for use in the methods disclosed herein are:

Chemical formula

[0134] Further PTC readthrough agents suitable for use in the methods disclosed herein include, but are not limited to, isepamicin, tobramycin, RTC#1, RTC#2, RTC#3, RTC#4, RTC#7, RTC#9, RTC#10, RTC#11, RTC#16, RTC#17, critocin, macrolide spiramycin, macrolide josamycin, macrolide tylosin, NB30, streptomycin, hygromycin, promicon, ribidomycin, TC001, TC003, TC032, JL022, JL023, hygromycin B, kanamycin A, kanamycin B and its "JL" derivatives, neomycin and its "TC" derivatives, paromamine and its synthetic derivatives, paromomycin and its "NB" derivatives, or oleandomycin, negamycin, sisomicin, garamine, 2-deoxystreptamine, gentamicin, gentamicin B1, gentamicin C1, gentamicin C1a, gentamicin C2, gentamicin C2a, and gentamicin C2b.

[0135] In yet other embodiments, the compounds for promoting PTC lead-through for use in the methods disclosed herein include, but are not limited to, [Chemical formula] and may be such as negamycin derivatives.

[0136] In some embodiments, the compound for promoting PTC lead-through is of Formula V: [Chemical formula] wherein:

[0137] In the formula: A1 is C, CH or N, V and X are independently selected from N or C, W is selected from N, C, or CH, wherein at least one of V, W, or X is N, and when W is N, at least one of V or X is also N; Y and Z are independently selected from N, C, C-Rc, C=O, C=S, where Rc is H, CH3, or NH2, provided that when one of Y or Z is C=O or C=S, the other can also be selected from NH, S, or O, R1 is a carbonyl group optionally substituted with carboxy, cyano, or a C1-C4 alkoxy group, R2 is absent or nitro, Ar1 is C1-C4 alkyl optionally substituted with an R group; C6-C10 aryl optionally substituted with 1, 2, or 3 independently selected R groups; a 5-10 membered heterocycle optionally substituted with 1, 2, or 3 independently selected R groups; together with the heterocycle to which Ar2 and Ar1 and Ar2 are attached, forms a ring structure selected from Ar1-2, or together with the heterocycle to which Ar3 and Ar1 and Ar3 are attached, forms a ring structure selected from Ar1-3, Ar2 is absent or, together with the heterocyclic ring to which Ar1 and Ar1 and Ar2 are attached, forms a ring structure selected from Ar1-2, Ar3 is absent or, together with the heterocyclic ring to which Ar1 and Ar1 and Ar3 are attached, forms a ring structure selected from Ar1-3, Ar4 is absent or is C1-C4 alkyl, C1-C4 alkoxy, or C1-C4 thioalkyl, any of which together with A1 forms a 4- to 7-membered carbocyclic or heterocyclic ring, R is hydrogen; an a-Ra group, or two R groups (R may contain an oxy group) together with the phenyl or heterocyclic ring to which they are attached form a ring structure selected from RR; wherein: Ar1-2 and Ar1-3 are selected from 11- to 14-membered hetero tricyclic ring structures optionally substituted with one or more halogens, C1-C4 alkyl groups, C1-C4 haloalkyl groups, C1-C4 alkoxy groups optionally substituted with halogen, or are amino groups optionally substituted with a carbonyl group substituted with a C1-C4 alkoxy group, a C1-C4 haloalkoxy group, or a C1-C4 alkyl group, RR is a 9- to 10-membered bicyclic ring structure optionally substituted with one or more halogens, C1-C4 alkyl groups, C1-C4 haloalkyl groups, C1-C4 alkoxy groups, oxo groups, or C1-C4 haloalkoxy groups, Ra is selected from the group consisting of: a hydroxy group; a halogen; C1-C4 alkyl optionally substituted with one or more independently selected halogens or hydroxy groups; C1-C4 alkoxy optionally substituted with one or more independently selected halogens or phenyl groups; C4-C8 cycloalkyl optionally substituted with one or more independently selected C1-C4 alkyl groups; -Rb group; -O-Rb group; a 4- to 6-membered heterocycle optionally substituted with one or more independently selected C1-C4 alkyl, oxo, or -Rb groups; a 9- to 10-membered heterocycle having two ring structures; carbonyl optionally substituted with hydroxy, C1-C4 alkyl, or C1-C4 alkoxy groups; carbamoyl optionally substituted with one or two C1-C4 alkyl groups; nitro group; cyano group; thio optionally substituted with hydroxy, C1-C4 alkyl, or -Rb group; sulfonyl optionally substituted with hydroxy, C1-C4 alkyl, or -Rb group; or amino, and aminocarbonyl groups optionally substituted with an aminosulfonyl group optionally substituted with hydroxy, C1-C4 alkyl, or -Rb group, and optionally substituted with one or two independently selected C1-C4 alkyl, sulfonyl, or carbonyl groups, and optionally substituted with C1-C4 alkyl, C1-C4 haloalkyl, benzyloxy, or -Rb groups, and optionally substituted with an amino group, -Rb is hydroxy, halogen, a C1-C4 alkyl group, a C1-C4 haloalkyl group, a C1-C4 alkoxy group, or an amino group optionally substituted with one or more C1-C4 alkyl groups; or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, racemate, stereoisomer, or polymorph of the compound,

[0138] In some embodiments, the compound that promotes PTC lead-through is

Chemical formula

[0139] In some embodiments, the compound that promotes PTC readthrough is of formula VI:

Chemical formula

[0140] In certain embodiments, Z is a substituted aryl. In another embodiment, Z is a halo-substituted aryl. In another embodiment, Z is a fluoro-substituted aryl. In another embodiment, Z is a substituted phenyl. In another embodiment, Z is a halo-substituted phenyl. In another embodiment, Z is a fluoro-substituted phenyl.

[0141] In some embodiments, the compound that facilitates PTC lead-through is of formula VII:

Chemical formula

[0142] In some embodiments, the compound that promotes PTC lead-through is of formula VIII:

Chemical formula

[0143] In some embodiments, the compound that promotes PTC lead-through is of formula IX:

Chemical formula

[0144] In one embodiment, the compound that promotes PTC lead-through is of formula X:

Chemical formula

[0145] In some embodiments, the following compounds are compounds that promote PTC readthrough:

Chemical formula

Chemical formula

Chemical formula

[0146] In certain embodiments, the compounds that facilitate PTC lead-through are of formula XI:

Chemical formula

[0147] In certain embodiments, the compound that promotes PTC lead through is of formula XII:

Chemical formula

[0148] Other compounds suitable for promoting PTC lead-through for use in the present invention can be found in U.S. Patent Application Publication Nos. 2015 / 0274674, 2015 / 0051251, 2013 / 0217717, 2012 / 0087896, 2011 / 0046136, 2011 / 0003843, 2010 / 0093867, 2008 / 0207538, 2007 / 0203123, 2006 / 0166926, 2006 / 0167263; International Application Publication Nos. WO2015 / 134711, WO2015 / 109248, WO2013 / 142346, WO2012 / 016930, WO2008 / 101935, WO2004 / 009558, WO2004 / 009610, WO2004 / 009533, and WO2014 / 055644, and U.S. Patents Nos. 8,163,782 and 6,992,096, the disclosures of each of which are incorporated herein by reference in their entirety.

[0149] In some embodiments, a method of treating a subject's cancer comprises administering a compound that promotes PTC lead-through as disclosed herein in combination with a molecule that inhibits at least one immune checkpoint protein as disclosed herein. In some embodiments, the method comprises administering a therapeutically effective amount of a compound that promotes PTC lead-through and a therapeutically effective amount of at least one checkpoint inhibitor.

[0150] In some embodiments, the method comprises administering a compound that promotes PTC lead-through in combination with an anti-PD-1 antibody. In some embodiments, the method comprises administering a compound that promotes PTC lead-through in combination with an anti-PD-L1 antibody. In some embodiments, the method comprises administering a compound that promotes PTC lead-through in combination with an anti-CTLA-4 antibody. In some embodiments, the method comprises administering a compound that promotes PTC lead-through in combination with anti-PD-1 and anti-CTLA-4 antibodies. In some embodiments, the method comprises administering a compound that promotes PTC lead-through in combination with anti-PD-L1 and anti-CTLA-4 antibodies.

[0151] In some embodiments, the amount of the compound for promoting PTC lead-through administered to the individual is in the following ranges: about 0.5 to about 5 mg / kg, about 5 to about 10 mg / kg, about 10 to about 15 mg / kg, about 15 to about 20 mg / kg, about 20 to about 25 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 50 to about 75 mg / kg, about 50 to about 100 mg / kg, about 75 to about 100 mg / kg, about 100 to about 125 mg / kg, about 125 to about 150 mg / kg, about 150 to about 175 mg / kg, about 175 to about 200 mg / kg, about 200 to about 225 mg / kg, about 225 to about 250 mg / kg, about 250 to about 300 mg / kg, about 300 to about 350 mg / kg, about 350 to about 400 mg / kg, from about 400 to about 450 mg / kg, or from about 450 to about 500 mg / kg.

[0152] In some embodiments, the checkpoint inhibitor is administered at a dose of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.3 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 33.3 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg.

[0153] In preferred embodiments, the antibodies against PD-1, PD-L1 and CTLA-4 are administered at a dose of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, or 30 mg / kg. The dosage is daily, every two days, every three days, every four days, every five days, weekly, every two weeks, every three weeks, every four weeks, etc. until remission.

[0154] In some embodiments, a method of generating an immune response in an individual in need thereof comprises administering to the individual a therapeutically effective amount of a compound that inhibits the NMD complex. In some embodiments, a method of treating cancer in a subject comprises administering to the individual a therapeutically effective amount of a compound that inhibits the NMD complex.

[0155] As used herein, the term "NMD degradosome" refers to any one of the intracellular proteins involved in NMD of mRNAs having a PTC (including but not limited to one or more of UPF1, UPF2, UPF3, UPF3BI, RNPS1, eIF4AIII, MLN51, Y14 / MAGOH heterodimer, RENT1, RENT2, SMG-1, SMG-5, SMG-6, and / or SMG-7). As such, a compound inhibits the function of one or more NMD degradosome proteins, thereby allowing PTC-bearing mRNAs to be translated into polypeptides.

[0156] Candidate compounds can be, but are not limited to, small molecule chemical compounds (such as any of the small molecules described above), antibodies, proteins, or any combination thereof.

[0157] In one embodiment, an inhibitor of the NMD complex is a compound of formula XIII:

Chemical formula

[0158] In one embodiment, an inhibitor of the NMD complex is a compound of formula XIV:

Chemical formula

[0159] Other non-limiting examples of NMD complex inhibitors include the following:

Chemical formula

[0160] In one aspect, the compound is not an inhibitory nucleic acid (such as, but not limited to, an antisense oligonucleotide or a small interfering RNA (siRNA)). In another embodiment, the compound is any of the compounds disclosed in U.S. Patent Application Publication No. 2013 / 0224237.

[0161] Embodiments of the invention include the generation of a multi-domain molecule comprising a target-specific domain and at least one domain that modulates the expression and function of molecules associated with the nonsense-mediated decay pathway.

[0162] A method of treating a patient includes administering a therapeutically effective amount of a multi-domain bioactive molecule. In a preferred embodiment, the multi-domain molecule comprises an oligonucleotide, e.g., an interfering RNA (RNAi), and a cell-binding ligand that binds to cells in the tumor stroma (such as endothelial cells, fibroblasts or immune cells, etc.) for specifically targeting the desired cells in vivo. The cell-binding ligand is, for example, an integrin, glucose-regulated protein 78, neuropilin, a growth factor receptor, such as a VEGF receptor, and the oligonucleotide is generated against a specific product expressed by the target cells that is specific for inhibition of the nonsense-mediated decay pathway and related molecules. Inhibition of the nonsense-mediated decay pathway allows for upregulation of existing antigens and / or induction or enhancement of antigenicity of the target cells and / or induction of new antigens not previously expressed by the target cells, ultimately leading to destruction by the immune system.

[0163] In other embodiments, a composition that inhibits the nonsense-mediated decay (NMD) pathway in a patient in vivo comprises at least one first domain that specifically binds to at least one tumor cell target or normal cell target of the tumor stroma and a second domain that is specific for a molecular component of at least one nonsense-mediated decay pathway. Here, the second domain includes an antisense oligonucleotide molecule, a peptide, a protein, a nucleic acid, an organic or inorganic molecule, and inhibits the nonsense-mediated decay pathway.

[0164] In some embodiments, the oligonucleotide molecule of the second domain comprises at least one of small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide, small temporal RNA (stRNA), short hairpin RNA (shRNA), or a combination thereof.

[0165] In some embodiments, the oligonucleotide molecule of the second domain inhibits the function and / or expression of at least one factor associated with the nonsense-mediated mRNA decay (NMD) pathway, which comprises at least one of RENT1, RENT2, eIF4A, UPF1, UPF2, UPF3B, RNPS1, Y14, MAGOH, NMD1, SMG, or a combination thereof.

[0166] In some embodiments, the first domain specifically or selectively binds to any desired target. Preferably, the target is a tumor cell target, a normal cell target, a cell in the tumor stroma, or a combination thereof. Preferably, the first domain specifically binds to a tumor or normal cell target having vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR-2), Tie2; fibronectin, vitronectin, collagen, laminin, fibroblast antigen, fibroblast activation protein (FAP), glucose-regulated protein 78 (GRP78), stromal cell-derived factor 1 (SDF-1), MCP-1, MIP-1α, MIP-1β, RANTES, exotaxin IL-8, C3a, P-selectin, E-selectin, LFA-1, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, neuropilin, MAdCAM-1, neuropilin-1, α4 integrin, α5 integrin, or beta defensins 3 and 4.

[0167] In some scenarios, the compounds / MAG0H heterodimer, RENT1, RENT2, SMG-1, SMG-5, SMG-6 and / or SMG-7 that bind (such as preferentially bind) to one or more NMD degradation complex proteins (UPF1, UPF2, UPF3, UPF3BI, RNPS1, eIF4AIII, MLN51, Y14, etc.) are antibodies. In some embodiments, the antibodies are NMD degradation complex protein antagonists and can inhibit NMD.

[0168] Variants of the antibody can also be created based on information known in the art without substantially affecting the activity of the antibody. For example, in the case of an antibody, at least one amino acid residue within the antibody molecule may be replaced with a different residue. In the case of antibodies, the hypervariable regions are generally the sites of greatest interest for substitution mutagenesis, although changes in the framework region (FR) are also contemplated.

[0169] In the case of an antibody, one type of substitution variant involves the substitution of one or more hypervariable region residues of the parental antibody (e.g., a humanized antibody or a human antibody). Generally, the variants resulting from selection for further development will have improved biological properties compared to the parental antibody from which they are generated. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (such as 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibodies thus generated are presented from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding.

[0170] Nucleic acid molecules encoding amino acid sequence variants of antibodies can be prepared by a variety of methods known in the art. These methods include isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and preparation by cassette mutagenesis of variants or non-variant versions of an initially prepared antibody, but are not limited thereto.

[0171] In some aspects, it may be desirable to introduce one or more amino acid modifications into the Fc region of the immunoglobulin polypeptide of the present invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions including the amino acid position of the hinge cysteine.

[0172] In some aspects, compounds / MAGOH heterodimers, RENT1, RENT2, SMG-1, SMG-5, SMG-6 and / or SMG-7) that bind (e.g., preferentially bind) to one or more NMD degradation complex proteins (UPF1, UPF2, UPF3, UPF3BI, RNPS1, eIF4AIII, MLN51, Y14, etc.) are non-antibody binding polypeptides. In some embodiments, the non-antibody binding polypeptide is an NMD degradation complex protein antagonist and can inhibit NMD.

[0173] The binding polypeptide can be chemically synthesized using known polypeptide synthesis methodologies or prepared and purified using recombinant techniques. The binding polypeptide is typically at least about 5 amino acids in length, or at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length and can bind to a target such as any component of the NMD degradation complex, such binding polypeptides are discussed herein.

[0174] In some embodiments, a method of treating cancer in a subject comprises administering a compound that inhibits the NMD complex disclosed herein in combination with a molecule that inhibits at least one immune checkpoint protein disclosed herein. In some embodiments, the method comprises administering a therapeutically effective amount of an NMD complex inhibitor and a therapeutically effective amount of at least one checkpoint inhibitor.

[0175] In some embodiments, the method comprises administering a compound that inhibits the NMD complex in combination with an anti-MD-1 antibody. In some embodiments, the method comprises administering a compound that inhibits the NMD complex in combination with an anti-PD-L1 antibody. In some embodiments, the method comprises administering a compound that inhibits the NMD complex in combination with an anti-CTLA-4 antibody. In some embodiments, the method comprises administering a compound that inhibits the NMD complex in combination with anti-PD-1 and anti-CTLA-4 antibodies. In some embodiments, the method comprises administering a compound that inhibits the NMD complex in combination with anti-PD-L1 and anti-CTLA-4 antibodies.

[0176] In some embodiments, the amount of the compound that inhibits the NMD complex administered to the individual is in the range of any of the following: about 0.5 to about 5 mg / kg, about 5 to about 10 mg / kg, about 10 to about 15 mg / kg, about 15 to about 20 mg / kg, about 20 to about 25 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 50 to about 75 mg / kg, about 50 to about 100 mg / kg, about 75 to about 100 mg / kg, about 100 to about 125 mg / kg, about 125 to about 150 mg / kg, about 150 to about 175 mg / kg, about 175 to about 200 mg / kg, about 200 to about 225 mg / kg, about 225 to about 250 mg / kg, about 250 to about 300 mg / kg, about 300 to about 350 mg / kg, about 350 to about 400 mg / kg, about 400 to about 450 mg / kg, or about 450 to about 500 mg / kg.

[0177] In some embodiments, the checkpoint inhibitor is administered at a dose of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.3 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 33.3 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg.

[0178] In a preferred embodiment, the antibodies against PD-1, PD-L1, and CTLA-4 are administered at a dose of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, or 30 mg / kg. The dosage is daily, every two days, every three days, every four days, every five days, weekly, every two weeks, every three weeks, every four weeks, etc. until remission.

[0179] In some embodiments, a method of treating a subject having cancer may include administering a therapeutically effective amount of a compound that promotes PTC readthrough. In some embodiments, a method of treating a subject having cancer may include administering a therapeutically effective amount of a compound that inhibits the NMD complex. In some embodiments, a method of treating a subject having cancer may include administering a combination of a compound that promotes PTC readthrough and a compound that inhibits the NMD complex. In some embodiments, a method of treating a subject having cancer may include administering a combination of a compound that promotes PTC readthrough and a compound that inhibits the NMD complex, and further administering at least one checkpoint inhibitor disclosed herein.

[0180] In some embodiments, provided herein are methods for generating an immune response and / or inducing the expression of one or more neoantigens on the surface of abnormal cells in an individual in need thereof. NMD is an evolutionarily conserved mRNA surveillance pathway in eukaryotic cells that detects and removes mRNAs containing premature termination codons (PTCs). Without wishing to be bound by theory, upregulation of gene expression when NMD is inhibited in tumor cells contributes to therapeutically useful enhancement of tumor antigenicity, i.e., new products function as effective tumor antigens and can induce an immune response that contributes to tumor rejection. Inhibition is achieved by administering to the individual in need thereof an effective amount of one or both of the compounds that promote the above-described PTC readthrough and inhibition of the NMD degradation complex. In one embodiment, the protein translated from the mRNA after inhibition of PTC readthrough and the NMD degradation complex is a non-functional protein. The effective amount may provide the functionality described below and herein.

[0181] In some embodiments, the amount of the compound that promotes PTC readthrough administered to an individual and the amount of the compound that inhibits the NMD complex are included in any of the following ranges: about 0.5 to about 5 mg / kg, about 5 to about 10 mg / kg, about 10 to about 15 mg / kg, about 15 to about 20 mg / kg, about 20 to about 25 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 50 to about 75 mg / kg, about 50 to about 100 mg / kg, about 75 to about 100 mg / kg, about 100 to about 125 mg / kg, about 125 to about 150 mg / kg, about 150 to about 175 mg / kg, about 175 to about 200 mg / kg, about 200 to about 225 mg / kg, about 225 to about 250 mg / kg, about 250 to about 300 mg / kg, about 300 to about 350 mg / kg, about 350 to about 400 mg / kg, about 400 to about 450 mg / kg, or about 450 to about 500 mg / kg. In some embodiments, the amount of the telomerase inhibitor in a therapeutically effective amount administered to an individual (e.g., in a unit dosage form) ranges from about 5 mg to about 500 mg, such as from about 30 mg to about 300 mg, or from about 50 mg to about 200 mg or from about 10 mg to about 100 mg.

[0182] In some embodiments, the checkpoint inhibitor is administered at a dose of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.3 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 33.3 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, or 50 mg / kg.

[0183] In a preferred embodiment, the antibodies against PD-1, PD-L1 and CTLA-4 are administered at a dose of 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, or 30 mg / kg. The dosing is daily, every two days, every three days, every four days, every five days, weekly, every two weeks, every three weeks, every four weeks, etc. until remission.

[0184] In a further embodiment, treatment with one or more compounds comprising anlexanox, a PTC read-through compound, an NMD complex inhibitor, and a checkpoint inhibitor, by any of the methods disclosed herein, results in at least about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 33.3%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, 99%, or 100% decrease in tumor size compared to tumors not treated with the compounds disclosed herein.

[0185] In some embodiments, treatment with one or more compounds, including anleraxanox, a PTC lead-through compound, an NMD complex inhibitor, and a checkpoint inhibitor, according to any of the methods disclosed herein, results in at least about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 33.3%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any of the CD4+, CD8+, CD3+, and / or CD45+ effector T cell responses (e.g., intratumoral T cell infiltration), as compared to the T cell response of tumors not treated with the compounds disclosed herein.

[0186] In one embodiment, the PTC read-through inhibitor is ataluren (PTC124), and the compound that inhibits nonsense-mediated decay is NMDI14. In another embodiment, the PTC read-through inhibitor (e.g., ataluren) and the compound that inhibits nonsense-mediated decay (e.g., NMDI14) are administered in combination with an antibody against PD-1. In another embodiment, the PTC read-through inhibitor (e.g., ataluren) and the compound that inhibits nonsense-mediated decay (e.g., NMDI14) are administered in combination with an antibody against CTLA-4. In a further embodiment, the combination of a PTC read-through inhibitor (e.g., ataluren) and a compound that inhibits nonsense-mediated decay (e.g., NMDI14) and a single compound that inhibits an immune checkpoint protein (e.g., anti-PD-1 antibody or anti-CTLA-4 antibody) inhibits only tumors more effectively or is effective in inhibiting tumor growth compared to a combination of two or more compounds that inhibit an immune checkpoint protein (e.g., a combination of anti-PD-1 antibody and anti-CTLA-4 antibody).

[0187] Epigenetic regulatory compound In some aspects of any of the methods disclosed herein, the method further comprises administration of one or more epigenetic regulatory compounds. As used herein, "epigenetic" refers to physical changes imposed on the cell on chromosomes and genes, which changes affect the function of DNA and genes within the chromosome and do not change the nucleotide sequence of the DNA in the gene. Representative examples of epigenetic modulation include, but are not limited to, covalent chemical modifications of DNA such as methylation and acetylation, and non-covalent and non-chemical modifications of the binding of DNA-DNA supercoiling and chromosomal proteins such as histones. Representative non-limiting examples of the results of epigenetic changes include increases or decreases in the level of RNA, thereby changing the protein product produced by a particular gene and / or the way in which a transcription factor binds to a gene promoter.

[0188] Epigenetic regulatory compounds suitable for use in the methods of the present invention include, but are not limited to, one or more of histone deacetylase (HDAC) inhibitors, azacitidine, BET inhibitors, EZH2 inhibitors, and / or dotlL. In some embodiments, the epigenetic regulatory compound is one or more of vorinostat (Merck), romidepsin (Celgene), decitabine (Otsuka), and 5-azacitidine (Celgene), panobinostat (Novartis), or belinostat (Spectrum).

[0189] Cancer treatment The methods of the present invention can be carried out in an adjuvant setting. An "adjuvant setting" refers to a clinical setting in which an individual has a history of a proliferative disease, particularly cancer, and has generally (but not necessarily) been treated with therapy, including surgery, radiation therapy, and / or chemotherapy. However, due to the history of the proliferative disease, these individuals are considered to be at risk of developing the disease or may have a detectable and / or microscopic disease. Treatment or administration in an "adjuvant setting" refers to subsequent treatment modalities.

[0190] The methods provided herein can also be carried out in a "neoadjuvant setting", i.e., the method can be carried out prior to primary / definitive therapy. In some aspects, the individual has been previously treated. In other aspects, the individual has not been previously treated. In some embodiments, the treatment is a first-line therapy.

[0191] In some embodiments, any of the methods described herein includes administering to a subject in need thereof a therapeutically effective amount of an anti-cancer therapy. As used herein, a "therapeutically effective amount" or "therapeutically effective dose" of an anti-cancer therapy is an amount sufficient to produce a beneficial or desired result. For therapeutic use, beneficial or desired results include alleviation of one or more symptoms attributable to cancer, improvement in the quality of life of people suffering from cancer, reduction in the dosage of other medications required for the treatment of cancer, enhancement of the effect of another drug such as targeting, delay in the progression of the disease, and / or clinical outcomes such as prolongation of survival. The effective dosage can be administered in one or more administrations. For the purposes of the present invention, an effective dose of an anti-cancer therapy is an amount sufficient to directly or indirectly achieve a therapeutic treatment. As understood in a clinical context, a therapeutically effective amount of an anti-cancer therapy may or may not be achieved in combination with another anti-cancer therapy.

[0192] In some embodiments, any of the treatment methods described herein can further include administering to the subject one or more additional anti-cancer therapies. A variety of types of anti-cancer agents can be used. Non-limiting examples include radiation therapy, alkylating agents (such as cisplatin, carboplatin, or oxaliplatin), antimetabolites (such as azathioprine or mercaptopurine), anthracyclines, plant alkaloids (such as vinca alkaloids (such as vincristine, vinblastine, vinorelbine, etc.) or vindesine) and taxanes (such as paclitaxel, taxol, or docetaxel)), topoisomerase inhibitors (such as camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, or teniposide), podophyllotoxins (and derivatives such as etoposide and teniposide), antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormonal therapy, soluble receptors, and other anti-tumor agents (e.g., dactinomycin, doxorubicin, epirubicin, bleomycin, mechlorethamine, cyclophosphamide, chlorambucil, or ifosfamide).

[0193] T cell agonist In some embodiments of any of the methods disclosed herein, the method further comprises administration of one or more compounds that activate T cells. These are typically agonist antibodies and often target co-stimulatory molecules such as members of the tumor necrosis factor (TNF) receptor superfamily and the B7-CD28 superfamily. Non-limiting examples of T cell agonists suitable for use in the present invention include activators of CD27 (e.g., CDX-1127 (Celldex Therapeutics)), GITR, B7-H3, CD28 (e.g., TGN1412), CD40, interleukin-2 receptor subunit beta (ILR2P; also known as CD122; e.g., NKTR-214), CD137 (also known as TNFRSF9, 4-1BB, and induced by lymphocyte activation (ILA)), ICOS, and / or OX40 (also known as CD134 and TNFRSF4; MEDI0562, MEDI6469, and MEDI6383 (AstraZeneca)), but are not limited thereto. In some embodiments, the co-stimulatory molecule can also be an Ig fusion protein such as OX40-IgG.

[0194] Molecular adjuvant In some embodiments of any of the methods disclosed herein, the method further comprises administration of one or more molecular adjuvants. As used herein, "molecular adjuvant" refers to a molecule that enhances an immune response, including but not limited to agents that activate dendritic cells. Molecular adjuvants can include proteins, lipids, nucleic acids, carbohydrates, or compounds having receptors that, by their occupancy, result in changes in intracellular signaling and antigen-presenting cell phenotype, resulting in an improvement in the quantity or quality of the resulting immune response. Non-limiting examples of molecular adjuvants include agonists of the TNF receptor superfamily, Toll-like receptor (TLR) ligands, and intracellular DNA sensor agonists.

[0195] TNFR agonist as a molecular adjuvant The TNF receptor superfamily includes many important receptors on dendritic cells, macrophages, and T cells. For example, Cluster of Differentiation 40 (CD40) is a co-stimulatory protein found on antigen-presenting cells and is required for its activation. The binding of CD154 (CD40L) on TH cells to CD40 activates antigen-presenting cells and induces various downstream effects. CD40L strongly upregulates the expression of CD80 and CD86 on DCs and differentiates CD4+ T cells towards Th1 cells.

[0196] Other TNFR agonists that have shown important potential as molecular adjuvants include, but are not limited to, 4-1BB, CD30, herpes virus entry mediator, CD40, CD27, OX40, and glucocorticoid-induced TNFR-related protein (GITR), and their ligands are 4-1BBL, CD30L, LIGHT, CD27L / CD70, GITRL, and ICOS.

[0197] TLR agonist As used herein, the term "Toll-like receptor" (or "TLR") refers to a member of the Toll-like receptor family of proteins or a fragment thereof that senses microbial products and / or initiates an adaptive immune response. In one embodiment, the TLR activates dendritic cells (DCs). Toll-like receptors (TLRs) are a family of pattern recognition receptors first identified as sensors of the innate immune system that recognize microbial pathogens. TLRs constitute a family of conserved transmembrane molecules that include an extracellular domain of leucine-rich repeats, a transmembrane domain, and an intracellular TIR (Toll / IL-IR) domain. TLRs recognize different structures of microorganisms, often called "PAMPs" (pathogen-associated molecular patterns). Ligand binding to TLRs triggers a cascade of intracellular signaling pathways that induce the production of factors involved in inflammation and immunity.

[0198] In humans, 10 TLRs are identified. TLRs expressed on the cell surface include TLR-1, -2, -4, -5, and -6, while TLR-3, -7 / 8, and -9 are expressed in the ER compartment. Human dendritic cell subsets can be identified based on different TLR expression patterns. As an example, the myeloid or “conventional” subset of DC (mDC) expresses TLR1–8 when stimulated, generating a cascade of activation markers (CD80, CD86, MHC class I and II, CCR7), pro-inflammatory cytokines, and chemokines. The result of this stimulation and the resulting expression is antigen-specific CD4+ and CD8+ T cell priming. These DCs enhance the ability to take up antigen and present it to T cells in an appropriate form. In contrast, the plasmacytoid subset of DC (pDC) expresses only TLR7 and TLR9 upon activation, leading to the activation of NK cells and T cells. Since dying tumor cells can have an adverse effect on DC function, activating DCs with TLR agonists is suggested to be beneficial for the preparation of anti-tumor immunity in the immunotherapy approach of cancer treatment. Also, it is suggested that activation of TLR4 is required to successfully treat breast cancer using radiation and chemotherapy.

[0199] The TLR agonists known in the art and used in the present invention include, but are not limited to: Pam3Cys, a TLR-1 / 2 agonist, CLR, a TLR-2 agonist, MALP2 which is a TLR-2 agonist, Pam2Cys, a TLR-2 agonist, FSL-1 which is a TSL-2 agonist, Hib-OMPC which is a TLR-2 agonist, polyriboinosinic:polyribocytidylic acid (poly I:C), a TLR-3 agonist, polyadenosine-polyuridylic acid (poly AU) which is a TLR-3 agonist, poly-L-lysine, and polyinosinic-polycytidylic acid stabilized with carboxymethylcellulose (Hiltonol (registered trademark)), a TLR-3 agonist, monophosphoryl lipid A (MPL), an LPS, a TLR-4 agonist, bacterial flagellin, a TLR-5 agonist; sialyl-Tn (STn), carbohydrates associated with the MUC1 mucin of many human cancer cells, and a TLR-4 agonist, imiquimod which is a TLR-7 agonist, resiquimod which is a TLR-7 / 8 agonist, loxoribine, a TLR-7 / 8 agonist, and unmethylated CpG dinucleotides (CpG-ODN), a TLR-9 agonist.

[0200] Intracellular DNA sensor agonist The cGAS-STING pathway is a component of the innate immune system that functions to detect the presence of cytosolic DNA and, in response, induce the expression of inflammatory genes. DNA is normally in the cell nucleus. The localization of DNA to the cytosol is associated with tumor formation or viral infection. The cGAS-STING pathway acts to detect cytosolic DNA and induce an immune response.

[0201] When binding to DNA, the protein cyclic GMP-AMP synthase (cGAS) causes dimerization of AMP and GMP, forming cyclic GMP-AMP (cGAMP). cGAMP and other cyclic dinucleotides bind to the stimulator of interferon genes (STING), trigger TBK1, phosphorylate the downstream transcription factor IRF3 that induces a type I IFN response, and STAT6 induces chemokines such as CCL2 and CCL20 independently of IRF3 (Burdette et al., 2011, Nature 478, 515-18). The signaling pathway activated by STING binds to induce an innate immune response in cells with ectopic DNA in the cytosol. Loss of STING activity inhibits the ability of mouse embryonic fibroblasts to fight infection by certain viruses and is more generally required for the type I IFN response to introduced cytosolic DNA.

[0202] DNA has been shown to be a potent adjuvant that enhances the immune response to antigens encoded by vaccines. cGAMP stimulates the transcription of interferon through the activation of IRF3 by STING. Thereby, cGAMP becomes a potential vaccine adjuvant that can promote the inflammatory response. Studies have shown that a vaccine encoding the chicken antigen ovalbumin (OVA) in combination with cGAMP can activate antigen-specific T and B cells in a STING-dependent manner in vivo. When stimulated with OVA peptides, T cells from mice inoculated with OVA + cGAMP were shown to have increased IFN-γ and IL-2 compared to animals administered OVA alone. Furthermore, due to the improved stability of cGAMP by the unique 2'-5' phosphodiester bond, it may become an adjuvant for DNA suitable for in vivo use.

[0203] Microenvironment modulator In any other embodiment of the methods disclosed herein, the method further comprises administration of one or more microenvironment modulators. A "microenvironment modulator" refers to a factor capable of generating an immunosuppressive tumor microenvironment that supports tumor growth. One such modulator is indoleamine (2,3)-dioxygenase (IDO), also identified as a checkpoint protein (see above). IDO is an enzyme with two isoforms (IDO1 and IDO2) that acts at the first step of a metabolic pathway that degrades the essential amino acid tryptophan. IDO exerts an immunomodulatory effect by shutting down effector T cells of the immune system. IDO expression also directly activates regulatory T cells, a subset of T cells whose main function is to block T cell-mediated immunity at the end of an immune response.

[0204] Another microenvironment modulator is tryptophan 2,3-dioxygenase (TDO). TDO plays a central role in the physiological regulation of tryptophan flux in the human body. It catalyzes the first rate-limiting step of tryptophan degradation along the kynurenine pathway, thereby regulating systemic tryptophan levels. Tryptophan 2,3-dioxygenase has been shown to be expressed in a significant proportion of human tumors. In the same study, tryptophan 2,3-dioxygenase expression by tumors prevented rejection by immune mice. Tryptophan 2,3-dioxygenase inhibitors developed by a group restored the ability of these mice to reject tryptophan 2,3-dioxygenase-expressing tumors, indicating that tryptophan 2,3-dioxygenase inhibitors have potential in cancer treatment.

[0205] Other microenvironment modulators suitable for use in the methods of the present invention include IDO, TDO, CD73, COX2 inhibitors, CD39 inhibitors, and A2A receptor agonists, as well as antibodies against CD73, CD39, and the A2A receptor.

[0206] Chemokine receptor antagonist In yet other embodiments of any of the methods disclosed herein, the method further comprises administering one or more chemokine receptor antagonists. Chemokine receptors are G protein-coupled receptors that mainly contain seven transmembrane domains on the surface of leukocytes. Chemokine receptors are classified into different families of CXC chemokine receptors, CC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors corresponding to four different subfamilies of binding chemokines.

[0207] In some embodiments, the method of the invention comprises one or more antagonists to chemokine receptors of the CXC chemokine receptor family. Targets of suitable CXC family members include CXCR1 (IL8RA or CD181) and CXCR4 (alias fusin or CD 184), which are thought to be involved in cell proliferation and angiogenesis necessary for tumor survival.

[0208] In other embodiments, the method of the invention comprises one or more antagonists to chemokine receptors of the CC chemokine receptor (or beta chemokine receptor) family, including but not limited to CCR2, CCR5, and / or CCR4.

[0209] Cytokine therapy In yet other embodiments of any of the methods disclosed herein, the method further comprises administering one or more cytokine therapies. Cytokines are a broad group of proteins produced by many types of cells present in tumors and have the ability to regulate the immune response. Due to these immunomodulatory effects, they can be used as drugs to trigger an immune response. Two commonly used groups of cytokines are interferons and interleukins.

[0210] Non-limiting examples of cytokine therapies suitable for use in the present invention include, but are not limited to, type I IFN (IFNα), IL-2, IL-7, IL-15, IFNγ, IL-10, IL-12, IL-21, IL-33, IL-35, FLT3, and / or anti-TGFβ. The receptors for these proteins (such as IL-2R, IL-7R, IL-15R, IL-10R, IL-12R, IL-21R, etc.) can also be targeted with activators such as (e.g., (small molecules), antibodies, or polypeptides).

[0211] Other immunotherapies Other immunotherapies suitable for use in the methods disclosed herein include immunogenic chemotherapy, XRT, oncolytic viruses, cryotherapy, TACE, intratumoral injection of immunomodulators, targeted therapies of oncogenic pathways (such as MAPK, β-catenin, PI3K / PTEN, FGFR3, etc.), epigenetic therapies, CSF1 / CSFR1 depletion antibodies and anti-CCR4 (e.g., mogamulizumab, Kyowa), anti-IL-8 / IL-8R, anti-CCR2, anti-CCR5, anti-CXCR1 / CXCR2, anti-CTLA4, anti-CCR4, anti-CCR8, anti-CD25, anti-KIR, anti-NKG2a, anti-NKG2DL (MICA), arginase, IDO / TDO, adenosine, A2AR, CD39, CD73, PI3K gamma, anti-NKG2D, CD94, and CD47 / SIRPa, Mer / Axl / Tyro3, TIM3, MFG-E8 / GAS6, and / or therapies for activating or inhibiting one or more of DD1alpha.

[0212] In some embodiments, the methods disclosed herein can be used in combination with any CAR-T therapy known in the art. A chimeric antigen receptor (CAR, also known as a chimeric T cell receptor) is a synthetic construct expressed in host T cells or NK cells and designed to induce an immune response against a specific target antigen and the cells expressing that antigen. A CAR typically includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is typically an antibody fragment such as an scFv or Fab fragment and can be targeted to bind any antigen. The transmembrane domain can be derived from the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. The intracellular domain is typically a selective T cell activation portion comprising the endodomains of CD3-zeta, CD28, ICOS, CTLA4, PD1, PTLA, HVEM, CD27, 4-1BB, OX40, DR3, DcR3, FAS (CD95), GITR, CD30, CD40, SLAM, CD2, 2B4, TIM1, TIM2, TIM3, TIM4, TNFR1 (CD120a), TNFR2 (CD120b), LTβR, Ly108, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, TIGIT, CD226, CRTAM (CD355), CD96, CD160, LAG3, LAIR1, B7-1, RANK (CD265), TACI, BAFFR, BCMA, TWEAKR, EDAR, XEDAR, RELT, DR6, TROY, NGFR, OPG, TRAILR1-4 and B7-H1. Various combinations of the extracellular domain, transmembrane domain, and intracellular domain can be used to construct a CAR.

[0213] In some embodiments, the methods disclosed herein can be used in combination with any adoptive cell transfer (ACT) therapy. ACT is a very effective form of immunotherapy and involves transplanting immune cells with anti-tumor activity into cancer patients. ACT includes the in vitro identification of lymphocytes with anti-tumor activity, the in vitro expansion of these cells, and the injection into a cancer-bearing host. The lymphocytes used for adoptive immunotransfer are derived from the stroma of the resected tumor (tumor-infiltrating lymphocytes or TILs). They are also blood-derived or derived from blood if they are genetically engineered to express an anti-tumor T cell receptor (TCR) or chimeric antigen receptor (CAR), enhanced in a mixed lymphocyte tumor cell culture (MLTC), or cloned using autologous antigen-presenting cells and tumor-derived peptides. ACT derived from a host with cancer into which the lymphocytes are injected is called autologous ACT.

[0214] All numerical upper limits given throughout this specification are intended to include all lower numerical limits, as if such lower numerical limits were expressly written herein. All numerical lower limits given throughout this specification are intended to include all higher numerical limits, as if such higher numerical limits were expressly written herein. All numerical ranges given throughout this specification include any narrower numerical ranges that fall within such broader numerical ranges, as if such narrower numerical ranges were all expressly written herein.

[0215] Pharmaceutical composition Also provided herein is a pharmaceutical composition comprising any of the compounds disclosed herein, including anlexanox, a PTC read-through compound, an NMD complex inhibitor, and a checkpoint inhibitor. The pharmaceutical compositions of the present invention can include tablets, capsules, granules, powders, pellets, caplets, mini-tablets, lozenges, capsules filled with mini-tablets and / or pellets, multilayer tablets, granules for suspension, granules or powders in sachets, or one or more of the foregoing. In other embodiments, the compositions of the present invention can be coated to obtain film-coated tablets.

[0216] The compositions of the present invention can be prepared by mixing pharmaceutical excipients and granulating, optionally with other pharmaceutically acceptable excipients and with sugar, in an aqueous or alcoholic solution of the compound. The granules can be dried, smoothed, and converted into a suitable dosage form.

[0217] Stable solid pharmaceutical compositions of compounds such as anlexanox, PTC read-through compounds, NMD complex inhibitors, and checkpoint inhibitors can be prepared by processes known to those skilled in the art of pharmaceutical technology, such as direct compression, wet or dry granulation, slugging, hot melt granulation, hot melt extrusion, fluidized bed granulation, extrusion spheronization, spray drying, and solvent evaporation. In one embodiment, a stable composition of a compound that promotes PTC read-through and a compound that inhibits the NMD complex or a pharmaceutically acceptable salt thereof is prepared by dry / wet granulating the compound with one or more sugars and one or more pharmaceutically acceptable compounds. The excipients, and optionally the granules, are mixed with other excipients.

[0218] Pharmaceutically acceptable excipients can include one or more binders, fillers, lubricants, solubilizers, stabilizers, disintegrants, glidants, and the like.

[0219] Suitable "diluents" may include one or more of lactose, microcrystalline cellulose, calcium phosphate, dextrin, dextrose, dextrose hydrate, mannitol, sorbitol, sucrose, etc. In particular, the diluents are lactose and microcrystalline cellulose. The diluent may be present in the extragranular and / or intragranular portions of the composition.

[0220] Suitable "disintegrants" may include one or more of crospovidone (Polyplasdone), low-substituted hydroxypropyl cellulose, carmellose, sodium carboxymethyl starch, calcium carmellose, corn starch, pregelatinized starch, croscarmellose sodium, sodium starch glycolate, etc. In particular, the disintegrant is crospovidone. The disintegrant may be present in the extragranular and / or intragranular portions of the composition.

[0221] Suitable "binders" may include one or more of hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone (Povidone K30), polyvinyl alcohol, their partial saponification products, starch, etc. In particular, the binder is polyvinyl pyrrolidone.

[0222] Suitable "solubilizers" include poloxamer, polyethylene glycol, polysorbate, sodium lauryl sulfate, glyceryl monostearate, glyceryl monooleate, lecithin, polyoxyethylene alkyl ester, polyoxyethylene castor oil derivative, polyoxyethylene fatty acid ester, etc. In particular, the solubilizers are poloxamer and glyceryl monooleate.

[0223] Suitable "stabilizers" may include one or more of citric acid, tartaric acid, fumaric acid, maleic acid, vitamin E acetate, etc. In particular, the stabilizer is vitamin E acetate.

[0224] Suitable "lubricants / flow promoters" include one or more of magnesium stearate, stearic acid, palmitic acid, calcium stearate, zinc stearate, sodium stearyl fumarate, glyceryl behenate, talc, and the like.

[0225] Any of the compounds according to the present invention can be formulated by conventional methods using one or more pharmaceutically acceptable carriers or excipients. In some embodiments, multiple routes of administration can be used for drug administration in a given treatment plan. For example, a compound that promotes PTC readthrough can be administered orally, and a compound that inhibits NMD can be administered intravenously. Thus, the compounds for use according to the present invention can be in a form suitable for one or more of, for example, oral, sublingual, buccal, parenteral, rectal, vaginal, or intranasal administration, or for administration by inhalation or insufflation (via the mouth or nose), or in a form suitable for topical administration, preferably for topical application to the eye. In another embodiment, the compound is formulated for topical or subcutaneous administration.

[0226] In the case of oral administration, the pharmaceutical composition may be in the form of tablets or capsules prepared by conventional means together with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (magnesium stearate, talc, silica, etc.); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may be in the form of, for example, solutions, syrups or suspensions, or may be provided as dry products to be constituted with water or other suitable vehicles before use. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats), emulsifying agents (lecithin, acacia, etc.); non-aqueous vehicles (almond oil, oily esters, ethyl alcohol, etc.); preservatives (e.g., methyl or propyl - p-hydroxybenzoic acid or sorbic acid).

[0227] For buccal administration, the composition may be in the form of tablets or lozenges formulated by conventional methods.

[0228] Compounds such as amlexanox, PTC lead-through compounds, NMD complex inhibitors, checkpoint inhibitors, etc. can be formulated for parenteral administration by injection, convenient intravenous, intramuscular, intratumoral, or subcutaneous injection, such as bolus injection or continuous intravenous injection. Injectable formulations may be presented in unit dosage forms, for example, placed in ampoules or multi-dose containers with preservatives added as required. Compositions for parenteral administration may take forms such as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient may be in a dry form such as a powder, crystalline, or lyophilized solid, and may be in a suitable vehicle, for example, sterile water or isotonic saline free of pyrogens before use. They may be presented, for example, in sterile ampoules or vials.

[0229] The compounds may also be formulated for rectal administration, such as suppositories or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, and may be formulated intranasally.

[0230] Tablets for sublingual administration can be formulated by conventional methods.

[0231] In the case of intranasal administration, the compounds can be used, for example, in the form of a liquid such as a solution, suspension, or emulsion provided in the form of a spray, drops, or as a powder. Preferably, preparations for intranasal administration are delivered in the form of a spray or aerosol from a dispenser, or from a pressurized pack or nebulizer using a suitable propellant.

[0232] For administration by inhalation, the compounds can be conveniently delivered in the form of an aerosol spray from a nebulizer, pressurized pack or metered-dose inhaler using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, tetrafluoroethane, heptafluoropropane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges Gelatin formulations for use in an inhaler or insufflator can be formulated containing a powder mixture of a compound of the invention and a suitable powder base such as lactose or starch.

[0233] For topical administration, the pharmaceutical composition may be in the form of a cream, gel, lotion, foam or drops suitable for topical application to the eye, a liquid such as a solution, suspension or emulsion (including nanoparticles or liposome-containing emulsions).

[0234] The composition can be formulated into unit dosage forms containing an active ingredient in which each dosage is from about 5 mg to about 100 mg or more, for example from about 1 mg to about 5 mg, 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 30 mg, about 1 mg to about 40 mg, about 1 mg to about 50 mg, about 1 mg to about 60 mg, about 1 mg to about 70 mg, about 1 mg to about 80 mg, or from about 1 mg to about 90 mg, including any range between these values. The term "unit dosage form" refers to physically discrete units suitable as individual unit doses, each unit containing a predetermined quantity of active substance calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical excipient or carrier.

[0235] The present invention can be further understood by reference to the following examples, which are provided by way of illustration and not of limitation.

Examples

[0236] Example 1: In vivo study using PTC lead-through compounds, NMD inhibitors, checkpoint inhibitors

[0237] Materials and methods Animals: Female C57BL / 6 mice at 6 - 8 weeks of age (estimated age at inoculation) were obtained from Shanghai Lingchang Bio-Technology Co., Ltd (LC, Shanghai, China). The animals were housed at 20 - 26 °C and fed for 12 hours under a 12-hour light / dark cycle.

[0238] Cell culture: MC38 tumor cells were maintained in vitro as monolayer cultures in DMEM medium supplemented with 10% fetal bovine serum at 37 °C in an atmosphere of 5% CO2 in air. The tumor cells were usually passaged twice a week. Cells in the logarithmic growth phase were harvested and counted for tumor inoculation.

[0239] Therapeutic compounds: Anti-PD-1 and anti-CTLA-4 antibodies were obtained from BioXCell, similar to the anti-CD8 antibody. The nonsense-mediated decay inhibitor NMDI14 (4,5-dimethyl-1-2-[[2-(1,2,3,4-tetrahydro-6,7-dimethyl-3-oxo-2-quinoxalinyl)acetyl]amino]-3-thiophenecarboxylic acid ethyl ester, ethyl 2-{[((6,7-dimethyl-3-oxo-1,2,3,4-tetrahydro-2-quinoxalinyl)acetyl)amino]-4,5-dimethyl-3-thiophenecarboxylate} was obtained from ChemBridge Corp. (La Jolla, California). RTC Ataluren (PTC124) was obtained from Selleck Chemicals (Houston, Texas). The compounds were formulated as shown in Table 2. Table 2: Formulations of therapeutic compounds.

Table 2

[0240] Tumor inoculation: For tumor development in each mouse, 0.1 mL of MC38 tumor cells (1x10 6 ) in PBS was subcutaneously inoculated into the right lower flank region. The average tumor size was approximately 50 mm 3Treatment was initiated when [the specified condition] was reached. The compound was administered, and the number of animals in each study group is shown in Table 2. The date of tumor cell inoculation was designated as Day 0.

[0241] Group Assignment: Before grouping and treatment, the body weights of all animals were measured, and tumor volume was measured using calipers. Tumor volume was used as a numerical parameter for randomizing the selected animals into specific groups to minimize systematic error. Grouping was performed using StudyDirector(tm) software (Studylog Systems, Inc., California, USA). One optimal randomization design (generated by the uniform distribution) showing the minimum inter-group variation in tumor volume was selected for group assignment. Table 3: Administration of Test Articles and Number of Animals in Each Study Group

Table 3

[0242] FACS Analysis: Tumor cells were isolated from each treatment group, and FACS analysis was performed according to methods well-known in the art. The reagents used for FACS analysis are shown in Table 4 below. Table 4: Reagents Used for FACS Analysis of Tumor Cells.

Table 4

[0243] Immunohistochemistry (IHC): Formalin-fixed paraffin-embedded (FFPE) tissue from tumor samples was sectioned at 4 μm. Antigen retrieval (AR) was performed at 100 °C for 20 minutes in EDTA buffer, pH 9.0. Primary antibody (diluted at the effective concentration), RT 60 minutes + secondary antibody (ready to use), RT 60 minutes + Bond Polymer Refine Detection. The antibodies and reagents used are shown in Table 5. Table 5: Antibodies and reagents used in the IHC experiment.

Table 5

[0244] After tumor cell inoculation, the morbidity and mortality of the animals, as well as tumor growth and treatment, were checked daily for mobility, visual estimation of food and water consumption, weight gain / loss, eye / hair matting and other abnormal effects. Tumor volume was measured at least two-dimensionally twice a week using calipers, and the volume was expressed in mm3 using the formula: V = 0.5 x a x b2 where a and b are the major and minor diameters of the tumor, respectively.

[0245] The average tumor volume of each treatment group during the study period is shown in Table 6, and the inhibition rate of tumor volume is shown in Table 7. Table 6: Average tumor volume (mm3) (+ / - standard error of the mean)

Table 6

Table 7

[0246] As shown in Table 7, the combination of PTC124, nonsense-mediated decay inhibitor, and anti-PD-1 and anti-CTLA-4 immunotherapies inhibited tumor volume by nearly 75% (see also Figure 1), which is greater than the results achieved by either immunotherapy alone or their combinations (Figure 2).

[0247] Immunohistochemistry analysis demonstrated that the combination of a PTC readthrough inhibitor and an NMD-blocking compound resulted in significant numbers of CD3+ immune cells infiltrating tumor tissue, as shown in Figure 3. Figure 3 also shows that this effect was enhanced when the treatment was combined with antibodies against PD-1 and CTLA-4. [Example]

[0248] Example 2: In vivo study using amlexanox and checkpoint inhibitors Animals and cell cultures were maintained as described in Example 1. For tumor development, C57BL / 6 mice were inoculated with MC38 tumor cells (1 × 10) in 0.1 mL of PBS. 6 ) was subcutaneously inoculated into the right lower abdomen. The average tumor size was approximately 50 mm 3 Treatment was initiated when the tumor cell count reached 0. The compounds were administered and the number of animals in each test group is shown in Table 8. The date of tumor cell inoculation was designated as day 0. All compounds were administered intraperitoneally. Table 8 [Table 8]

[0249] After tumor cell inoculation, animals were checked daily for morbidity and mortality, as well as tumor growth and treatment, through visual assessment of exercise, food and water consumption, weight gain / loss, eye / hair matting, and other abnormal effects. Tumor volume was measured twice weekly in at least two dimensions using calipers, and volume is expressed in mm3 using the formula: V = 0.5ax b2, where a and b are the long and short diameters of the tumor, respectively. The mean tumor volume for each treatment group during the study period is shown in Table 9, and the tumor volume inhibition rates are shown in Table 10. Table 9 [Table 9] Table 10. Percent inhibition of tumor volume for each treatment group (negative values indicate an increase in tumor volume). [Table 10]

[0250] As shown in Table 10, the combination of anlexanox with anti-PD-1 and anti-CTLA-4 immunotherapies inhibited tumor volume by nearly 84% (see also Figure 4) and was compared with the combination of PTC124 and PD-1 / CTLA-4.

Example

[0251] Example 3: Xenograft studies using other cancer cell lines Syngeneic immunocompetent tumor models can be created with mouse cancer cell lines, and the examples include pancreas (Pan02), prostate (RM1), colon (CT-26, Colon-26, MC38-26), kidney (Renca), bladder (MBT-2), lung (LL / 2, KLN205), melanoma (B16BL6, B16F10, S91), breast (4T1, EMT6, JC), fibrosarcoma (WEHI-164), leukemia (C1498, L1210), liver (H22, Hepal-6), lymphoma (A20, EL-4, E.G&-OVA, L5178-R, P388D1), mastocytoma (P815), myeloma (MPC-11), neuroblastoma (Neuro-2a), etc. As shown in Examples 1 and 2, mice receive a suspension of subcutaneously injected tumor cells, which develop into tumors approximately 4 - 6 weeks after injection.

[0252] To test the efficacy of treatment against early-established mouse tumors, a cohort of 10 mice per group receives the drug (or sham control) on days 3 - 7 or when the tumor is palpable. The drug is administered twice a day, several times a week, or continuously until the mice are sacrificed at 4 - 6 weeks, or the tumor reaches 3 - 5 cm or ulcerates. During treatment, the volume of the tumor is determined using three-dimensional measurements with calipers three times a week. As shown in Examples 1 and 2, the tumors are harvested after sacrifice and weighed to determine the final tumor volume.

Example

[0253] Example 4: Combination of amelxanox with epigenetic regulators This example shows the administration of anleranox and an epigenetic regulator. The addition of one or more epigenetic regulators may enhance the immune recognition of neoantigens.

[0254] As shown in Examples 1 and 2, mice receive a suspension of subcutaneously injected tumor cells and develop tumors approximately 4 - 6 weeks after injection. Drug administration, tumor size evaluation, intratumoral immune response, CD4 and CD8 effector T cell responses, and Treg responses are performed as described above.

[0255] Treatment of tumors with epigenetic regulators can remove the suppression of genes involved in the immune response. The combination of RTC and NMDI with epigenetic regulators enhances the recognition of neoantigens. Non - limiting examples of epigenetic regulators include, but are not limited to, HDAC inhibitors, azacitidine, BET inhibitors, EZH2 inhibitors, and / or dot1L inhibitors (such as pinometostat), and DNA methyltransferase (DNMT) inhibitors.

Example

[0256] Example 5: Combination of Anleranox and Radiation Therapy This example shows the effect of tumor treatment in syngeneic immunocompetent mice by the combination of anleranox and radiation therapy. Treatment with anleranox drug before radiation therapy (RT) increases the expression of neoantigens in tumors and enhances neoantigen presentation before immunostimulatory cell death.

[0257] As shown in Examples 1 and 2, mice receive a suspension of subcutaneously injected tumor cells and develop tumors approximately 4 - 6 weeks after injection. Drug administration, tumor size evaluation, intratumoral immune response, CD4 and CD8 effector T cell responses, and Treg responses are performed as described above.

[0258] Continuous treatment with anlequinox during tumor-targeted radiotherapy (RT) may generate neoantigens through mutagenesis and DNA damage. Release of these antigens during cell death associated with inflammatory signals that induce an immune response activating tumor-specific T cells. Radiotherapy affects the tumor microenvironment, promotes infiltration of activated T cells, and overcomes barriers to tumor rejection. The combination of anlequinox with immunotherapeutic agents and RT will enhance the effect of radiation on both the priming (antigen presentation) and effector phases of the immune response in individual patients by increasing neoantigen expression.

Example

[0259] Example 6: Combination of Anlequinox and Chemotherapy This example shows the effect of tumor treatment in syngeneic immunocompetent mice by the combination of RTC and NMDI and chemotherapy. Treatment with RTC and NMDI drugs before chemotherapy increases the expression of neoantigens in the tumor and enhances neoantigen presentation prior to immunostimulatory cell death.

[0260] As shown in Examples 1 and 2, mice receive a suspension of subcutaneously injected tumor cells and develop tumors approximately 4 - 6 weeks after injection. Drug administration, tumor size evaluation, intratumoral immune response, CD4 and CD8 effector T cell responses, and Treg responses are performed as described above.

[0261] Continuous treatment with anlequinox during chemotherapy may generate neoantigens through mutagenesis and DNA damage and may release these antigens during cell death in conjunction with inflammatory signals that trigger an immune response activating tumor-specific T cells. Chemotherapy affects the tumor microenvironment, promotes infiltration of activated T cells, and overcomes barriers to tumor rejection. The combination of anlequinox with immunotherapeutic agents (Example 1) and chemotherapy enhances the effect of chemotherapy on both priming and the effector phase of the immune response in individual patients by enhancing neoantigen expression.

Example

[0262] Example 7: Combination of Amlexanox with Oncolytic Virus This example shows the effect of tumor treatment in syngeneic immunocompetent mice by the combination of amlexanox and oncolytic virus.

[0263] As shown in Examples 1 and 2, mice received a suspension of subcutaneously injected tumor cells, which developed into tumors approximately 4 - 6 weeks after injection. Administration of the drug, evaluation of tumor size, intratumoral immune response, CD4 and CD8 effector T cell responses, and Treg response were performed as described above.

[0264] Other approaches to inducing immunogenic cell death include the use of oncolytic viruses to selectively kill tumor cells. Thus, pretreatment of patients with amlexanox enables improvement of antigen presentation of neoantigens induced by oncolytic viruses and subsequent enhancement of T cell responses.

Example

[0265] Example 8: Combination of Amlexanox and Vaccination Therapy This example shows the effect of tumor treatment in syngeneic immunocompetent mice by the combined use of amlexanox and vaccination therapy.

[0266] As shown in Examples 1 and 2, mice received a suspension of subcutaneously injected tumor cells, and tumors developed approximately 4 - 6 weeks after injection. Administration of the drug, evaluation of tumor size, intratumoral immune response, CD4 and CD8 effector T cell responses, and Treg response were performed as described above.

[0267] Neoantigen vaccination emerges as a potentially effective vaccine approach in cancer. To date, these neoantigens contain amino acid substitutions, but amlexanox broadens the range of neoantigens beyond single amino acid substitutions. Aberrant peptides generated from amlexanox or the DNA or RNA encoding these products are components of individual vaccines. Transcription profiling of tumors from patients treated with amlexanox provides candidate aberrant lead-through proteins that can be used to generate such vaccines. Whole tumors treated with amlexanox can also be used as the basis for whole cell vaccines. Such vaccines against the induced neoantigens can be combined with any of the agents of these examples in addition to other vaccines.

Example

[0268] Example 9: Combination of Amlexanox with CAR-T Cells or Patient-Derived Tumor-Infiltrating Lymphocytes (TILs) This example shows the effect of the combination of amlexanox with CAR-T cells or the treatment of tumors in syngeneic immunocompetent mice with patient-derived tumor-infiltrating lymphocytes.

[0269] As shown in Examples 1 and 2, mice receive a suspension of subcutaneously injected tumor cells and develop tumors approximately 4 - 6 weeks after injection. Drug administration, tumor size evaluation, in-tumor immune response, CD4 and CD8 effector T cell responses, and Treg responses are performed as described above.

[0270] T cells induced in patients treated with amlexanox are induced to recognize neoantigens generated by these drugs. These specific T cells can be expanded ex vivo and reinjected directly into the patient or their TCR can be cloned and used to engineer CAR-T cells for reinjection.

Example

[0271] Example 10: Combination of Amlexanox with Immunomodulatory Agents This example shows the effect of tumor treatment in syngeneic immunocompetent mice by combining an immunomodulatory agent with an anrexinox such as a CDN, a TLR agonist, a TNFR superfamily agonist, and an epigenetic regulatory compound.

[0272] As shown in Examples 1 and 2, mice receive a suspension of subcutaneously injected tumor cells and develop tumors approximately 4 - 6 weeks after injection. Administration of the drug, evaluation of tumor size, intratumoral immune response, CD4 and CD8 effector T cell responses, and Treg responses are performed as described above.

[0273] Other approaches to inducing immunogenic cell death include the use of immunomodulatory agents such as CDNs, TLR agonists, TNFR superfamily agonists, and epigenetic regulatory compounds to selectively kill tumor cells. Thus, pre - treatment of patients with anrexinox can improve the antigen presentation of induced neoantigens by immunomodulatory agents and subsequently enhance the T cell response.

Claims

**Claim 1** A method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of anlequinox and a therapeutically effective amount of at least one checkpoint inhibitor. **Claim 2** The method of claim 1, wherein the at least one checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-A2AR antibody, an anti-KIR antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, and combinations thereof. **Claim 3** The method of claim 1, wherein the method comprises administering anlequinox in combination with an anti-PD-1 and an anti-CTLA-4 antibody. **Claim 4** The method of claim 1, wherein the method comprises administering anlequinox in combination with an anti-PD-L1 and an anti-CTLA-4 antibody. **Claim 5** The method of claim 1, wherein the anlequinox is administered at a dose of about 1 mg / kg to 50 mg / kg per day. **Claim 6** The method of claim 1, wherein the checkpoint inhibitor is administered at a dose of about 3 mg / kg to about 10 mg / kg every three days. **Claim 7** The method of claim 1, wherein the route of administration is selected from the group consisting of oral, topical, subcutaneous, intramuscular, intraperitoneal, intracavitary, intrathecal, transdermal, and intravenous injection. **Claim 8** The method according to claim 1, wherein the cancer is selected from the group consisting of colon carcinoma, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, Merkel cell carcinoma, craniopharyngioma, epithelioma, pineal body, hemangioblastoma, acoustic neuroma, anaplastic glioma, meningioma, melanoma, neuroblastoma, retinoblastoma, acute lymphocytic leukemia, acute myeloid leukemia, chronic leukemia, polycythemia vera, lymphoma, multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, and combinations thereof.

9. The method according to claim 1 includes the step of administering an anti-cancer agent selected from the group consisting of tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, megestrol acetate, anastrozole, letrozole, vorazole, exemestane, flutamide, nilutamide, bicalutamide, cyproterone acetate, goserelin acetate, leuprolide, finasteride, Herceptin, methotrexate, 5-fluorouracil, cytosine arabinoside, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mitramycin, cisplatin, carboplatin, melphalan, chlorambucil, busulfan, cyclophosphamide, ifosfamide, nitrosourea, thiotepa, vincristine, taxol, taxotere, etoposide, teniposide, amsacrine, irinotecan, topotecan, epothilone, gefitinib, erlotinib, sorafenib, angiogenesis inhibitor, EGF inhibitor, VEGF inhibitor, CDK inhibitor, cytokine, Her1 and Her2 inhibitor, and monoclonal antibody.

10. The method according to claim 1, further comprising the step of administering an immunomodulatory agent selected from co-stimulatory molecules, TLR agonists, TNF receptor superfamily agonists, cyclic dinucleotides, T cell agonists, cytokines, chemokines, oncolytic viruses, and combinations thereof.

11. The method according to claim 1, further comprising the step of administering an epigenetic regulatory compound selected from vorinostat, romidepsin, decitabine, 5-azacytidine, panobinostat, belinostat, and combinations thereof.

12. A method for killing cancer cells, comprising the step of contacting the cancer cells with a composition comprising anlecanox in combination with at least one checkpoint inhibitor.

13. The method according to claim 12, wherein the at least one checkpoint inhibitor is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-A2AR antibody, anti-KIR antibody, anti-LAG3 antibody, anti-B7-H3 antibody, and combinations thereof.

14. The method according to claim 12, wherein the cancer cells are selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer, liver cancer, kidney cancer, melanoma, gastrointestinal cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, sarcoma, glioblastoma, T cell and B cell lymphoma, endometrial cancer, cervical cancer, and combinations thereof.

15. A method for killing cancer stem cells, comprising the step of contacting the cancer stem cells with a composition comprising anlecanox in combination with at least one checkpoint inhibitor.

16. The method according to claim 15, wherein the at least one checkpoint inhibitor is selected from the group consisting of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, A2AR antibody, anti-KIR antibody, anti-LAG3 antibody, anti-B7-H3 antibody, and combinations thereof.

17. A method for generating an immune response in an individual by inducing the expression of neoantigens on the surface of cancer cells, comprising the step of administering a therapeutically effective amount of anlecanox in combination with at least one checkpoint inhibitor.

18. The method according to claim 17, wherein the at least one checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-A2AR antibody, an anti-KIR antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, and combinations thereof.

19. The method according to claim 17, wherein the method comprises administering anlecanox in combination with an anti-PD-1 and an anti-CTLA-4 antibody.

20. The method according to claim 17, wherein the method comprises administering anlecanox in combination with an anti-PD-L1 and an anti-CTLA-4 antibody.

21. The method according to claim 17, wherein the anlecanox is administered at a dose of about 1 mg / kg to 50 mg / kg per day.

22. The method according to claim 17, wherein the checkpoint inhibitor is administered at a dose of about 3 mg / kg to about 10 mg / kg every three days.

23. The method according to claim 17, wherein the route of administration is selected from the group consisting of oral, topical, subcutaneous, intramuscular, intraperitoneal, intracavitary, intrathecal, transdermal, and intravenous injection.

24. The method according to claim 17, wherein the cancer cells are selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer, bladder cancer, liver cancer, kidney cancer, melanoma, gastrointestinal cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, sarcoma, glioblastoma, T cell and B cell lymphoma, endometrial cancer, cervical cancer, and combinations thereof.

25. The method according to claim 17, further comprising administering an immunomodulatory agent selected from the group consisting of an immunostimulatory molecule, a TLR agonist, a TNF receptor superfamily agonist, a cyclic dinucleotide, a T cell agonist, a cytokine, a chemokine, an oncolytic virus, and combinations thereof.

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