Methods and compositions for cancer immunotherapy

Abstract

In general, in an aspect, methods and compositions of cancer immunotherapy are disclosed herein. Methods include administration of two or more immunotherapies to a subject where at least one immunotherapy provokes an anti-infection cell response within a tumor or at distant metastatic sites, such that the administered immunotherapies have a synergistic anticancer effect in a subject. Compositions include novel therapeutic or prophylactic agents that present at least a portion of an infectious microorganism indicium efficient to prime T cells against cancer cells (in a tumor or at distant metastatic sites) to that indicium.

Description

Methods and Compositions for Cancer ImmunotherapyCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of United States Provisional Application Ser. No. 63 / 790,643, filed April 17, 2025, the contents of which are hereby incorporated by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTINGThe contents of an XML electronic sequence listing (6809_0002PCT.xml; 5,485 bytes; created April 15, 2026) are herein incorporated by reference in its entirety.BACKGROUND

[0001] Innate and adaptive immunity work to restrict the replication and spread of infections.Pattern recognition of the indicia of infectious microorganisms (be they bacterial, fungal, viral, or parasitic) results in a multifactorial signal that drives an inflammatory cascade to inhibit infection and primes T and B cells via presentation of infectious microorganism epitopes to antigen-presenting cells (APCs). Of importance are primed CD8+T cells, for once primed, they can recognize epitopes that bind to major histocompatibility complex (MHC) class-1 molecules presenting on the surface of infected cells. There is also a growing realization that CD4+T cells can provide significant effector function in addition to their well-known immune recruiting functions, particularly when presented with epitopes that bind to MHC class-11 molecules; for example, CD4+T cells are known to exert antiviral effector function when differentiated to T helper 1 (Thl) cells or when further differentiated to become cytotoxic, APC-lyzing CD4+phenotypes. Specific recognition of infected cells helps to eliminate niches of e.g., viral replication in an antigen-specific manner in the short term; and in the long term, this capability is a major hallmark of immunological memory. In short, such "anti-infection T cells" [driven by benign or pathogenic infectious agents] provide for potent effector and memory responses throughout the life of a person. CD4+or CD8+"anti-infection T cell" response, raised on manifold occasions through exposure to infections, are likely prevalent in a large proportion of the global population and therefore represent a major effector response against infection.

[0002] T cells also constitute a major effector entity in cancer. "Antitumor T cells" are known to identify and kill malignant cells that express epitopes associated with cancer. Onceactivated, antitumor CD8+T cells are capable of attacking not only existing but also recurrent cancer cells, while leaving non-malignant cells intact; and they are efficacious against not only cancer cells within the local tumor microenvironment (TME) but also in distant sites of metastasis.

[0003] Antitumor T cells also form an important part of modern cancer immunotherapy, particularly in therapeutic immune checkpoint inhibitors (ICIs) such as antibodies designed to target programmed death 1 (PD-1), programmed death-ligand 1 (PDL-1), or cytotoxic T- lymphocyte-associated protein 4 (CTLA-4). PD-1 and its ligand, PD-L1, are monomeric type I transmembrane proteins that play critical roles in T cell inhibition and exhaustion. PD-L1 is composed of two extracellular immunoglobulin (Ig)-like domains whereas PD-1 is composed of a single extracellular Ig like domain and an intracellular tail. The crystal structure of the PD-1 / PD-L1 complex reveals that PD-1 binds to PD-L1 with a 1:1 stoichiometry to form a monomeric complex. Monoclonal antibodies developed for cancer immunotherapy binding to either PD-1 or PD-L1 have demonstrated significant response rates in patients, particularly for melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC) and bladder cancer. Such a strategy can be enhanced with adjuvants or in combination with ICIs currently approved for clinical use; immune checkpoint blockade leads to an enhancement in T cell cytotoxic activity at the tumor site. The profusion of ICI therapies within cancer therapy has led to increased emphasis on identifying the antitumor T cell responses within a given tumor type or even within a given patient's tumor. ( I nt rigu ingly, immune checkpoint blockade has also shown promise for the control or elimination of chronic viral infections, such as hepatitis B virus.)

[0004] The interplay between anti-infection and antitumor T cell response is still not well understood. In both anti-infection and antitumor T cells, the T cell receptors (TCRs) recognize MHC-presented ligands / epitopes, but the recognition signatures are distinct. Nevertheless, immunotherapy strategies exist that repurpose anti-infection T cell response for antitumor functions. For example, synthetic immunogenic viral epitopes can be fashioned that bind to MHC-I molecules on tumor cell surfaces, thereby simulating a reinfection event and tricking antiviral CD8+T cells into attacking a cancer cell.

[0005] Since they are generated against non-self epitopes, anti-infection T cells can in some ways be thought superior to antitumor T cells, in that anti-infection CD8+T cells often show increased functionality, polyclonality, breadth, and frequency relative to antitumor CD8+T cells. Moreover, thymic tolerance, systemic tolerance, and immunosuppression in the TME- all potential negative host factors for antitumor T cell strategies - appear to be negligible for anti-infection T cell strategies. Due to previously encountered viral infections in a subject, such as infections caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), or influenza, antiviral CD8+T cells have been found in human tumors. Human tumors also show evidence ofT cells that bearTCRs recognizing bacterial (e.g., E. coli, Pseudomonas sp., Streptococcus sp.) or parasitic (e.g., Plasmodium sp.) indicia. However, for common infections, particularly those caused by long-extant, widely circulating viruses, a diversity of epitopes makes it challenging to identify an appropriate target. Further complicating the identification of immunotherapies is that the body's response to its first exposure to an infectious microorganism - its "antigenic sin" - affects its response across the diachronic evolution of such microorganisms.

[0006] Novel strategies that activate T cells continue to be of high interest in cancer immunotherapy and are urgently needed.SUMMARY

[0007] Embodiments described herein are based, at least in part, on utilizing anti-infection therapies to treat cancer. Methods of treatment comprising the administration of novel combinations of existing anti-infection immunotherapy and / or anticancer immunotherapy (e.g., ICIs) are herein presented that synergistically work to enhance anti-tumor response.

[0008] In general, in an aspect, a method of treating cancer in a subject having a malignant tumor is disclosed. The method includes administering a therapeutically effective amount of a first immunotherapy, administering a therapeutically effective amount of a second immunotherapy after a predetermined first duration, and optionally, administering a therapeutically effective amount of a third immunotherapy after a predetermined second duration; wherein at least one of the administered immunotherapies provokes an antiinfection T cell response within a tumor.

[0009] Implementations may include one or more of the following. The first immunotherapy is an anti-infection immunotherapy and the second immunotherapy is an antitumor immunotherapy. The first immunotherapy is an antitumor immunotherapy and the second immunotherapy is an anti-infection immunotherapy. The anti-infection immunotherapy comprises an infectious microorganism indicium that is not associated with oncogenesis. The infectious microorganism indicium is a viral indicium selected from a coronavirus, influenza virus, respiratory syncytial virus, Epstein-Barr virus, cytomegalovirus, denguevirus, or combinations thereof. The infectious microorganism indicium is a viral indicium selected HIVl, HIV2, HTLV1, Epstein Barr virus, cytomegalovirus, adenovirus, adeno- associated virus, BK virus, Human Herpesvirus 6, Human Herpesvirus 8, influenza A virus, influenza B virus parainfluenza virus, avian flu virus, MERS coronavirus, SARS coronavirus, SARS-CoV-2, Crimean Congo Hemorrhagic fever virus, rhino virus, enterovirus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Lassa fever virus, zika virus, RSV, measles virus, mumps virus, rhino virus, varicella virus, herpes simplex virus 1 and 2, varicella zoster virus, HIV-l, HTLV1, Hepatitis virus, enterovirus, hepatitis B virus, Hepatitis C virus, Nipah and Rift valley fever viruses, Japanese encephalitis virus, or combinations thereof.Expression of the infectious microorganism indicium by tumor cells results in recruitment of anti-infection CD8+T cells into the tumor microenvironment. At least one of the administered immunotherapies is an immunostimulatory agent derived from a microbe. The immunostimulatory agent is derived from Bacillus Calmette-Guerin, Mycobacterium tuberculosis, atypical mycobacteria species, Pneumocystis jirovecii, toxoplasmosis, Rickettsia, Nocardia, Aspergillus, Mucor, or Candida. At least one of the administered immunotherapies is an immune checkpoint inhibitor. The immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody. At least one immunotherapy is administered intratumorally. At least one immunotherapy is administered according to a prime-and-boost regimen. The predetermined first duration or the predetermined second duration corresponds to a vaccine dosing interval used in a public health immunization schedule. The cancer is selected from melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, colon cancer, or sarcoma.

[0010] In general, in an aspect, a novel composition is disclosed. The composition includes a nucleic acid encoding at least a portion of an infectious microorganism indicium, wherein the composition is capable of causing tumor cells to express the infectious microorganism indicium and thereby prime or activate anti-infection T cells within a tumor.

[0011] Implementations may include one or more of following. The composition also includes a nucleic acid encoding at least one tumor-associated antigen. Infectious microorganism indicia are selected to correspond to prevalent anti infection T cell receptor repertoires in ahuman population. The composition also includes a pharmaceutically acceptable carrier. The composition also includes a lipid nanoparticle that encapsulates the nucleic acid. BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 shows Western immunoblots for SI and S2 expression in various cancer cell lines after exposure to COVID-19 vaccine produced by Moderna "MOD" or Pfizer "PFIZ", as discussed in Example 1.

[0013] FIG. 2 shows graphs of cell viability at increasing concentrations treated according to Example 1.

[0014] FIG. 3 shows proportion ofT cell stimulation in spleens treated according to Example 2.

[0015] FIG. 4 shows the immunological profile ofT cells in spleens treated according to Example 3.

[0016] FIG. 5 shows the proportion of functionally competent T cells in spleens and tumors according to Example 4.

[0017] FIG. 6 shows the anticancer effects in co-culture with combined in vivo and in vitro vaccine therapy according to Example 5.

[0018] FIG. 7 shows the anticancer effects of a therapy in vivo according to Example 6.

[0019] FIG. 8 shows the anticancer effects of a therapy in vivo according to Example 7.

[0020] FIG. 9 shows characterization data of genetically modified cancer cell lines according to Example 8B.

[0021] FIG. 10 shows the anticancer effects of a therapy in vivo according to Example 9.

[0022] FIG. 11 shows tumor volume over time in an experiment described in Example 9.

[0023] FIG. 12 shows tumor volume over time and survival over time in an experiment described in Example 10.

[0024] FIG. 13 shows tumor volume over time and survival over time in an experiment described in Example 11.

[0025] FIG. 14 shows CD8+ T-cell sequencing results side-by-side (left: tumor specific; right: virus specific) upon a VDJ antigen overlay in a uniform manifold approximation and projection.

[0026] FIG. 15 shows CD4+ T-cell sequencing results side-by-side (left: tumor specific; right: virus specific) upon a VDJ antigen overlay in a uniform manifold approximation and projection.DETAILED DESCRIPTION

[0027] DEFINITIONS

[0028] "Administration" or "administering" a composition as used herein should be understood to mean providing a composition disclosed herein to a subject in a form that can be introduced into that individual's body in an amount effective for prophylaxis, treatment, or diagnosis, as applicable. Such forms may include e.g., oral dosage forms, injectable dosage forms, transdermal dosage forms, inhalation dosage forms, and rectal dosage forms.

[0029] "Anti-infection immunotherapy" is used herein to refer to any composition that can be therapeutically or prophylactically administered to prime anti-infection T cells. T cells primed may be CD4+or CD8+and may respond to any infectious agent indicium or indicia presented in such immunotherapy. Species of anti-infection immunotherapy herein are "antiviral immunotherapy", "antibacterial immunotherapy", "antifungal immunotherapy", or "antiparasitic immunotherapy". Anti-infection immunotherapy may also include T cells having endogenous TCR signatures arising from natural infections.

[0030] "Antitumor immunotherapy" is used herein to broadly refer to any therapy that utilizes immune response to kill tumor cells, whether in the TME, in circulation, or in distant metastases. The term is also used herein more specifically to refer to any composition that can be therapeutically or prophylactically administered to prime antitumor T cells.

[0031] "Deoxyribonucleic acid" and "DNA" as used herein mean a polymer composed of deoxy ribonucleotides.

[0032] "Excipients" include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and / or manufacture can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005). Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. As would be appreciated by one of skill in this art, the excipients may be chosen based on what the composition is useful for. For example, with a pharmaceutical composition, the choice of the excipient will depend on the route of administration, the agent being delivered, time course of delivery of the agent, etc., and can be administered to humans and / or to animals, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), buccally, or as an oral or nasal spray.

[0033] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 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 higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in acandidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0034] "Infectious agent indicium" is used herein in the context of anti-infection immunotherapy to describe material, protein, peptide or oligonucleotide sequences or portions thereof that are derived from one or more infectious microorganisms, are included within the immunotherapy, and have the potential to be recognized by a subject's immune system as non-self; thereby provoking an immune response that primes or otherwise activates anti-infection CD4+or CD8+T cells. Species of infectious agent indicium include "virus (or viral) indicium", "bacteria (or bacterial) indicium", "fungus (or fungal) indicium", and "parasite (or parasitic indicium)", or indicia specific to a given genera or species of infectious microorganism.

[0035] "Nucleic acid" as used herein means a polymer composed of nucleotides, e.g.deoxyribonucleotides or ribonucleotides.

[0036] "Oligonucleotide" is used herein to denote single- or double-stranded nucleotide multimers of from about 2 to up to about 100 nucleotides in length. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using e.g., a commercial automated oligonucleotide synthesizer. When oligonucleotides are referred to as "double-stranded," it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen- bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term "double-stranded," as used herein is also meant to refer to those forms.

[0037] "Predetermined duration" is used herein to refer to a length of time between administrations of immunotherapy in a subject, whether the time interval is between two different immunotherapies, two doses of the same immunotherapy, or some othersequence of immunotherapy as described herein or motivated by, e.g., known public health vaccine schedules or regimens. Sometimes this term is used herein to refer to the length of time between the first sequence dose of a first immunotherapy and the first sequence dose of a second immunotherapy.

[0038] "Ribonucleic acid" and "RNA" as used herein mean a polymer composed of ribonucleotides. Species of RNA include ribosomal RNA (rRNA) and transfer RNA (tRNA), among others. Of note is messenger RNA (mRNA), a species key to the process of protein synthesis in a cell; mRNA is commonly used in vaccine strategies to cause a subject's cells to produce a microorganism protein that will attract interest in the immune system for the purpose of conferring immunity to a given microorganism. Self-amplifying RNA (saRNA) is a subspecies of mRNA with potential for improved protein expression in vaccine therapeutics over conventional mRNA.

[0039] Unless otherwise indicated, a "therapeutically effective amount" of a composition is an amount sufficient to treat a disease or condition, or one or more symptoms associated with the disease or condition.

[0040] SUBJECTS

[0041] Any of the compositions and methods of treatment provided may be used to treat a subject (e.g., human) who has been diagnosed with or is suspected of having cancer. In some embodiments, the subject may be a human who exhibits one or more symptoms associated with cancer or hyperproliferative disease. In some embodiments, the subject may be a human who exhibits one or more symptoms associated with cancer. In some embodiments, the subject is at an early stage of a cancer. In other embodiments, the subject is at an advanced stage of cancer.

[0042] As used herein, a subject refers to a mammal, including, for example, a human."Mammal" includes, without limitation, human, monkeys, rabbits, mice, domestic animals, such as dogs and cats, farm animals, such as cows, horses, or pigs, and laboratory animals.

[0043] Veterinary applications are contemplated in some embodiments.

[0044] Methods and compositions disclosed herein are suitable for treating cancer in a subject.The specific method or composition selected for cancer immunotherapy according to embodiments disclosed herein can be motivated by mapping of a subject's tumor with respect to anti-infection T cell response.

[0045] In some embodiments, one or more immunotherapies is selected for a subject based upon a pretreatment evaluation of a biopsied tumor, a pretreatment evaluation of acirculating tumor cell, a peri-operative assessment of TME T cell response, a pretreatment diagnostic readout of T cell response within the TME or metastases, or some other methodology allowing a qualified medical practitioner to learn which viral epitopes might present with a subject's cancer cells to be recognizable by a subject's anti-infection T cells.

[0046] In some embodiments, a subject's immunotherapy is selected based on the infectious disease burden in the area in which the subject lives or has lived. For example, a subject may have grown up in a world region where dengue or typhoid are common and can thus be expected to have T cells bearing TCRs responsive to dengue or typhoid indicia, respectively. A qualified medical practitioner can then use this information to select a cancer immunotherapy from the methods and compositions described herein.

[0047] In some embodiments, a subject's immunotherapy is a personalized therapy manufactured on small scale locally based upon the results of analysis of tumor or blood samples. In some embodiments, analysis includes WES, WGS, RNAseq, or neoantigen prediction using machine learning. In some embodiments, a subject's immunotherapy comprises a lipid nanoparticle having mRNA whose sequence or translation product is an infectious disease indicium expected to be recognized by the subject's T cells on the basis of the analysis.

[0048] In some embodiments, a subject's immunotherapy is selected from off-the-shelf cancer components.

[0049] In some embodiments, a personalized cancer vaccine workflow enhances response to therapy, including to ICI therapy. In some embodiments, a personalized cancer vaccine workflow reduces delay of therapy delivery. In some embodiments, a personalized cancer vaccine workflow reduces risk of toxicity of therapy.

[0050] In some embodiments, a subject's cancer cells are contacted by mRNA from an anticoronavirus therapy, e.g., Moderna or Pfizer anti-COVID-19 therapy, such that the cancer cells produce spike protein and thereby recruit antiviral T cells for anti-cancer activity. Cancer cells killed by antiviral T cells in a tumor then induce long-term antitumor immunity, since by lyzing they present their antigen signatures to T cells within the TME and promote effector and long-term memory functions against the tumor. In some embodiments, the anticoronavirus therapy comprises one or more of mRNA-1273, mRNA- 1273.351, mRNA-1273.211, mRNA-1283, elasomeran, imelasomeran, tozinameran, famtozinameran, or raxtozinameran.

[0051] The exact amount of an active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult. Useful dosages of the compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0052] In some embodiments, a composition (e.g., vaccine) for use in a method of vaccinating a subject is administered to the subject as a single dosage of between 0.051 pg / kg and 400 pg / kg of the nucleic acid vaccine (in an effective amount to vaccinate the subject). In some embodiments, a composition (e.g., vaccine) for use in a method of vaccinating a subject is administered to the subject as a single dosage of between 0.05 pg and 400 pg of the nucleic acid vaccine (in an effective amount to vaccinate the subject). In some embodiments, a composition (e.g., vaccine) for use in a method of vaccinating a subject is administered to the subject as a single dosage of 0.1-1000 pg (e.g., a single dosage of mRNA encoding an infection agent and a mRNA encoding T-cell epitope, or an mRNA encoding an infection agent and at least one universal T-cell epitope). In some embodiments, a composition (e.g., vaccine) is administered to the subject as a single dosage of 1, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 pg. For example, a composition (e.g., vaccine) may be administered to a subject as a single dose of 0.05-100, 0.1-100, 1-100, 5-100, 10- 100, 15-100, 20-100, 25-100, 25-500, 50-100, 50-500, 50-1000, 100-500, 100-1000, 250- 500, 250-1000, or 500-1000 pg. In some embodiments, a composition (e.g., vaccine) for use in a method of vaccinating a subject is administered to the subject as two dosages, the combination of which equals 25-1000 pg of the composition (e.g., vaccine).

[0053] METHODS

[0054] Methods of treatment of cancer are disclosed herein that comprise administration of a first immunotherapy followed by a second immunotherapy after a predetermined first duration. In some embodiments, the first immunotherapy is an anti-infection immunotherapy and the second immunotherapy is an antitumor immunotherapy. In someembodiments, the first immunotherapy is an antitumor immunotherapy and the second immunotherapy is an anti-infection immunotherapy. In some embodiments, the first immunotherapy and the second immunotherapy are both anti-infection immunotherapies. In some embodiments, a method of treatment of cancer further comprises administration of a third immunotherapy after a predetermined second duration. In some embodiments, at least one immunotherapy administered is provided on multiple occasions according to a prime + booster sequence. In some embodiments, two immunotherapies administered are provided on multiple occasions and the predetermined first duration is measured as from the date / time of the first administration of a given therapy; in such circumstances, administration of the first immunotherapy's sequence may not be complete before the second immunotherapy's sequence begins.

[0055] In some embodiments, an immunotherapy used as a first, second, or third immunotherapy is a vaccine intended to enhance immune response to a virus not known to cause cancer. In some embodiments, the virus is a coronavirus. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the virus is an influenza virus. In some embodiments, the virus is a seasonal flu virus. In some embodiments, the virus is a rotavirus. In some embodiments, the virus is measles. In some embodiments, the virus is chicken pox. In some embodiments, the virus is one or more viruses commonly immunized against as part of a vaccination schedule recognized by the field of public health. In some embodiments, the virus is selected from HIVl, HIV2, HTLV1, Epstein Barr virus (EBV), cytomegalovirus (CMV), adenovirus, adeno-associated virus, BK virus, Human Herpesvirus 6, Human Herpesvirus 8, influenza A virus, influenza B virus parainfluenza virus, avian flu virus, MERS and SARS coronaviruses, Crimean Congo Hemorrhagic fever virus, rhino virus, enterovirus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Lassa fever virus, zika virus, RSV, measles virus, mumps virus, rhino virus, varicella virus, herpes simplex virus 1 and 2, varicella zoster virus, HIV-l, HTLV1, Hepatitis virus, enterovirus, hepatitis B virus, Hepatitis C virus, Nipah and Rift valley fever viruses, and Japanese encephalitis virus.

[0056] In some embodiments, an immunotherapy used as a first, second, or third immunotherapy is an immunostimulatory agent derived from a microbe. In some embodiments, the immunostimulatory agent is derived from Bacillus Calmette-Guerin (BCG). In some embodiments, the bacterium is selected from BCG, Mycobacteriumtuberculosis, atypical mycobacteria species, Pneumocystis jirovecii,toxoplasmosis, Rickettsia, Nocardia, Aspergillus, Mucor, and Candida.

[0057] In some embodiments, an immunotherapy used as a first, second, or third immunotherapy is a vaccine intended to enhance immune response to presentation of one or more indicia of infectious microorganisms, where the microorganisms are selected from a list of the most common anti-infective TCRs found endogenously in the subject's species. In some embodiments, an immunotherapy used as a first, second, or third immunotherapy is a vaccine intended to enhance immune response to presentation of an infectious microorganism indicium, the subject is a human, and the infectious microorganism is selected from AdV, CMV, Coxsackie virus B, Dengue, EBV, HCV, HCoV-HKUl, HHV, HIV-l, HPV, HPV-16, HSV-2,HTLV-1, influenza A, influenza B, LCMV, MCM, MVPyV, RSV, rotavirus A, SARS-CoV, SARS-CoV-2, SIV, VSV, YFV, Plasmodium berghei, Plasmodium falciparum, E. coli, M. tuberculosis, Mtb, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Streptomyces kanamyceticus.

[0058] In some embodiments, an immunotherapy used as a first, second, or third immunotherapy comprises a composition effecting the production of both anti-infection and anti-tumor antigens. In some embodiments, the immunotherapy comprises mRNA encoding for antigenic transcripts from two or more major infectious microorganisms. In some embodiments, the immunotherapy comprises mRNA encoding for antigenic transcripts from CMV, EBV, influenza, RSV, and SARS-CoV-2. In some embodiments, the immunotherapy comprises mRNA encoding for antigenic transcripts from one or more major infectious microorganisms and one or more tumor types. In some embodiments, the tumor antigen is selected based on analysis of the subject's own tumor, e.g., via TCR sequencing of the subject's tumor-infiltrating CD8+T cells. In some embodiments, an immunotherapy containing both anti-infection and anti-tumor mRNA is intratumorally administered to a subject's cancer.

[0059] In some embodiments, an immunotherapy used as a first, second, or third immunotherapy is an ICI. In some embodiments, one or more anti-infection immunotherapies are administered to promote the immunologically desired T cell phenotype or phenotypes within the TME, and an ICI is administered thereafter. In some embodiments, the ICI is an anti-programmed cell death protein 1 (anti-PD-1) antibody, such as nivolumab, pembrolizumab , pidilizumab, PF-06801591, BGB-A317, GLS-010 (WBP-3055), AK-103 (HX-008), MGA-012, BI-754091, cemiplimab, tislelizumab,retifanlimab, toripalimab, AGEN-2034, JS-OO1, JNJ-63723283, genolimzumab, LZM-009, BCD-100, LY-3300054, SHR-1201, or BAT-1306. In some embodiments, the ICI is an antiprogrammed death-ligand 1 (anti-PD-Ll) antibody such as BMS-936559, atezolizumab, durvalumab, avelumab, CK-301, MSB0010718C, MEDI0680, CX-072, CBT-502, spartalizumab, dostarlimab, cosibelimab, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308, FAZ-053, or MDX1105-01. In some embodiments, the ICI is an anti-CTLA-4 antibody such as ipilimumab ortremelimumab. In some embodiments, the immune checkpoint is blockaded using anti-PDl or targets other immune checkpoint modulators such as PDL1, PDL2, TIM3, TIG IT, VISTA, CTLA-4, CD28, 0X40 (CD134), 4-1BB (CD137), GITR (Glucocorticoid-Induced TNFR-related protein), ICOS (Inducible T-cell CoStimulator), CD40, CD47, BTLA (B and T Lymphocyte Attenuator), Siglec-15, NKG2A, or combinations thereof. In some embodiments, an immunotherapy used as a first, second, or third immunotherapy is a metabolic checkpoint modulator, especially metabolic checkpoints that affect immune functions. In some embodiments, the metabolic checkpoint modulator is selected from Cd38, NAMPT, CD73, A2AR (Adenosine A2A Receptor), IDO1, TDO, Arginase 1, SLC7A11 (Cystine), GLUT1 , CD36, or combinations thereof.

[0060] In some embodiments, one or more anti-infection immunotherapies are administered pre-operatively to promote the immunologically desired T cell phenotype or phenotypes within the TME, followed by "repo-challenge" intratumorally.

[0061] In some embodiments, the cancer treated is pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer or colon cancer. In preferred embodiments, the cancer treated is non-small cell lung cancer, small cell lung cancer, melanoma, prostate cancer, or sarcoma.

[0062] In some embodiments, an immunotherapy used as a first, second, or third immunotherapy is a vaccine intended to enhance immune response to an oncolytic virus. In some embodiments, the oncolytic virus is a human papillomavirus. In some embodiments, the oncolytic virus is Epstein-Barr virus. In some embodiments, the oncolytic virus is Hepatitis B virus. In some embodiments, the oncolytic virus is Hepatitis C virus.

[0063] In some embodiments, described herein are methods of treating a subject with a cancer said method including administering to the subject a therapeutically effective amount of a nucleic acid described herein, a composition described herein, a nanoparticle described herein, a lipid nanoparticle described herein, a vaccine described herein, or a pharmaceutical composition described herein.

[0064] A composition (e.g., vaccine pharmaceutical composition) described herein can be formulated into a dosage form described herein or as understood in the art, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous). The compositions as used in the methods described herein can be administered by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the active components described herein can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral and parenteral routes of administering. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Intratumoral injection is also contemplated.

[0065] COMPOSITIONS

[0066] Compositions are disclosed herein comprising novel therapeutic or prophylactic agents that present at least a portion of an infectious microorganism indicium efficient to prime T cells within a tumorto that indicium. In some embodiments, the indicium is a viral indicium. In some embodiments, the indicium is a bacterial indicium. In some embodiments, the indicium is a fungal indicium. In some embodiments, the indicium is a parasitic indicium.

[0067] In some embodiments, a composition comprises an oligonucleotide encoding a portion of one or more non-natural TCR antigen binding domain(s), the one or more non-natural TCR antigen binding domain(s) correspondingly binding to their respective or similar disease-associated antigens that are associated with HIVl, HIV2,HTLV1, Epstein Barr virus (EBV), cytomegalovirus (CMV), adenovirus, adeno- associated virus, BK virus, Human Herpesvirus 6, Human Herpesvirus 8, influenza A virus, influenza B virus parainfluenza virus, avian flu virus, MERS and SARS coronaviruses, Crimean Congo Hemorrhagic fever virus, rhino virus, enterovirus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Lassa fever virus, zika virus, RSV, measles virus,mumps virus, rhino virus, varicella virus, herpes simplex virus 1 and 2, varicella zoster virus, HIV-l, HTLV1, Hepatitis virus, enterovirus, hepatitis B virus, Hepatitis C virus, Nipah and Rift valley fever viruses, Japanese encephalitis virus, Mycobacterium tuberculosis, atypical mycobacteria species, Pneumocystis jirovecii, toxoplasmosis, Rickettsia, Nocardia, Aspergillus, Mucor, and / or Candida.

[0068] In some embodiments, the composition comprises a vaccine platform having mRNA encoding for antigenic transcripts selected from two or more infectious microorganism indicia. In some embodiments, the indicia are selected from two or more of SARS-Cov-2, Influenza, RSV, EBV, CMV, HIV, HCV, Dengue, typhoid, and Strep.

[0069] In some embodiments, the composition comprises a vaccine platform having mRNA encoding for antigenic transcripts selected from one or more infectious microorganism indicia and transcripts selected from tumor antigens. In some preferred embodiments, the composition described above is administered intratumorally. In some embodiments, the tumor antigens are preselected with reference to TCR sequencing information from a subject's tumor infiltrating T cells.

[0070] Antigens from other pathogenic organisms may also be used in compositions described herein. Exemplary pathogens from which antigens may be obtained from include, but are not limited to, Bacillus, including Bacillus anthracis; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi;Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C. perfringens; Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. Y. lamblia, Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida; Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis; Treponema, e.g. T palladium; B. anthracis, Y. pestis, Brucella spp., F. tularensis, B. mallei, B. pseudomallei, B. mallei, B. pseudomallei, C. botulinum, Salmonella spp., SEB V. cholerae toxin B, E. coli O157:H7, Listeria spp., Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Enterobacter sp., Klebsiella sp., Listeria sp., Mycoplasma ssp., Francisella spp., Bartonella spp., Borrelia spp., Campylobacter spp., Chlamydia spp., Simkan ia spp., Ehrlichia spp., Enterococcus spp., Coccidioides spp., Bordetella spp., Coxiella spp., U reaplasma spp., Trichomatis spp., Helicobacter spp., Legionella spp., Mycobacterium spp., C orynebacterium spp., Rhodococcus spp., Rickettsia spp., Arcanobacterium spp., Listeria spp. , Treponema spp., Brucella spp., Campylobacter spp., Pasteurella spp., Pseudomonas ssp., Burkholderii spp. and the like, orthomyxoviruses, (e.g. influenza virus), paramyxoviruses (e.g., respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses (human and porcine), coronaviruses, reoviruses, togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variola virus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus), hepatitis viruses (including A, B, C and E), herpesviruses (e.g., Herpes simplex virus, HV-I and HV-II, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus), rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus (e.g. rabies virus), retroviruses (including HIV-l, HIV-2, HTLV-I and —II), papovaviruses (e.g. papillomavirus), polyomaviruses, picornaviruses, Dengue virus, Filoviruses (e.g., Marburg and Ebola viruses), Hantavirus, Rift Valley virus, HHV-8, Human papillomavirus, Bovine leukemia virus, Influenza virus, Guanarito virus, Lassa virus, Measles virus, Rubella virus, Mumps virus, Chickenpox (Varicella virus), Monkey pox, Epstein Bahr virus, Parvovirus B19, Hantaan virus, Sin Nombre virus, Venezuelan equine encephalitis, Sabia virus, West Nile virus, Yellow Fever virus, and the like, causative agents of transmissible spongiform encephalopathies, Creutzfeldt-Jakob disease agent, variant Creutzfeldt-Jakob disease agent, Candida strains including C. glabrata, C. albicans, C. krusei, C. lusitaniae and C. maltosa, as well as species of Aspergillus, Cryptococcus, Histoplasma, Coccidioides, Blastomyces, and Penicillium, Cryptcooccus, Cryptosporidium, Giardia lamblia, Microsporidia, Plasmodium vivax, Plasmodium falciparum, Pneumocystis carinii, Toxoplasma gondii, Trichophyton mentagrophytes, Enterocytozoon bieneusi, Cyclospora cayetanensis, Encephalitozoon hellem, Encephalitozoon cuniculi, Ancylostama, Strongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator, Enterobius, Strongyloides and Wuchereria;Acanthamoeba and other amoebae, Cryptosporidium, Fasciola, Hartmanella, Acanthamoeba, Giardia lamblia, Isospora belli, Leishmania, Naegleria,Plasmodium spp., Pneumocystis carinii, Schistosoma spp., Toxoplasma gondii,and Trypanosoma spp., among other viruses, bacteria, archaea, protozoa, fungi, and the like).

[0071] In an embodiment, the disease-associated antigens are selected from Hepatitis A virus cellular receptor 1; Hep B gpl20; human papilloma virus E6; human papilloma virus E7; HIV1 envelope glycoprotein; HIVl-gag; HTLVl-Tax: CMV pp65; EBV-EBNA3c; influenza A hemagglutinin (HA); herpes simplex virus (HSV) gB glycoprotein; HIV gpl20; HIV 11 IB gp 120V3 loop; human cytomegalovirus (HCMV); HSV gD glycoprotein; respiratorysyncytial virus (RSV) F; RSV Fgp; and GAD.

[0072] In some embodiments, a composition comprises a vaccine platform having mRNA encoding for an immunologically recognizable portion of each of CMV, EBV, influenza, RSV, and SARS-CoV-2. In some embodiments, vaccines include a composition including a single chain trimer nucleic acid (e.g., mRNA) encoding a first T cell epitope, a |32-microglobulin, and a first MHC heavy chain sequence. In some embodiments, also described herein are vaccines including a composition including a single chain nucleic acid dimer encoding a T cell epitope, and a primary MHC heavy chain sequence.

[0073] Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetra hydrofurfury I alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0074] Injectable compositions, for example, injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be an injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents for pharmaceutical or cosmetic compositions that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w / v) sodium carboxymethyl cellulose and 0.1% (v / v) Tween 80. The injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form ofsterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0075] Solid compositions include capsules, tablets, pills, powders, and granules. In such solid compositions, the particles are mixed with at least one excipient and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0076] Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the nanoparticles in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the particles in a polymer matrix or gel.

[0077] DELIVERY METHODS

[0078] For embodiments delivering nucleic acids, delivery vehicles can include lipid-based (e.g., a liposome formulation, lipoplexes, or lipid nanoparticles (LNP)), viral-based, or physical methods such as injection, microinjection, electroporation, ultrasound, gene gun, hydrodynamic applications, or any combination thereof.

[0079] In some embodiments, a composition comprises a nanoparticle. In some embodiments, the nanoparticle can be a lipid nanoparticle. In some embodiments, the nanoparticles can be a lipid-polycation complex, referred to as a cationic lipid nanoparticle. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and / or polyarginine. In some embodiments, the lipid nanoparticle can include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE). A lipid nanoparticle formulation may beinfluenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG- modified lipid. In some embodiments, a lipid nanoparticle formulation comprises at least one ionizable lipid, cationic lipid, or any combination thereof; a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic I ipid :5-25% neutral I ipid :25- 55% sterol; 0.5-15% PEG-lipid.

[0080] In some embodiments, a lipid nanoparticle formulation includes 25% to 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., 35 to 65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis. In some embodiments, lipid nanoparticle formulations include 25-75% of a cationic lipid, 0.5-15% of the neutral lipid, 5- 50% of the sterol, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 35-65% of a cationic lipid, 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 45-65% of a cationic lipid, 5-10% of the neutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 60% of a cationic lipid, 7.5% of the neutral lipid, 31% of the sterol, and 1.5% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid, 10% of the neutral lipid, 38.5% of the sterol, and 1.5% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid, 10% of the neutral lipid, 35% of the sterol, 4.5% or 5% of the PEG or PEG-modified lipid, and 0.5% of the targeting lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 40% of a cationic lipid, 15% of the neutral lipid, 40% of the sterol, and 5% of the PEG or PEG- modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations include 57.2% of a cationic lipid, 7.1% of the neutral lipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticleformulations include 57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA, 7.5% of the neutral lipid, 31.5% of the sterol, and 3.5% of the PEG or PEG-modified lipid on a molar basis. In some embodiments, lipid nanoparticle formulations including a lipid mixture in molar ratios of 20-70% cationic I ipid :5-45% neutral lipid : 20-55% cholesterol: 0.5- 15% PEG-modified lipid. In some embodiments, lipid nanoparticle formulations including a lipid mixture in a molar ratio of 20-60% cationic lipid :5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid. In some embodiments, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG), 57.2 / 7.1 / 34.3 / 1.4 (mol % cationic lipid / neutral lipid, e.g., DPPC / Chol / PEG-modified lipid, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 50 / 10 / 35 / 4.5 / 0.5 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DSG), 50 / 10 / 35 / 5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 40 / 10 / 40 / 10 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or 52 / 13 / 30 / 5 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG- cDMA).

[0081] In some embodiments, lipid nanoparticle formulations may comprise a cationic lipid, a PEG lipid and a structural lipid and optionally comprise a non-cationic lipid. As a nonlimiting example, a lipid nanoparticle may comprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid. As another nonlimiting example, the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yet another non-limiting example, a lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid.

[0082] In some embodiments, lipid nanoparticle formulations described herein may be 4 component lipid nanoparticles. The lipid nanoparticle may comprise a cationic lipid, a noncationic lipid, a PEG lipid and a structural lipid. As a non-limiting example, the lipid nanoparticle may comprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid. As another non-limiting example, the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yet another non-limiting example, the lipid nanoparticle maycomprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid.

[0083] In some embodiments, lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid. As a non-limiting example, the lipid nanoparticle comprises 50% of the cationic lipid DLin-KC2-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprises 50% of the cationic lipid DLin-MC3-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprises 50% of the cationic lipid DLin-MC3-DMA, 10% of the noncationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of the structural lipid cholesterol. As yet another non-limiting example, the lipid nanoparticle comprises 55% of the cationic lipid L319, 10% of the non-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of the structural lipid cholesterol.

[0084] In some embodiments, the nanoparticles may be such, or incorporate such ingredients in proportions described herein, as those described in U.S. Patent No. 12,208,288;11,622,972; 11,524,023; 11,485,972; 11,464,848; 10,933,127; 10,898,574; 10,703,789; 10,702,600; 10,577,403; 10,442,756; 10,266,485; 10,064,959; or 9,868,692; the contents of each of which is herein incorporated by reference in its entirety.

[0085] In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm.

[0086] The lipid nanoparticles described herein may be made in a sterile environment. In some embodiments, the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle. As a non-limiting example, the lipid particle may comprise one or more active agents or therapeutic agents; one or more cationic lipids comprising from 50 mol % to 85 mol % of the total lipid present in the particle; one or more non-cationic lipids comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and one or more conjugated lipids that inhibit aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle.

[0087] The nanoparticle formulations may comprise a conjugate to enhance the delivery of nanoparticles of the present disclosure in a subject.

[0088] In some vaccine embodiments, lipid nanoparticles can include 20% to 80% of an ionizable lipid, cationic lipid, or any combination thereof; greater than 0 % to 5 % polyethylene glycol-l ipid; greater than 0 % to 20 % helper lipids; 20 % to 80 % sterol; and a single chain trimer nucleic acid encoding a first T cell epitope, a |32-microglobulin, and a MHC heavy chain sequence encapsulated in the lipid nanoparticle. In some embodiments, lipid nanoparticles can include 20% to 80% of an ionizable lipid, cationic lipid, or any combination thereof; greater than 0 % to 5 % polyethylene glycol-l ipid; greater than 0 % to 20 % helper lipids; 20 % to 80 % sterol; and a single chain trimer nucleic acid encoding a first T cell epitope, a |32-microglobulin, and a MHC heavy chain sequence encapsulated in the lipid nanoparticle. In one embodiment, the nanoparticle can include from 40% to 60% of an ionizable lipid, cationic lipid, or any combination thereof; from 1% to 2% polyethylene glycol-lipid; from 8% to 12% helper lipids; from 35% to 40% sterol; and a single chain trimer nucleic acid encoding a first T cell epitope, a |32-microglobulin, and a MHC class I heavy chain sequence encapsulated in the lipid nanoparticle. In one embodiment, the nanoparticle can include from 40% to 60% of an ionizable lipid, cationic lipid, or any combination thereof; from 1% to 2% polyethylene glycol-lipid; from 8% to 12% helper lipids; from 35% to 40% sterol; and a single chain trimer nucleic acid encoding a first T cell epitope, a |32-microglobulin, and a MHC class I heavy chain sequence encapsulated in the lipid nanoparticle.

[0089] In some embodiments, a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include, without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments, the formulation includes 5% to 50% on a molar basis of the sterol (e.g., 15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis. A non-limiting example of a sterol is cholesterol. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 20% on a molar basis of the PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1,500 Da, around 1,000 Da, or around 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearoyl glycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA.

[0090] In some embodiments, the composition may be encapsulated in, linked to and / or associated with zwitterionic lipids. In some embodiments, the nanoparticle includes a sterol.

[0091] Suitable cationic lipids may include, but are not limited to, 2-amino-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-l-yloxy]-2-{[(9Z)-octadec- 9-en-l-yloxy]methyl}propan-l-ol; 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2- [(octyloxy)methyl]propan-l-ol; and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-l- yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-ol; or any pharmaceutically acceptable salt or stereoisomer thereof. As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)— N,N-dimethylnonacosa-20,23-dien-10- amine, (17Z,20Z)— N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)— N5N- dimethylpentacosa-16, 19-dien-8-amine, (13Z,16Z)— N,N-dimethyldocosa-13,16-dien-5- amine, (12Z,15Z)— N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N- dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)— N,N-dimethyltetracosa-15,18-dien-7- amine, (18Z,21Z)— N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)— N,N- dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)— N,N-dimethyltricosa-14,17-dien-4- amine, (19Z,22Z)— N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21 Z)-N,N- dimethylheptacosa-18,21-dien-8 amine, (17Z,20Z)— N,N-dimethylhexacosa-17,20-dien-7- amine, (16Z,19Z)— N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N- dimethylhentriaconta-22,25-dien-10-amine, (21 Z,24Z)— N,N-dimethyltriaconta-21,24-dien- 9-amine, (18Z)— N,N-dimetylheptacos-18-en-10-amine, (17Z)— N,N-dimethylhexacos-17- en-9-amine, (19Z,22Z)— N,N-dimethyloctacosa-19,22-dien-7-amine, N,N- dimethylheptacosan-10-amine, (20Z,23Z)— N-ethyl-N-methylnonacosa-20,23-dien-10- amine, l-[(llZ,14Z)-l-nonylicosa-ll,14-dien-l-y I] pyrrolidine, (20Z)— N,N- dimethylheptacos-20-en-10-amine, (15Z)— N,N-dimethyl eptacos-15-en-10-amine, (14Z)— N,N-dimethylnonacos-14-en-10-amine, (17Z)— N,N-dimethylnonacos-17-en-10-amine, (24Z)— N,N-dimethyltritriacont-24-en-10-amine, (20Z)— N,N-dimethylnonacos-20-en-10- amine, (22Z)— N,N-dimethylhentriacont-22-en-10-amine, (16Z)— N,N-dimethylpentacos- 16-en-8-amine, (12Z,15Z)— N,N-dimethyl-2-nonylhenicosa-12,15-dien-l-amine, (13Z,16Z)— N,N-dimethyl-3-nonyldocosa-13,16-dien-l amine, N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropyl]eptadecan-8-amine, l-[(lS,2R)-2-hexylcyclopropyl]-N,N- dimethylnonadecan-10-amine, N,N-dimethyl-l-[( IS, 2R)-2-octylcyclopropyl] nonadecan-10-amine, N, N-d imethy I-21-[(1S, 2R)-2-octylcyclopropyl] hen icosan-10-a mine, N,N-dimethyl-l- [(lS,2S)-2-{[(lR,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N- dimethyl-l-[(lS,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(lR,2S)-2- undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(lS,2R)-2- octylcyclopropyl]heptyl}dodecan-l-amine, l-[(lR,2S)-2-heptylcyclopropyl]-N,N- dimethyloctadecan-9-amine, l-[(lS,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6- amine, N, N-d imethyl-l-RlS,2R)-2-octylcyclopropyllpentadecan-8-a mine, R— N,N-dimethyl- l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, S— N,N-dimethyl-l- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, l-{2-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-l-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-l- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-[(5Z)-oct-5-en-l-yloxy]propan-2-amine, l-{2- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-l-[(octyloxy)methyl]ethyl}azetidine, (2S)-1- (hexyloxy)-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-l-yloxylpropan-2-amine, (2S)-1- (heptyloxy)-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-l-yloxylpropan-2-amine, N,N- dimethyl-l-(nonyloxy)-3-R9Z,12Z)-octadeca-9,12-dien-l-yloxylpropan-2-amine, N,N- dimethyl-l-[(9Z)-octadec-9-en-l-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-l- [(6Z,9Z,12Z)-octadeca-6,9,12-trien-l-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)- icosa-ll,14-dien-l-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-l-(hexyloxy)-3- [(llZ,14Z)-icosa-ll,14-dien-l-yloxy]-N,N-dimethylpropan-2-amine, l-[(llZ,14Z)-icosa- ll,14-dien-l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, l-[(13Z,16Z)-docosa-13,16- dien-l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-l-[(13Z,16Z)-docosa-13,16- dien-l-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-l-[(13Z)-docos-13-en-l- yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, l-[(13Z)-docos-13-en-l-yloxy]-N,N- dimethyl-3-(octyloxy)propan-2-amine, l-[(9Z)-hexadec-9-en-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(l-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca- 9,12-dien-l-yloxy]propan-2-amine, (2R)-l-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3- R9Z,12Z)-octadeca-9,12-dien-l-yloxylpropan-2-amine, N,N-dimethyl-l-(octyloxy)-3-({8- RlS,25)-2-{[(lR,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-l-l [8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,2OZ,23Z)— N,N-dimethylnonacosa-ll,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[0092] In some embodiments, the lipid may be a cationic lipid such as, but not limited to, DLin- DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids.

[0093] In some embodiments, the nanoparticle comprises an ionizable lipid in a molar ratio of from 0% to 80%. In some embodiments, the ionizable lipid can be present in a molar ratio of at least 0%, (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%). In some embodiments, the ionizable lipid can be present in a molar ratio of 80% or less, (e.g., 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, or 0.5% or less).

[0094] The ionizable lipid can be present in a molar ratio ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the ionizable lipid can be present in a molar ratio of from 0% to 80% (e.g., from greater than 0% to 80%, from greater than 0% to 70%, from greater than 0% to 60%, from greater than 0% to 50%, from greater than 0% to 40%, from greater than 0% to 30%, from greater than 0% to 20%, from greater than 0% to 10%, from greater than 0% to 5%, from greater than 0% to 1%, from greater than 0% to 0.5%, from 1% to 30%, from 1% to 20%, from 1% to 10%, from 1% to 5%, from 5% to 30%, from 5% to 20%, from 5% to 10%, from 10% to 30%, from 10% to 20%, from 20% to 30%, from 20% to 40%, or from 30% to 40%.

[0095] In some embodiments, the nanoparticle comprises a helper lipid. In some embodiments, the helper lipid can be a non-cationic lipid. In some embodiments, the noncationic lipid can include, but is not limited to, l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l-stearoyl-2-oleoyl-sn-glycero- 3-phosphoethanolamine (SOPE), DPPC (l,2-dipalmitoyl-sn-glycero-3- phosphocholine), 1,2- dioleyl-sn-glycero-3-phosphotidylcholine (DOPC), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), l,2-dioleoyl-5 / 7-glycero-3- phospho-(l'-rac-glycerol) (DOPG), or combinations thereof. In one embodiment, the non-cationic lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the non-cationic lipid is l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE), In one embodiment, the non-cationic lipid is 1,2-distearoyl- sn-glycero-3-phosphocholine (DSPC). In one embodiment, the non-cationic lipid is 1- stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). While several non-cationic lipids are described here, additional non-cationic lipids can be used in combination with the compounds disclosed herein.

[0096] In some embodiments, the nanoparticle comprises a helper lipid in a molar ratio of from 0% to 20%. In some embodiments, the nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of about 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 15%, or 20%.

[0097] In some embodiments, nanoparticles may be hydrophobic polymer particles.

[0098] In some embodiments, the composition of the present disclosure can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In some embodiments, the compositions may be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term "encapsulate" means to enclose, surround or encase. As it relates to the formulation of the compounds of the disclosure, encapsulation may be substantial, complete or partial. The term "substantially encapsulated" means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. "Partial encapsulation" means that less than 10, 10, 20, 30, 40, 50 or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the disclosure using fluorescence and / or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the disclosure are encapsulated in the delivery agent.

[0099] In some embodiments, the compositions described herein formulated for controlled release and / or targeted delivery may also include at least one degradable polyester which may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

[0100] BIOLOGICAL EXAMPLES

[0101] Example 1 - in vitro production of infectious agent-specific antigen in cancer cells

[0102] HEK293T cell line and B16F10, MCA205, TC-1, and LLC cancer cell lines were separately grown in appropriate media. Each cell type was exposed to either the Moderna Spikevax or Pfizer Comirnaty COVID-19 mRNA vaccine and compared to the negative control condition. After 16 h, Western blot and FACS cell sorting were performed to measure the level of the SARS-CoV-2 spike protein. It was determined that these cancer cells expressed spike protein following treatment by either vaccine in vitro. E.g., in FACS analysis, spike protein SI was found in 73.2% B16F10 cells treated with Moderna, 50.0% B16F10 cells treated with Pfizer, and none in the control condition. Western immunoblotting (FIG. 1) showed (1A) that S2 is expressed once HEK293T cells are at 1, 2, 3, and 6 ug dosing of Moderna and Pfizer vaccine; (IB) that SI is expressed at 3 and 6 ug for either vaccine in HEK293T; and (1C) that S2 is expressed in HEK293T, B16F10, MCA205, and LLC1. These findings show that spike protein antigens - a target of antiviral T cells - can be made within these cell lines.

[0103] Further, these cells were subjected to FACS analysis forthe presence of MHC epitopes and immune checkpoint components. B16F10 cells treated with Pfizer or Moderna expressed significantly more H2Db+ and H2Kb+ MHCs (*** p<0.001). MCA205 cells treated with Moderna, but not Pfizer, expressed significantly less PDL-1 (* p<0.05). These findings demonstrate that the production of spike protein in a cancer cell can have significant effects on immunologically relevant molecules.

[0104] Example 2 - cell viability of cancer cells in vitro

[0105] COVID-19 mRNA

[0106] MCA205 cells were exposed to 0, 1, 2, 4, 5, 10, or 15 uL of Moderna vaccine formulation and their live, dead, and Spike-2 positive populations were measured at 16 h and 24 h (FIG.2).

[0107] These findings demonstrate that in vitro Moderna vaccine treatment induces cell death in MCA205 cells in a concentration-dependent manner. In particular, there is an inverse relationship seen between the increase in dead cell % (middle graph) and the decrease in live cell % (top graph) as concentration increases.

[0108] Example 3 - ex vivo peptide stimulation for CD8+T cell activation

[0109] C57BL / 6 mice were given intramuscular injection of either Moderna or Pfizer COVID-19 mRNA vaccine, with two doses fifteen days apart. Seven days after the second dose (Day 22), the mice were sacrificed and their spleens harvested. These spleens were then exposed to various segments of the spike protein, or anti-CD28 antibody, or in the presence of anti-CD28 antibody or ConA immunostimulation. The sequences of thepeptides were as follows: CVNFNFNGL (S538-546) (SEQ ID NO:1); DLLFNKVTL (S82o-828) (SEQ ID NO:2); VNFNFNGL (S539-546) (SEQ ID NO:3); VVVLSFEL (S510-517) (SEQ ID NO:4);VTWFHAIHVSGTNGT (S62-76) (SEQ ID NO:5). See FIG. 3.

[0110] The results of this experiment showed that CD8+, I FN-y+and CD4+, I FN-y+cells were significantly increased relative to the control condition when exposed to several of these peptides, which illustrates that spleens from in vivo vaccine-treated mice were immunologically primed to respond to spike protein segments.

[0111] Further, using a phycoeythrin tetramer stain for quantification of antigen-specific CD8+T cells, it was established that spleens from Pfizer ("PFIZ" striped bars) or Moderna ("MOD" solid bars) treated animals had significantly greater antigen-specific T cells than the control condition ("NT" hollow bars); thus, treatment with antiviral therapy resulted in functionally competent T cells.

[0112] Vaccine-induced antiviral CD8+T cells in this experiment were also shown to express immune checkpoints that can be synergistically harnessed via ICI therapy. Specifically, spleen T cells were shown to express CD3, CD8, CD19, PD-1, and TIM3.

[0113] Vaccine-induced antiviral CD8+T cells in this experiment were also shown to have a differentiation profile known to correlate with good prognosis from cancers (FIG. 4). TN = naive; TCM = central memory; TEM = effector memory.

[0114] Example 4 - in vivo sarcoma model

[0115] C57BL / 6 were given intramuscular injection of either Moderna or Pfizer COVID-19 mRNA vaccine, with two doses fifteen days apart (a control group received no immunization). Seven days after the second dose (Day 22), 2 x 105MCA205 cancer cells were injected subcutaneously. Ten further days were allowed to elapse (Day 32), after which the mice were sacrificed and the tumors and spleens were harvested for tetramer stain as in the previous Example.

[0116] The results showed that, as previously shown in Example 3, the spleen was highly enriched in functionally competent, tetramer-positive T cells in vaccinated mice (either Moderna or Pfizer) relative to the control condition; this experiment showed that such cells persisted in the spleen even 17 days post immunization. Satisfyingly, T cells in the tumor were also highly enriched in functionally competent, tetramer-positive T cells in vaccinated mice relative to the control condition (FIG. 5). Within-subject levels of T cells in tumor corresponded to levels of T cells in spleen.

[0117] This result underscores that the antiviral CD8+T cells produced by immunization also infiltrate the TME.

[0118] Example 5 - co-culture cancer killing with antiviral CD8+T cells

[0119] C57BL / 6 mice were treated with Moderna, Pfizer, or no vaccine. Seven days later, the mice were sacrificed and splenocytes were isolated. On the day of sacrifice, CTV-labeled MCA205 cancer cells were separately grown in appropriate media, exposed to Moderna, Pfizer, or no treatment, and left for 16 h. The splenocytes were then added to the in vitro system in a 40:1 E:T ratio to form a co-culture. 24 hours later, the viability of the cancer cells was measured. It was determined that co-cultures containing ex vivo splenocytes from the mice immunized with a single dose of Pfizer anti-COVID-19 vaccine and treated in vitro with either the Moderna (**** p<0.001) or Pfizer vaccine (* p<0.05) had significantly more dead cancer cells in the co-culture system than any other experimental condition (FIG. 6).

[0120] These results show that antiviral T cells can be exploited within antitumor functions.

[0121] Example 6 - in vivo sarcoma model

[0122] C57BL / 6 were given intramuscular injection of either Moderna or Pfizer COVID-19 mRNA vaccine, with two doses fifteen days apart (a control group received no immunization). Seven days after the second dose (Day 22), 2 x 105MCA205 cancer cells were injected subcutaneously; these cancer cells had been treated ex vivo with Moderna, Pfizer, or nothing (control group). Mixed therapy (e.g., Moderna in vivo + Pfizer treated cells) was not evaluated. Thus there were seven groups (see FIG. 7): naive with nonpretreated tumor (red circle; depicted on all graphs); naive in vivo with Moderna-ex vivo treated tumor (blue square, top graphs); naive with Pfizer-ex vivo treated tumor (blue square, bottom graphs); Moderna in vivo vaccination with non-ex vivo pretreated tumor (purple triangle, top graphs); Moderna in vivo vaccination with Moderna-ex vivo treated tumor (green triangle, top graphs); Pfizer treated with non-pretreated tumor (purple triangle, bottom graphs); Pfizer treated with Pfizer-treated tumor (green triangle, bottom graphs)). Mean tumor volume and survival were charted throughout the study until 65 days post tumor inoculation. The results showed that groups with both vaccinated mice and immunized cancer cells had profoundly smallertumors. 100% of such groups (green triangles) survived to the end of the experiment. In fact, the Moderna vaccinated and ex vivo pre-treated group was rechallenged with cancer cells at day 112 and was found to produce much smaller tumors than age-matched naive controls, suggesting that this therapeutic approach drives further generation of antitumor immunity over the long term.

[0123] Example 7 - in vivo melanoma model

[0124] Analogous to Example 6, an in vivo model was performed using in vivo vaccination with COVID-19 mRNA vaccine and B16F10 cells that were immunized with COVID-19 mRNA vaccine prior to injection into mice. Mice that were both vaccinated in vivo and injected with cancer cells immunized ex vivo (either with Moderna or Pfizer vaccines) had profoundly smaller tumors and survived to the end of the experiment (FIG. 8).

[0125] Example 8 - CD8 dependence of efficacious immunotherapy in a melanoma in vivo model

[0126] C57BL / 6 mice were given intramuscular injection of Moderna COVID-19 mRNA vaccine, with two doses fifteen days apart. Two days after the second dose (Day 17) and on several days thereafter (Days 18, 19, 21), an antibody (either an anti-CD8 antibody or an isotope control antibody) was injected intraperitoneally. Seven days after the second dose (Day 22), 2 x 105B16F10 cancer cells were injected subcutaneously, these cells having been treated ex vivo with Moderna or negative control. Finally, the i.p. antibody injections were correspondingly repeated on Days 25, 28, 31, and 34. Thus, four groups were created: Moderna i.m., non-treated cancer cells, control antibody ( 10_l); Moderna i.m., nontreated cancer cells, anti-CD8 antibody ( 10_3); Moderna i.m., Moderna ex vivo-treated cancer cells, control antibody ( 10_5); Moderna i.m., Moderna ex vivo-treated cancer cells, anti-CD8 antibody (10_7).

[0127] The hypothesis of this experiment was that groups 10_l and 10_3 should have similar survival, since the cancer cells had not been induced to produce antigen, but 10_5 and 10_7 should be different provided the anti-cancer effect is mediated by CD8+T cells. And indeed, this is what was observed. The non-treated cancer cells in groups 10_l and 10_3 produce tumors that kill all mice by day 25 post tumor inoculation; mice in group 10_7 have their antiviral CD8+T cell response ablated by the anti-CD8 antibody treatment, resulting in 0% survival by day 39 post inoculation; but most mice in group 10_5 (Moderna ex vivo-treated cancer cells, control antibody) are still alive by day 40 post inoculation.

[0128] Example 8B - Model systems for studying vaccine-induced antitumor immunity

[0129] MCA205 and B16F10 cancer cells were genetically modified to produce the COVID-19 spike protein. These cells were shown to have a much larger expression of spike protein (FIG. 9, leftmost peaks) than wild-type cells (FIG. 9, rightmost peaks). Characterization of these cell lines demonstrated (as shown in the Western blot in FIG. 9 for MCA205) that spike protein was produced in vivo when these cells were implanted.

[0130] Example 9 - In vivo sarcoma model

[0131] In vivo experiments described above utilized cancer cell lines that were treated ex vivo with an mRNA anti-COVID-19 vaccine composition. As a demonstration of the broad applicability of the methods described herein, MCA205 cells were modified to overexpress spike protein (as described above). C57BL / 6 were given intramuscular injection of Moderna COVID-19 mRNA vaccine or PBS control on Day 0 and Day 14. On Day 21, 2 x 105MCA205 cancer cells were injected subcutaneously, either wild-type or spike protein overexpressing. Tumor size and survival were monitored post tumor inoculation and as expected, the vaccinated group exposed to spike protein expressing tumor (green squares) had the smallest (i.e., nearly absent) tumor volumes (FIG. 10). Rechallenge of the no tumor group with either WT MCA (pink triangle) or spike-expressing MCA (green inverted triangle) showed this effect was persistent, with 100% survival and no discernable tumor volume beyond day 40 while parallel naive groups (red circle, blue square) showed similar tumor growth and survival as in the first-described experiment in this Example (FIG. 11).

[0132] Example 10 - Evaluation of tumor kinetics in an in vivo sarcoma model including intratumoral injection and immune checkpoint blockade efficacy

[0133] C57BL / 6 mice (N=10) were given a subcutaneous injection of MCA205 cancer cells. Five days later, they were given either an intramuscular injection of Moderna COVID-19 mRNA vaccine (Group 1, N=5) or neutral vehicle (PBS buffered aqueous; Group 2, N=5). Both groups were then given three intratumoral injections of Moderna vaccine, one on each of days 8, 10, and 12. Tumor volume grew similarly across both groups, suggesting that intratumoral vaccine injection did not result in a change in tumor kinetics absent preimmunization.

[0134] As a follow-up experiment, C57BL / 6 mice (N=10) were given either an intramuscular injection of Moderna COVID-19 mRNA vaccine (Group 1, N=5) or neutral vehicle (PBS buffered aqueous; Group 2, N=5) on each of days -29 and -15. On day -6, they were given a subcutaneous injection of MCA205 cancer cells. Six days later, the tumor was palpable (this is denoted as Day 0 post palpable tumor). Both groups were then given three intratumoral injections of Moderna vaccine, one on each of days 0, 2, and 4. Mice were followed for 26 days. PBS i.m. injected mice showed a consistent increase in tumor volume and were all dead by day 22 post palpable tumor. Vaccine i.m. injected mice showed a slower and inconsistent rate of increase in tumor volume and survived until day 26.

[0135] As further, analogous follow-up experiments, C57BL / 6 mice were given either Moderna or PBS prior to cancer cell injection. Intratumoral injections on each of days 0, 2, and 4 were PBS; Moderna; or Moderna plus an immune checkpoint blockade (ICB) agent (an anti- PD-1 antibody). The Moderna vaccinated and Moderna+ICB intratumoral treated group had the lowest tumor volume and longest survival (day 35) (FIG. 12). Similar results were seen for a follow-up experiment using B16F10 cancer cells (FIG. 13).

[0136] Example 11 - in vivo sarcoma model

[0137] C57BL / 6 (N=20) were given intramuscular injection of either Moderna COVID-19 mRNA vaccine (N=10) or neutral vehicle (PBS buffered aqueous; N=100, with two doses fourteen days apart. Seven days after the second dose (Day 21), MCA205 cancer cells were injected subcutaneously; these cancer cells had been treated ex vivo with Moderna (N=10) or nothing (control group; N=10). The resulting groups were Moderna injected, wild-type MCA205 (N=5; Group 1); Moderna injected, Moderna-treated MCA205 (N=5; Group 2); PBS injected, wild-type MCA205 (N=5; Group 3); PBS injected, Moderna-treated MCA205 (N=5; Group 4). Tumor growth and immune response were measured through Day 26. Group 2 failed to generate tumors, and all mice survived throughout the study. This finding further confirms the anti-cancer effect of anti-infection immunization as described herein.

[0138] Example 12 - in vivo sarcoma re-challenge model

[0139] Group 2 mice from Example 11 (N=5) was challenged either with wild-type MCA205 subcutaneous injection (Group A, N=3) or with MCA205 that had been modified to overexpress spike protein (Group B, N=2). Six additional C57BL / 6 mice were likewise challenged at the same time (Group C, N=3 MCA205 WT; Group D, N=3 MCA205 Spike) as a treatment naive control. These mice were monitored for tumor volume over the next 30 days. Naive control mice developed measurable tumors. By contrast, those animals that had previously shown tumor remission also failed to generate tumors when re-challenged, suggesting long term immunity is present.

[0140] Example 13 -T cell receptor sequencing analysis

[0141] TCR sequencing analysis of human non-small cell lung cancer (NSCLC) infiltrating T cell populations (n = 18; 9 Treated, 9 Normal) showed a robust, systemic immune response characterized by infiltration of infection-specific CD8 and CD4 T cells in the tumor; see side- by-side views FIG. 14 and FIG. 15. The frequency of viral-specific T cells (various colored circles) was much greater than that of the tumor-specific T cells within the tumormicroenvironment (red circles). This finding supports the potential of tumor-infiltrating virus-specific T cells to enhance antitumor immunity in a clinical context.

Claims

CLAIMS1. A method of treating cancer in a subject having a malignant tumor, the method comprising: administering a therapeutically effective amount of a first immunotherapy; administering a therapeutically effective amount of a second immunotherapy after a predetermined first duration;optionally, administering a therapeutically effective amount of a third immunotherapy after a predetermined second duration;wherein at least one of the administered immunotherapies provokes an anti-infection T cell response within a tumor.

2. The method of claim 1, wherein the first immunotherapy is an anti-infection immunotherapy and the second immunotherapy is an antitumor immunotherapy.

3. The method of claim 1, wherein the first immunotherapy is an antitumor immunotherapy and the second immunotherapy is an anti-infection immunotherapy.

4. The method of any of the preceding claims, wherein the anti-infection immunotherapy comprises an infectious microorganism indicium that is not associated with oncogenesis.

5. The method of claim 4, wherein the infectious microorganism indicium is a viral indicium selected from a coronavirus, influenza virus, respiratory syncytial virus, Epstein-Barr virus, cytomegalovirus, dengue virus, or combinations thereof.

6. The method of claim 4, wherein the infectious microorganism indicium is a viral indicium selected HIVl, HIV2, HTLV1, Epstein Barr virus, cytomegalovirus, adenovirus, adeno-associated virus, BK virus, Human Herpesvirus 6, Human Herpesvirus 8, influenza A virus, influenza B virus parainfluenza virus, avian flu virus, MERS coronavirus, SARS coronavirus, SARS-CoV-2, Crimean Congo Hemorrhagic fever virus, rhino virus, enterovirus, Dengue virus, West Nile virus, Ebola virus, Marburg virus, Lassa fever virus, zika virus, RSV, measles virus, mumps virus, rhino virus, varicella virus, herpes simplex virus 1 and 2, varicella zoster virus, HIV-l, HTLV1, Hepatitis virus, enterovirus, hepatitis B virus, Hepatitis C virus, Nipah and Rift valley fever viruses, Japanese encephalitis virus, or combinations thereof.

7. The method of claim 4, wherein expression of the infectious microorganism indicium by tumor cells results in recruitment of anti-infection CD8+T cells into the tumor microenvironment.

8. The method of any of the preceding claims, wherein at least one of the administered immunotherapies is an immunostimulatory agent derived from a microbe.

9. The method of claim 8, wherein the immunostimulatory agent is derived from Bacillus Calmette-Guerin, Mycobacterium tuberculosis, atypical mycobacteria species, Pneumocystis jirovecii, toxoplasmosis, Rickettsia, Nocardia, Aspergillus, Mucor, or Candida.

10. The method of any of the preceding claims, wherein at least one of the administered immunotherapies is an immune checkpoint inhibitor.

11. The method of claim 10, wherein the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-Ll antibody, or an anti-CTLA-4 antibody.

12. The method of any of the preceding claims, wherein at least one immunotherapy is administered intratumorally.

13. The method of any of the preceding claims, wherein at least one immunotherapy is administered according to a prime-and-boost regimen.

14. The method of any of the preceding claims, wherein the predetermined first duration or the predetermined second duration corresponds to a vaccine dosing interval used in a public health immunization schedule.

15. The method of any of the preceding claims, wherein the cancer is selected from melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer, pancreatic cancer, colorectal cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, colon cancer, or sarcoma.

16. A composition comprising a nucleic acid encoding at least a portion of an infectious microorganism indicium, wherein the composition is capable of causing tumor cells to express the infectious microorganism indicium and thereby prime or activate anti-infection T cells within a tumor.

17. The composition of claim 16 further comprising a nucleic acid encoding at least one tumor-associated antigen.

18. The composition of claim 16, wherein infectious microorganism indicia are selected to correspond to prevalent anti-infection T cell receptor repertoires in a human population.

19. The composition of any of claims 16-18 further comprising a pharmaceutically acceptable carrier.

20. The composition of claim 19, wherein the composition further comprises a lipid nanoparticle that encapsulates the nucleic acid.

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