Multi-antigenic cancer vaccines, dendritic cell targeting agents and associated methods

Abstract

The present invention provides compositions including nanoparticles and at least two polynucleotide or protein antigens separated by a linker. The fusion polynucleotides or fusion polypeptides may encode or may be cancer antigens. The nanoparticles are engineered to target dendritic cells. Methods of using the compositions and cells treated with the compositions are also provided.

Description

[0001] MULTI-ANTIGENIC CANCER VACCINES, DENDRITIC CELL TARGETING AGENTS AND ASSOCIATED METHODS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 611,679 filed on December 18, 2023, the contents of which are incorporated by reference in their entireties.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (18543600004. xml; Size: 168,324 bytes; and Date of Creation: December 18, 2024) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Traditional treatments for cancer like surgery, chemotherapy, and radiation each have limitations in that these treatments may not target specific cancer cells and may cause damage to noncancerous cells and tissue. Cancer vaccines have emerged as a promising alternative therapy for treating cancer, in that they engage the immune system to selectively recognize and attack cancer cells. Cancer-based vaccines and immune-based therapies may therefore be effective in preventing certain cancers, managing the disease more effectively relative to traditional treatments, and potentially eradicating cancer cells by engaging the immune system to recognize specific cancer antigens.

[0008] SUMMARY

[0009] The present invention provides compositions and methods to increase immunosurveillance in subjects in need of the same. Compositions of the present disclosure comprise an antigen presenting cell targeted nanoparticle which comprise antigens, including cancer antigens. The antigens may be provided as proteins, peptides, DNA or RNA. The DNA or RNA may encode a protein or peptide antigen (s).

[0010] One aspect of the present invention provides a composition comprising a nanoparticle and a polynucleotide. In some embodiments the nanoparticle comprises a ligand for targeting dendritic cells (DC), and wherein the polynucleotide encodes at least two antigens separated by a linker. In some embodiments, the ligand for targeting DC is selected from the group consisting of mannose, CDla, CDlc (BOCA1), CDl lb (ITGAM), CDllc (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD 172a (SIRPa), CD 180 (RP105), CD205 (DEC-205), CD206 (MRC1), CD209 (DC- SIGN), FceRl, HLA-DR, TLR2, TLR3, TLR4 , TLR7 / 8, and TLR9 and combinations thereof. In some embodiments, the polynucleotide encodes 3 to 750 antigens each of which are separated by a linker. In some embodiments, the antigens are between about 8 amino acids and 50 amino acids in length. In some embodiments, the antigens are cancer antigens. In some embodiments, the cancer antigens are selected from the cancer antigens in Tables 1-14, SEQ ID NO: 100-157 or are encoded by at least one of SEQ ID NO: 95-99. In some embodiments, the cancer antigens are selected from the group consisting of KRAS, TP53, PIK3CA, EGFR, NRAS, IDH2, FLT3, nucleophosmin, BRCA1, BRCA2, and DNMT3A, and combinations thereof. In some embodiments, the cancer antigens are selected from SEQ ID NOs: 18-67. In some embodiments, the polynucleotide is DNA or RNA, in some embodiments, the polynucleotide comprises modifications.

[0011] In some embodiments, a composition comprising a lipid nanoparticle and a plurality of cancer antigen peptides, wherein the lipid nanoparticle comprises a ligand for targeting dendritic cells (DC) is provided. In some embodiments, the plurality of cancer antigens is selected from the cancer antigens in Tables 1-14, SEQ ID NO: 100-157 or are encoded by at least one of SEQ ID NO: 95-99. In some embodiments, the cancer antigens are selected from SEQ ID NOs: 68-92. In some embodiments, the at least two cancer antigens are part of a fusion peptide with a linker separating the cancer antigens. In some embodiments, the plurality of cancer antigen peptides includes 3 to 750 peptides each of which are separated by a linker.

[0012] In some embodiments, a pharmaceutical composition comprising the composition of any one of the preceding claims and a pharmaceutically acceptable carrier is provided.

[0013] Another aspect of the present disclosure provides a method of preventing or treating cancer or slowing the rate at which a pre-cancer develops into a cancer in a subject. In some embodiments, the method comprises administering a composition or pharmaceutical composition described herein or combinations thereof to the subject.

[0014] Another aspect of the present invention provides a method of inducing an anti-tumor immune response in a subject in need thereof. In some embodiments, the method comprises administering a composition or pharmaceutical composition described herein or combinations thereof to the subject.

[0015] Another aspect of the present invention provides a method of inducing a dendritic cell mediated anti-tumor immune response. In some embodiments, the method comprises administering a composition or pharmaceutical composition described herein or combinations thereof to the subject.

[0016] Another aspect of the present invention provides a method of inducing an immune response to more than one antigen. In some embodiments, the method comprises administering a composition or pharmaceutical composition described herein or combinations thereof to the subject.

[0017] In some embodiments, the subject in need is diagnosed with a genetic condition, wherein the genetic condition is associated with an increased risk or predisposition for the development of cancer.

[0018] Another aspect of the present invention provides a method of preventing or treating cancer in a subject with a cancer-associated genetic mutation. In some embodiments, the method comprises administering a composition or pharmaceutical composition described herein or combinations thereof to the subject.

[0019] In some embodiments, the genetic condition is selected from the group consisting of Lynch syndrome, Von Hippel-Lindau disease (VHL), Li-Fraumeni syndrome (LFS), Hereditary Breast and Ovarian Cancer (HBOC) syndrome, Peutz-Jeghers syndrome (PJS), Familial adenomatous polyposis (FAP), Hereditary leiomyomatosis and renal cell cancer (HLRCC), DICER1 syndrome, Gorlin syndrome, Cowden Syndrome, Hereditary Leukemia and Hematologic Malignancies Syndromes, Multiple endocrine neoplasias (MEN), PTEN Hamartoma tumor syndrome, MUTYH associated polyposis, juvenile polyposis syndrome, PALB2 gene, Birt-Hogg Dube syndrome, ataxia-telangiectasis, Familial GIST syndrome, and Carney Complex.

[0020] In some embodiments, the subject has or has been diagnosed with having Lynch syndrome and the at least two antigens comprise at least two of SEQ ID NO: 101-157. In some embodiments, the subject in need is diagnosed with a cancer selected from the group consisting of bone cancer, testicular cancer, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, squamous cell head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, etc.), ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma. In some embodiments, the subject in need is diagnosed with cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Tables 1-10. In some embodiments, the subject in need is diagnosed with colon cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 1. In some embodiments, the subject in need is diagnosed with lung cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 2. In some embodiments, the subject in need is diagnosed with pancreatic cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 3 or 9. In some embodiments, the subject in need is diagnosed with Diffuse large B-cell lymphoma (DLBCL)cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 4. In some embodiments, the subject in need is diagnosed with Acute myeloid leukemia (AML) cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 5. In some embodiments, the subject in need is diagnosed with melanoma cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 6. In some embodiments, the subject in need is diagnosed with bladder cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 7. In some embodiments, the subject in need is diagnosed with glioblastoma cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 8. In some embodiments, the nanoparticle is administered intramuscularly, intravenously, subcutaneously, or intratumorally.

[0021] Another aspect of the present disclosure provides a method of delivering cargo to a dendritic cell, the method comprising contacting a dendritic cell with a nanoparticle comprising a dendritic cell targeting ligand and the cargo. In some embodiments, the dendritic cell targeting ligand is selected from the group consisting of mannose, CDla, CDlc (BOCA1), CDl lb (ITGAM), CDllc (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD 180 (RP105), CD205 (DEC-205), CD206 (MRC1), CD209 (DC-SIGN), FcaRl, HLA-DR, TLR2, TLR3, TLR4 , TLR7 / 8, and TLR9 and combinations thereof. In some embodiments, the cargo comprises DNA, RNA, protein, gRNA, and enzymes.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, no is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention.

[0024] FIG. 1A illustrates T-cell memory measured as a percentage of CD3+CD62L+CD197+T- cell populations in cells stimulated with different peptide antigens using DC cells (left bars) relative to cells that were stimulated with the same peptide antigens in the absence of DC cells (right bars).

[0025] FIG. IB shows memory post stimulation in health donors stimulated with RNA of the 21 most common neoantigens in Cancer. Graphs shows memory panel % of CD3+ Cells Post 21 neoantigens.

[0026] FIG. 2 shows that a single production run yields a T cell product reactive to multiple mutations. The data shows an IFNy ELISpot analysis from an experiment where three different normal donors were prepared with our small-scale SMART-T™ manufacturing protocol. The results of the ELISpot showed that in all three (3) production runs, targeting 20 mutations, we achieved specific T cell reactivity to at least 18 of those mutation in our model construct.

[0027] FIG. 3 shows SMART-T™ cells selectively killing the target cells in in vitro killing assay with mutation to KRAS G12D and EGFR T790M peptides compared to the wild type (WT) control peptides. The results show our SMART-T™ selected T cells can recognize a single amino acid difference compared to the WT gene.

[0028] FIG. 4 Results of the Incucyte real-time cytotoxicity assay shows an increase in cell death over time while the target cells expressed surface presentation of the 20 target mutations. Target cells are human cells introduced with 20 target mutations. Four ratio of SMART-T™ vs target cells were tested (SMART-T™ : Target ratios include Target cells only, 4: 1; 2: 1;1: 1 and 0.5: 1; total target cells of 15,000. While there was an increase with the dead cells, the live target cells decreased overtime as the cells died and leaked the cytoplasm dye. There was high potency of the specific T cells as seen with a titration of SMART-T cells to target cell ratio. There was still strong results as low as the 0.5: 1 ratio with nearly 100% of cells killed.

[0029] FIG. 5 Shows the percentage of T cells producing IFNy at levels comparable to viral protein to neoantigens KRAS G12D) but not to healthy KRAS. The flow cytometry based intercellular cytokine assay shows the percent of cells expressing IFNy over CD8 cytotoxic T cells. The top row results show percentages comparable to viral proteins (22.3%) to neoantigen KRAS G12D (23.7%), but not to wild-type KRAS. When IFNy was stained with TNFa the double positive results showed comparable percentages with viral proteins expressing 11.7% , while the neoantigen KRAS G12D expressed 11.3%. Cells positive for both IFNy and TNFa is a phenotype suggesting highly potent T-cells. These T cells can kill as well as remodel the tumor microenvironment.

[0030] FIG. 6 Shows presence of T cells responsive to multiple tumor antigens predicts overall survival. Graph shows overall survival in melanoma correlates with the number of antigens targeted by T cells (Melan A, NY-ESO-1). Results of a survival curve where 0 targeted antigenic T cells, 1 targeted antigenic T cell, and 2 or more targeted antigenic T cells were infused into mice in a model of Melanoma. The 2 tumor associated antigens tested included Melan A, and NY-ESO- 1. The results overtime (months) showed 0 targeted T cells had the shortest overall mouse survivability of 0 mice at less than 12 months. One (1) targeted T cell had an overall mouse survivability time of -15% over 24 months. Two (2) targeted T cells had an overall mouse survivability time of -40% at 36 months. The Expanding T cell to additional targeted antigens should further increase the T cell diversity and overall survival

[0031] FIG 7A shows pancreatic and glioblastoma cancer patient T cell responses to NY-ESO-1. Graphs shows the results of a flow cytometry recall / restimulation (restim.) assay of Pancreatic and Glioblastoma cancer patient T cell response to NY-ESO-1. The marker CD107a (LAMP1) is a surrogate marker for Granzyme B degranulation. If the T cells are challenged with NY-ESO-1 Pepmix, the Granzyme B will be released and the CD107a should increase within the cell. TNFa was also stained at the same time. The pancreatic day 14 cells were challenged with a NY-ESO-1 Pepmix restim. The results showed a 2.3X increase in CD107a expression while TNFa had a 7.3X when compared to the no Pepmix control. The Glioblastoma day 14 cells were challenged with a NY-ESO-1 Pepmix restim. The results showed a 2.9X increase in CD107a expression while TNFa had a 3.6X when compared to the no Pepmix control.

[0032] FIG.7B Shows the results of a flow cytometry recall / restim assay of Pancreatic and Glioblastoma cancer patient T cell response to Survivin. The pancreatic day 14 cells were challenged with a Survivin Pepmix restim. The results showed a 10.3X increase in CD107a expression while TNFa had a 8.8X when compared to the no Pepmix control. FIG. 8 Shows the results of 2 Glioblastoma patients restimulation challenge to NY-ESO or viral proteins. Patient A expressed CD107a of 7.7% when restimed with NY-ESO-1 and 8.7% when challenged with CMV-pp65 Pepmix. Patient B expressed CD 107a of 17% when restimed with NY-ESO-1 and 6.7% when challenged with CMV-pp65 Pepmix. The Geneius GMP process produces T cells that degranulate (CD 107) to tumor associated antigens at a level comparable to or better than viral antigens.

[0033] FIG. 9A illustrates lipid nanoparticles delivering green fluorescent protein (GFP) RNA to Dendritic Cells (DCs). FIG. 9B and FIG 9C illustrate lipid nanoparticles delivering 6 Neoantigen to DCs. Example 4 illustrates lipid nanoparticles delivering 6 different Tumor Associated Antigens (TAA).

[0034] FIG. 10 Depicts a prototypical GFP levels in DCs transfected with nanoparticles comprising GFP RNA targeting a Mannose (2), CD180 (3), CD209 (4), or HLA-DR (5) targeting ligands relative to cells transfected with the same dose of GFP RNA formulated with LNP only.

[0035] FIG. 11 Examples illustrate a schematic of an exemplary polynucleotide or fusion peptide of the present disclosure. Exemplary polynucleotides or fusion peptides may comprise 2 to 50 individual antigens. FIG. 11A is a mRNA with a signal peptide. FIG. 1 IB is a mRNA without signal peptide. FIG. 11C pcDNA vaccine construct with signal peptide. FIG. 11 D pcDNA vaccine construct without signal peptide. FIG. HE and FIG. 1 IF illustrate examples with combinations of neo antigens and tumor associated antigens, and combinations with viral tumor associated antigens. Antigen sequences may comprise neoantigens, TAA (NY-ESO-1, MART-1, or any combination of TAA) and, EBV viral proteins (LMP1, LMP2a, EBNA-1). Additional modification may comprise 5-Methoxyuridine-5'-Triphosphate a modified UTP for the linear mRNA , a 162 poly A tail, Clean Cap® reagent AG as a 5’ cap. Numbering within the illustrations boxes are: 1. Kozak Consensus sequence; 2. Signal peptide for MHC Class I or MHC Class II; 3. Antigen sequence; 4. Linker sequence and 5. STOP codon.

[0036] DETAILED DESCRIPTION

[0037] The present invention provides compositions and methods to increase immunosurveillance in subjects in need of the same. Immunosurveillance is the process by which the immune system looks for and recognizes antigens. Compositions of the present disclosure comprise an antigen presenting cell targeted nanoparticle which comprise antigens, including cancer antigens. The antigens may be provided as proteins, peptides, DNA or RNA. The DNA or RNA may encode a protein or peptide antigen (s).

[0038] The methods and compositions provided herein are also useful to target cargo molecules such as proteins, DNA or RNA to dendritic cells. In some methods, those dendritic cells may act as antigen presenting cells as noted above, but in other methods the cargo targeted to the dendritic cells may be for an additional or distinct purpose such activating the dendritic cells or stimulating them to become or develop into a particular type of dendritic cell or maturation level. The targeted dendritic cells may express a particular set of cytokines or have phenotype distinct from nontargeted dendritic cells.

[0039] Nanoparticles have become an important delivery tool for bringing biomolecules (DNA, RNA, CRISPER products, peptides and proteins), as well as other drug compounds into the human body. In the past four years, nanoparticles have become mainstream news with the development of the SARS-CoV2 vaccines for the Covid- 19 respiratory disease. The biopharma companies Modema, and BioNTech with Pfizer formulated their mRNA vaccine delivery using lipid nanoparticles to deliver mRNA into muscle tissue and the cells. Once the mRNA gets into the cell cytoplasm, the mRNA gets translated into the virus Spike protein. Those muscle cells can then produce the Spike protein with some of the Spike protein being processed and expressed for immune recognition.

[0040] We propose to use nanoparticles to deliver multi-antigenic RNAs from cancer neoantigens. Neoantigens are new cancer antigens that result from somatic cellular mutations. The use of multi- antigenic RNAs will generate T cell responses that will have cytotoxicity properties to damage and kill the cancer cells. The expression of multiple antigens and multiple T cells clones will be important to have overlapping antigen coverage to successfully prevent cancer cells from escaping the initial T cell response.

[0041] The use of nanoparticles with specific binding motifs can be used to specifically target the tissues and cells of interest. Lipid nanoparticles loaded with mRNA encoding antigens and with different dendritic cells binding motifs will be generated and used to specifically target dendritic cells, by delivering the lipid nanoparticles to specific tissue and cell types, manufacturing the LNPs can be scaled down to personalized medicine dosages resulting in lower costs, better control of dosing, and less off target tissue expression. This system can be used in combination with an ex vivo T cell manufacturing process, called SMART-cells to expand antigen specific T cells. The manufacturing involves the use of autologous dendritic cells and mRNA encoding neoantigens found in a patient’s liquid biopsy.

[0042] The advantages of the current system include separation of the antigenic peptide from the oncogenic protein such that the whole protein is not ever expressed and so there is no oncogenic tertiary structure of the proteins. The peptides selected are not near active sites of the proteins. The same mutated gene is not put near another counterpart mutation. A non-antigenic linker is used between antigens to break up the sequence and maintain the antigenic nature of the peptides. The antigens used are mutations associated with cancer and by using smaller portions of the proteins the immune response is less likely to be directed to wild-type proteins thus reducing the risk of autoimmune issues. By targeting DCs the dose of the compositions provided here can also be lower and prevent side-effects by decreasing the biodistribution of the compositions provided here. Nanoparticles

[0043] One aspect of the present disclosure provides a composition comprising nanoparticles. A nanoparticle or ultrafine particle is a particle of matter 1 to 100 nanometers (nm) in diameter. The term is also sometimes used for larger particles, up to 500 nm, or fibers and tubes that are less than 100 nm in only two directions. Nanoparticles of the present disclosure can be of any composition. By way of example and not limitation nanoparticles useful in the instant disclosure comprise carbon nanotubes, metallic nanoparticles, polymer nanoparticles, lipid nanoparticles, magnetic nanoparticles, nonogels and nanocrystals. In some embodiments the nanoparticle may be lipid nanoparticles. Lipid nanoparticles (LNPs) are spherical particles made of lipids that are used to deliver therapeutic agents and nucleic acids. LNPs are used to deliver therapeutic agents like small molecules, nucleic acids, and monoclonal antibodies. They can protect drugs or molecules including but not limited to nucleotides or proteins, from degradation, increase their solubility, and enable targeted delivery. LNPs can be made up of various types of lipids, including but not limited to ionizable lipids, helper or neutral lipids, cholesterol, and lipids attached to polyethylene glycol (PEG). Exemplary lipid nanoparticles are described in WO2022115641A2 and WO2024130254A3, the contents of which are incorporated in their entirety.

[0044] The lipid composition may comprise proteolipids (e.g., protamine), carrier proteins, and / or small molecules. The lipid composition may comprise a single lipid group or multiple lipid groups. Nonlimiting examples of lipid groups include cationic lipids, anionic lipids, neutral lipids, polyethylene glycol (PEG)ylated lipids, ionizable lipids, helper lipids, stealth lipids, or cholesterols.

[0045] Nonlimiting examples of lipids include DOSPA 2,3 -dioleyloxy -N-[2- (sperminecarboxamido)ethyl]-N,N-dimethyl-l -propanaminium trifluoroacetate, DOTMA 1,2- di-O-octadecenyl-3 -trimethyl ammonium propane, DOTAP l,2-Dioleoyl-3- trimethyalammoniumpropane, and DC-Cholesterol 313-[N-(N',N' -dimethylaminoethane) carbamoyl] cholesterol.

[0046] Nonlimiting examples of ionizable lipids include SM-102 9-Heptadecanyl 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, ALC-0315 4- hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate) Dlin-MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,2823icol23htraen-19-yl4-(dimethylam ino) butanoate, and DODMA l,2-Dioleyloxy-3-dimethylamino propane.

[0047] Nonlimiting examples of helper lipids include cholesterol (!R,3aS,3bS,7 S,9aR,9bS, 1 laR)-9a,l la-Dimethyl-l-[(2R)-6-methylheptan-2- yl]- 2, 3, 3a, 3b, 4, 6, 7, 8, 9, 9a, 9b, 10,11,11 a-tetradecahydro -lH-cyclopen23icoll23hrenethren- 7-ol DSPC l,2-distearoyl-sn-glycero-3-phosphocholine, and DOPE 1,2-Dimyristoyl- sn glycerophosphoethanolamine.

[0048] Nonlimiting examples of stealth lipids include PEGIG (R)-2,3- bis(myristoyloxy)propyl-l -(methoxy poly (ethylene glycol) 2000) carbamate and ALC- 0159 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide.

[0049] Table 17: Components of lipid nanoparticles

[0050] In some embodiments, the nanoparticles may be polymer based, metal (e.g., silver, gold, palladium, titanium, zinc, or copper) based, silica based, or lipid based.

[0051] In some embodiments, the nanoparticles are a multilayer nanoparticle. Nonlimiting examples of multilayer nanoparticles (e.g., a nanoparticle having two or more polymer based layers; a nanoparticle having two or more metal based layers; a nanoparticle having two or more silica based layers; a nanoparticle having two or more lipid-based layers; or a nanoparticle having a first layer selected from the group consisting of a polymer-based layer, a metal based layer, a silica-based layer, and a lipid-based layer, and a second layer selected from the group consisting of a polymer-based layer, a metal based layer, a silica-based layer, and a lipid-based layer, wherein the second layer composition is different from the first layer composition).

[0052] Nanoparticles of the present invention may be modified by any means known in the art. In some embodiments, the nanoparticle may be modified to decrease degradation or filtration or to increase evasion, function or targeting. Nanoparticles may also be modified to increase contact, binding or internalization by antigen presenting cells. Nanoparticles may be modified with a variety of ligands such as small molecules, surfactants, dendrimers, polymers, and biomolecules.

[0053] In some embodiments nanoparticles can be carbon-based forming carbon nanotubes, graphene, and carbon nanofibers. Carbon nanoparticles can be linear, 2-dimensional (Graphene), and 3-dimensional (Carbon nanotubes and carbon nanifibers). DNA or RNA can be delivered

[0054] In some embodiments nanoparticles can be metallic in nature and include gold, silver, iron oxides, and ferric iron oxides. Gold nanoparticles have been used for immunizing mice and inserting CRISPR using gene gun high pressure helium. Gene guns, also called a biolistic particle delivery system can be used to deliver exogenous DNA transgenes, RNA, proteins, and peptides.

[0055] In some embodiments, the nanoparticle may comprise a ligand for targeting antigen presenting cells. The nanoparticle ligand may bind to a target on an antigen presenting cell such that the nanoparticle contacts and binds to the antigen presenting cell specifically. In some embodiments, the antigen presenting cell comprises a dendritic cell (DC).

[0056] In some embodiments, lipid nanoparticles incorporate antigen presenting cell (APC) binding moieties, such as DC binding moieties. Nonlimiting examples of APC binding moieties include antibodies and / or single-chain variable fragment (scFv) to surface molecules expressed on dendritic cells (e g., CDla, CDlc (B0CA1), CDl lb (ITGAM), CDl lc (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD180 (RP105), CD205 (DEC -205), CD206 (MRC1), CD209 (DC-SIGN), Fci:Rl, HLA-DR, TLR2 (e.g., glycolipids and phospholipids), TLR3 (double-stranded RNA), TLR4 (lipopolysaccharide), TLR7 / 8 (single-stranded RNA), and TLR9 (unmethylated CpG DNA)). In some embodiments, the ligand is selected from mannose, CD 180, CD209, HLA-DR and combinations thereof. In some embodiments, the APC binding moieties are conjugated to a lipid in the lipid composition. The APC binding moieties may be identified or designed using methods including, but not limited to, critical process parameters (CPPs), high-throughput sequencing, or predictive modeling.

[0057] The APC binding moieties may be a ligand that binds a surface marker (e.g., peptide, glycoprotein, carbohydrate) of the APC cell (e.g., a DC, a B cell, a T cell, a macrophage). In some embodiments, the surface marker is a molecule that is upregulated during maturation of the APC cell.

[0058] In some embodiments, nanoparticles comprise opsonization elements. The opsonization elements may engage APCs (e.g., bind or interact with APCs). The opsonization elements may induce the phagocytic process of macrophages, neutrophils, and dendritic cells. In some embodiments, the opsonization elements incorporate mannose or other carbohydrates.

[0059] In some embodiments, the nanoparticles comprise Fe binding fragments. The Fe binding fragments bind Fe receptors on APCs. In some embodiments, the nanoparticle incorporates complement factors C3b, C4 and or Clq.

[0060] In some embodiments, the nanoparticle may comprise a ligand to target the nanoparticle to a dendritic cell (DC). A dendritic cell is a phagocyte and a type of antigen-presenting cell. DC phagocytose pathogens and antigens and present them to T and B lymphocytes to stimulate and immune response. DC also produce cytokines and co-stimulatory and co-inhibitory molecules to regulate lymphocyte activation. Dendritic cells are commonly categorized as convention dendritic cell or myeloid dendritic cell and plasmacytoid dendritic cell. Dendritic cells can be identified by cell surface markers, which can change with maturity, as well as cellular origin. Common markers of DC include, but are not limited to CD11c, HLA-DR, CD 123, CD la, CD207, CD68, CD 14, and CD 19 and combinations thereof. In some embodiments, combinations of one or more targeting ligands may be used. In some embodiments, combinations of markers may be used to target specific subsets of DC. By way of example, combinations of ligands may include HLA-DR, CDl lc, CDl lb, EpCAM, CD103, F4 / 80; XCR1, CLEC9A and CD141; CD45A, CD123, CD2, CD303 and CD304; CDlc, CD1 lb, CD172a, CDla, CD14 and CD5; CDlc CD1 lb, CD14, CDla, CCR2, CD206 and CD209. Proteins on antigen presenting cells that could be targeted include DEC-205 (CD205), which is found on dendritic cells and can be targeted using antibodies or antibody fragments specific to DEC-205, as well as certain glycoproteins with mannose-rich oligosaccharides. DC-SIGN (CD209) is found on dendritic cells and especially on immature dendritic cells and can be targeted using mannose-rich glycans, fucose-containing carbohydrates, and various pathogen-derived carbohydrates. Synthetic carbohydrate ligands or DC-SIGN- targeting antibodies. The mannose receptor (CD206) can also be targeted and is found on macrophages as well as dendritic cells and some endothelial cells and may be targeted using mannosylated ligands (mannose-, fucose-, and N-acetylglucosamine-terminated glycans) are frequently used to direct cargo to cells expressing the mannose receptor. Langerin (CD207) may also be targeted. It is found on Langerhans cells and some dermal dendritic cells and can be targeted using mannose and fucose containing glycans.

[0061] The APC targeting ligand may be attached or integrated into the nanoparticle by any means known in the art. Common methods include, but are not limited to covalent bonding through chemical reactions with functional groups on the nanoparticle surface (for example amine coupling, thiol coupling and carboxylic acid coupling), non-covalent interactions like electrostatic attraction, hydrogen bonding or hydrophobic interactions, and bioconjugation using specific linking agents to attach biomolecules like antibodies or peptides to the nanoparticle surface, often targeting specific receptors on cells. In some embodiments, the APC targeting ligand may be attached to the nanoparticle using click chemistry or CLIP chemistry.

[0062] In some embodiments, the linkers used in the ligand-nanoparticle conjugation process can be carbodiimide (EDC / NHS) linkers, where EDC activates carboxyl group to form an O- acylisourea intermediate and NHS stabilizes this intermediate, allowing the eventual formation of an amide bond with an amine-containing ligand.

[0063] In some embodiments, maleimide-thiol coupling can be used to link ligands to nanoparticles, where maleimide groups react specifically and efficiently with thiol groups to form stable thioether bonds under mild conditions.

[0064] In some embodiments, click chemistry (e.g., Copper-Catalyzed Azide-Alkyne Cycloaddition, CuAAC) can be used to form ligand-nanoparticle conjugation. In this instance, azide and alkyne groups undergo a cycloaddition reaction to form a stable triazole linkage in the presence of a copper catalyst.

[0065] In some embodiments, bio-orthogonal chemistry (strain-promoted Alkyne-Azide Cycloaddition (SPAAC), Tetrazine-Norbornene Ligation) is used to attach ligands to nanoparticles. This type of conjugation can be applied in instances where copper catalysts cannot be used. In some embodiments, Aldehyde-Amine or Aldehyde-Hydrazide / Hydroxylamine Conjugation can be used to form ligand-nanoparticle conjugations. This process of conjugation can be used to achieve reversible protein conjugation.

[0066] In some embodiments, avidin-biotin binding can be used to attach ligands to nanoparticles. This conjugation process is used in instances that necessitate the formation of a strong non- covalent bond.

[0067] In some embodiments, affinity tags (e.g., His-Tag / Ni-NTA) can be used in the process of ligand-nanoparticle conjugate formation. Ni-NTA chelates divalent nickel ions which strongly bind to the histidine-rich tag on proteins, yielding a stable but reversible affinity binding. This process is used to attach proteins to metal nanoparticles.

[0068] In some embodiments, PEG (Polyethylene Glycol) spacers can be used in the conjugation process of ligands to nanoparticles. Specifically, in the case of polymeric nanoparticles, PEG spacers are used to cover the nanoparticles in order to facilitate the binding of targeting ligands.

[0069] Linkers used in ligand-nanoparticle conjugation:

[0070] Carbodiimide (EDC / NHS) Chemistry o EDC activates a carboxyl group to form an O-acylisourea intermediate. NHS stabilizes this intermediate, allowing the eventual formation of an amide bond with an amine-containing ligand. o Commonly used to attach proteins, peptides, or antibodies (which have amine groups) to nanoparticle surfaces functionalized with carboxyl groups (e.g., on polymeric nanoparticles or certain metal nanoparticle coatings).

[0071] - Maleimide-Thiol Coupling o Maleimide groups react specifically and efficiently with thiol groups to form stable thioether bonds under mild conditions. o Ideal for conjugating antibodies or peptides that can be site-specifically thiolated, or for attaching thiolated oligonucleotides to gold nanoparticles.

[0072] "Click" Chemistry (e.g., Copper-Catalyzed Azide-Alkyne Cycloaddition, CuAAC) o In the presence of a copper catalyst, azide and alkyne groups undergo a cycloaddition reaction to form a stable triazole linkage. o Attaching sugars, peptides, or small molecules to nanoparticles when both can be easily functionalized with "clickable" groups. Bio-orthogonal Chemistries (Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC), Tetrazine-Norbornene Ligation) o Particularly useful for sensitive biological cargo or in vivo conjugation strategies where copper catalysts might be undesirable.

[0073] Aldehyde-Amine or Aldehyde-Hydrazide / Hydroxylamine Conjugation o Common in protein conjugation, can be tuned to achieve reversible or more stable linkages depending on conditions.

[0074] PEG (Polyethylene Glycol) Spacers

[0075] Avidin-Biotin Binding o The biotin-avidin interaction is extremely strong (one of the strongest non-covalent interactions known), providing a stable but non-covalent linkage. o Useful for modular systems where you might want to exchange ligands easily. However, non-covalent and can be disrupted under harsh conditions.

[0076] - His-Tag / Ni-NTA or Other Affinity Tags o Ni-NTA chelates divalent nickel ions which strongly bind to the histidine-rich tag on proteins, yielding a stable but reversible affinity binding. o Commonly used to attach recombinant proteins to metal nanoparticle surfaces (especially gold or quantum dots coated with Ni-NTA).

[0077] Polynucleotides

[0078] The composition of LNP of the present disclosure may further comprise at least one polynucleotide. In some embodiments, the polynucleotide encodes at least one antigen. As used herein, the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand). The polynucleotides may be cDNA or genomic DNA or RNA. Polynucleotides homologous to the polynucleotides described herein are also provided. Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide.

[0079] The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Nucleic acids generally refer to polymers comprising nucleotides or nucleotide analogs joined together through backbone linkages such as but not limited to phosphodiester bonds. Nucleic acids include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) such as messenger RNA (mRNA), transfer RNA (tRNA), Circular RNA, self-replicating RNA (srRNA) etc. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g. nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or include non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g. adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadeno sine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, and 2 -thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e g., phosphorothi oates and 5'-N-phosphoramidite linkages).

[0080] Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA Nucleic acid as used herein also refers to nucleic acids that have the same basic chemical structure as a naturally occurring nucleic acid. Such analogues have modified sugars and / or modified ring substituents but retain the same basic chemical structure as the naturally occurring nucleic acid. A nucleic acid mimetic refers to chemical compounds that have a structure that is different the general chemical structure of a nucleic acid, but that functions in a manner similar to a naturally occurring nucleic acid. Examples of such analogues include, without limitation, phosphorothiolates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0081] In some embodiments, the polynucleotide may be modified or comprise additional elements to alter the stability, function or expression of the polynucleotide. Modifications include, but are not limited to chemically or synthetically modified nucleotides, including nucleosides and nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications, chemically modified bases; biologically modified bases, intercalated bases; modified sugars, and / or modified phosphate groups. Polynucleotides described herein may also comprise elements including, but not limited to promoters, a 5’ cap, signal sequences (2), secretion sequences, restriction sites, kozak sequences (1), start and stop codons (5), untranslated regions (UTRs), polyA signals, insulators, and enhancer sequences. DNA polynucleotides may be provided as a construct and include elements for the transcription of the DNA construct. RNA polynucleotides may be circularized or otherwise modified to increase stability and include elements for the translation of the RNA. RNA polynucleotides may be mRNA polynucleotides. RNA polynucleotides may be self-replicating RNA. In some embodiments, each of the nucleotide sequences in the composition are present on different nucleotide chains.

[0082] In some embodiments, the RNA sequence is a messenger RNA (mRNA) sequence. In some embodiments, the RNA sequence is a self-replicating RNA. In some embodiments, the RNA sequence is a circular (circRNA) sequence. The circRNA may have increased half-life and / or increased expression relative to a non-circular RNA. In some embodiments, the RNA sequence is a messenger CRISPR RNA (crRNA) sequence or a gRNA.

[0083] In Vitro Transcription (IVT) to make linear mRNA

[0084] Methods for the synthesis of messenger RNA (mRNA) by in vitro transcription incorporating a 5' terminal cap structure, wherein said cap structure comprises a 7-methyl guanosine (m7G) cap, or pharmaceutically acceptable salts thereof, including but not limited to CleanCap Reagent AG or structurally equivalent cap analogs are provided. The invention further contemplates the incorporation of modified nucleotides, such as pseudouridine and 5- methoxyuridine 5'-triphosphate salts, within the mRNA structure to enhance stability, translational efficiency, and immunological compatibility.

[0085] The use of the 5' terminal cap structure, in combination with these modified nucleotides, enables the production of mRNA molecules with improved pharmacological profiles, rendering them particularly suitable for vaccine and therapeutic applications. By employing these modifications, the disclosed methodology mitigates immunogenicity, increases cellular uptake, and extends RNA half-life, thereby enhancing the efficacy and reliability of mRNA-based biomedical interventions.

[0086] Methods to Make Circular RNA

[0087] In Vitro Transcription (IVT) Followed by Circularization

[0088] In vitro transcription (IVT) followed by circularization is a widely used and versatile method for synthesizing circular RNA (circRNA). This approach begins with transcribing linear RNA from a DNA template, using enzymes like T7 RNA polymerase. The transcribed RNA is then circularized through enzymatic or chemical methods. Enzymatic techniques such as T4 RNA ligase or ribozyme-mediated reactions facilitate the joining of the RNA’s 5’ and 3’ ends to form a covalently closed circular structure. Chemical methods, while less common, can achieve similar results by leveraging selective reactions between the RNA termini. Following circularization, purification is crucial to isolate the circRNA from linear precursors and byproducts, ensuring high-quality material for downstream applications.

[0089] Plasmid-based approaches enhance IVT by introducing specialized DNA vectors that promote efficient circRNA formation. These engineered plasmids incorporate specific genetic elements — such as complementary sequences, introns, and motifs — that enable natural folding and splicing into circular RNA. By fine-tuning the plasmid design, researchers can optimize circRNA yield, stability, and antigenic or immunomodulatory properties, making this method highly attractive for vaccine and immunotherapy development. The flexibility in customizing plasmids allows for tailored circRNA molecules with improved translation efficiency and targeted immune responses.

[0090] Rolling Circle Transcription (RCT)

[0091] Rolling circle transcription (RCT) represents a scalable and efficient method for producing circular RNA (circRNA). This approach utilizes circular DNA templates to generate extended RNA transcripts through continuous RNA synthesis. These templates are transcribed by RNA polymerase to produce long RNA molecules, which are then processed into monomeric circular RNAs using enzymatic or ribozyme-mediated reactions. RCT allows for high-yield production and molecular consistency, making it ideal for industrial-scale applications in vaccine and immunotherapy development.

[0092] The method leverages specialized circular DNA constructs designed with specific genetic elements to promote efficient RNA circularization. By creating RNA templates that are enzymatically processed into circRNAs, RCT enables the production of uniform populations of therapeutic RNA molecules. This process provides advantages in scalability, consistency, and the ability to generate large quantities of circRNA with desired properties. However, precise enzymatic processing and comprehensive molecular characterization are required to ensure high- quality circRNA is suitable for immunotherapy and vaccine applications.

[0093] Endogenous Circularization Using Cell Systems

[0094] Endogenous circularization mimics natural cellular mechanisms to produce circRNA using host cell splicing machinery. DNA templates with inverted repeat sequences or specific splicing signals are introduced into cells, where back-splicing processes join a downstream splice donor to an upstream splice acceptor, forming circular RNA. This biogenesis pathway allows for incorporating natural modifications, such as methylation, that enhance stability and functionality, offering advantages for therapeutic applications.

[0095] Direct RNA backsplicing builds upon this concept, strategically engineering RNA sequences with specific splicing signals to encourage non-canonical splicing. By manipulating splice donor and acceptor sites, researchers can create artificial circRNAs encoding antigenic or immunomodulatory sequences. This approach leverages the precision of natural cellular processes while requiring the optimization of RNA secondary structures and extensive experimental validation. It offers significant potential for reducing synthetic complexity and improving the biocompatibility of circRNA molecules for vaccines and immunotherapy. Enzymatic Assembly of Circular RNA

[0096] Enzymatic ligation techniques utilize specific ligases to join RNA ends, creating circular structures with high precision. T4 RNA ligase and similar enzymes catalyze the formation of phosphodiester bonds between the 5’ and 3’ ends of linear RNA. The process allows for controlled circularization and generates circRNA with defined sequences and structural integrity. Reaction optimization and purification steps ensure the production of high-quality circRNA for therapeutic use.

[0097] This approach is complemented by RNA ligase methods, which provide a biologically inspired pathway to create molecular structures. By selecting optimal RNA ligases and substrates, researchers can design reactions that produce circRNAs with enhanced cellular compatibility and immunological properties. The method strikes a balance between synthetic control and biological authenticity, making it ideal for small-scale production of therapeutic circRNA molecules with tailored immunomodulatory features.

[0098] Biotechnological Platforms

[0099] Biotechnological platforms, such as cell-free systems and microfluidics, represent cutting-edge approaches to circRNA synthesis. Cell-free systems optimize reaction conditions for in vitro transcription and circularization, enabling high-throughput production. Microfluidic systems provide precise control over enzymatic reactions and purification, streamlining the workflow for efficient circRNA generation. These technologies reduce production time, enhance reproducibility, and enable scalability.

[0100] CRISPR / Cas-based methods are a complementary innovation, allowing precise genome editing to engineer genetic constructs that naturally generate circRNAs. By targeting splice sites, introducing flanking sequences, and manipulating genetic architectures, researchers can produce circRNAs with enhanced stability, targeted delivery, and specific immunological properties. These advanced techniques offer unparalleled control over circRNA structure and function, paving the way for sophisticated vaccine and immunotherapy applications.

[0101] Chemical Synthesis

[0102] Chemical synthesis of circRNA provides unmatched control over molecular architecture, enabling the creation of circular RNA with specific antigenic designs and optimized structural features. Researchers use chemical ligation techniques to join RNA strand ends, creating stable circular structures. This approach allows for the direct incorporation of modified nucleotides and chemical modifications to enhance stability and therapeutic potential.

[0103] Chemical synthesis methods are resource-intensive but offer significant flexibility for designing circRNAs with improved cellular penetration, reduced immunogenicity, and tailored therapeutic functions. By leveraging advanced organic chemistry protocols, scientists can create circRNA molecules with precise molecular weights and enhanced efficacy. This method is particularly valuable for small-scale production and experimental research, where customized circRNA designs are critical for vaccine and immunotherapy development.

[0104] Methods to Make Self-Amplifying RNA

[0105] Plasmid-Based Approach: Genetic Engineering of Self-Amplifying RNA

[0106] The plasmid-based approach to self-amplifying RNA synthesis represents a sophisticated genetic engineering technique that enables precise control over saRNA production. Researchers meticulously design DNA vectors containing comprehensive genetic elements necessary for RNA replication and antigen expression. These engineered plasmids incorporate multiple critical components, including viral RNA-dependent RNA polymerase (RdRp) genes, specific promoter sequences, and the target antigen's genetic instructions, creating a complete genetic blueprint for self-amplifying RNA production.

[0107] The complexity of plasmid-based saRNA synthesis lies in its intricate molecular design and strategic genetic arrangement. Scientists carefully optimize the plasmid's genetic architecture to ensure efficient transcription, RNA replication, and antigen expression. By manipulating sequence elements, regulatory regions, and replication machinery, researchers can create highly customized saRNA constructs capable of robust self-amplification within cellular systems. This method offers significant advantages in terms of genetic precision, reproducibility, and the ability to generate complex RNA molecules with enhanced immunological potential.

[0108] In Vitro Transcription Method: Controlled RNA Synthesis

[0109] In vitro transcription represents a powerful biochemical approach to self-amplifying RNA synthesis, providing researchers with unprecedented control over RNA molecule production.

[0110] This method involves using purified RNA polymerase enzymes to generate RNA molecules from precisely designed DNA templates, incorporating specialized genetic elements that enable selfreplication. Scientists meticulously craft these templates to include viral replication machinery genes, antigen-encoding sequences, and additional regulatory elements that facilitate efficient RNA amplification and expression.

[0111] The technical sophistication of in vitro transcription allows for remarkable molecular manipulation and optimization of self-amplifying RNA constructs. Researchers can strategically incorporate modified nucleotides, enhance RNA stability, and fine-tune the molecular characteristics of the synthesized RNA. By carefully controlling reaction conditions, enzyme selection, and template design, scientists can produce saRNA molecules with enhanced cellular penetration, improved immunogenicity, and more consistent amplification properties. This approach provides a flexible and precise platform for developing advanced vaccine and immunotherapy technologies.

[0112] Viral Vector-Based Approach: Leveraging Natural Viral Mechanisms

[0113] Viral vector-based self-amplifying RNA production harnesses the sophisticated genetic machinery of attenuated viruses to create powerful RNA constructs for vaccine and immunotherapy applications. By utilizing viral vectors, particularly those from alphaviruses like Venezuelan Equine Encephalitis virus (VEEV), researchers can exploit natural viral replication mechanisms to generate highly efficient self-amplifying RNA molecules. These vectors are engineered to incorporate specific genetic elements that enable robust RNA replication while eliminating pathogenic components, creating a safe and effective platform for RNA-based therapeutic strategies.

[0114] The viral vector approach offers unique advantages in saRNA production, capitalizing on evolutionary-refined molecular systems that have developed intricate mechanisms for genetic replication and cellular interaction. Scientists carefully modify viral genetic architectures to remove virulence factors while preserving essential replication machinery. By integrating antigen-encoding sequences with viral replication genes, researchers can develop saRNA constructs that trigger potent immune responses, potentially offering more effective and doseefficient vaccine technologies compared to traditional RNA delivery methods. CRISPR / Cas-Mediated Approach: Precision Genome Editing

[0115] CRISPR / Cas-mediated self-amplifying RNA synthesis represents a cutting-edge molecular engineering technique that provides unprecedented precision in genetic manipulation. By leveraging the revolutionary CRISPR / Cas genome editing technologies, researchers can create highly customized genetic constructs designed to generate self-amplifying RNA molecules with exceptional accuracy. This approach allows scientists to systematically design, modify, and optimize RNA replication machinery, introducing specific genetic elements that enhance RNA stability, amplification efficiency, and immunological potential.

[0116] The transformative power of CRISPR / Cas methods lies in their ability to perform molecular-level genetic interventions with remarkable specificity and control. Researchers can precisely target and modify genetic sequences responsible for RNA replication, introducing novel genetic architectures that optimize self-amplification mechanisms. By carefully engineering genetic elements, introducing targeted modifications, and developing sophisticated computational models, scientists can create saRNA constructs with enhanced cellular penetration, improved immune system activation, and potentially more effective vaccine and immunotherapy strategies.

[0117] Enzymatic Synthesis Method: Biochemical RNA Production

[0118] Enzymatic synthesis of self-amplifying RNA represents a sophisticated biochemical approach that utilizes specialized enzymes to generate complex RNA molecules with precise molecular characteristics. Researchers employ RNA-dependent RNA polymerases and other advanced enzymatic tools to catalyze the synthesis of self-amplifying RNA constructs, carefully controlling reaction conditions to optimize RNA production. This method allows for the incorporation of modified nucleotides, strategic molecular engineering, and the creation of RNA molecules with enhanced stability and amplification properties.

[0119] The enzymatic synthesis approach offers a nuanced and highly controlled method for saRNA production, providing researchers with exceptional molecular precision and flexibility. By selecting specific enzymes, optimizing reaction parameters, and designing sophisticated molecular templates, scientists can create self-amplifying RNA molecules with tailored characteristics for vaccine and immunotherapy applications. The technique enables the generation of RNA constructs with improved cellular compatibility, enhanced immunological properties, and potentially more efficient genetic delivery mechanisms.

[0120] Reverse Genetics Approach: Reconstructing Viral RNA Systems

[0121] Reverse genetics methods for self-amplifying RNA production represent an innovative approach that systematically deconstructs and reconstructs viral RNA replication mechanisms to develop advanced therapeutic RNA molecules. By comprehensively analyzing viral genetic systems, researchers can identify and isolate the critical genetic elements responsible for RNA replication, creating minimal yet functional genetic constructs capable of robust selfamplification. This approach allows scientists to develop sophisticated saRNA molecules that capture the essential replication capabilities of viral systems while eliminating pathogenic components.

[0122] The reverse genetics technique provides a powerful framework for understanding and manipulating RNA replication mechanisms at their most fundamental level. Researchers can meticulously map viral genetic architectures, identify key replication elements, and reconstruct these systems into safe and effective RNA molecules for therapeutic applications. By breaking down complex viral genetic systems into their core components and reassembling them with strategic modifications, scientists can develop self-amplifying RNA constructs with enhanced stability, improved immunological characteristics, and potentially more effective vaccine and immunotherapy strategies.

[0123] In some embodiments, each of the nucleotide sequences in the composition are present in a polycistronic sequence. In some embodiments, the polycistronic sequence comprises a nucleotide linker sequence. Nucleotide linker sequences may be used to connect or separate different nucleotide sequences that encode antigens, create spacers, enhance nucleotide sequence packaging into lipid compositions, or to facilitate molecular manipulations. In some embodiments, the polycistronic sequence comprises two or more antigens separated by a nucleotide linker sequence.

[0124] In some embodiments, the polynucleotide may encode at least one antigen. In some embodiments, the antigens are epitopes. An epitope is a small area on the surface of an antigen that can trigger an immune response. Epitopes are molecular regions that bind to antigen-specific receptors on the surface of immune cells.

[0125] In some embodiments, the polynucleotide may encode at least one antigen. In some embodiments, the polynucleotide encodes antigens in the range of between 2 to 1000 antigens. In some embodiments, the polynucleotide encodes 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, or more and any number in-between these set numbers of antigens (3). In some embodiments, the polynucleotides may comprise a nucleotide linker sequence (4) that is nonimmunogenic or comprises low immunogenicity (e.g., polyG linker sequence). In some embodiments the nucleotide linker sequence (4) is any sequence that encodes the protein linker sequence of GGSGGGSGG (SEQ ID NO: 93). Examples of nucleotide linker sequences (4) that encode SEQ ID NO: 2 include those shown in Tables 11-14. Exemplary linker sequences include, SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 56, 58, 60, 62, 64, and 66. In some embodiments, the linker sequence is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 56, 58, 60, 62, 64, and 66. In some embodiments, the nucleotide linker sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 56, 58, 60, 62, 64, and 66. The nucleotide linker sequence may disrupt an active site in a cancer peptide encoded by a nucleotide sequence of the present technology. In some embodiments, the nucleotide linker sequence may prevent tertiary structure formation of the cancer antigen and / or may prevent function of the cancer antigen. Polynucleotides of the present invention may be any size to accommodate the antigens and other elements necessary for their function. Polynucleotides may be in the range of about 100 bases or base pairs, 200 bases or base pairs (bp), 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more bases or bp and any size in-between. Each antigen encoded by the polynucleotide may be between 5 and 50 amino acids in length, between 8 and 50 amino acids in length, between 10 and 45 amino acids in length, between 15 and 40 amino acids in length or any number therebetween. The polynucleotides include coding regions for cancer antigens separated by linkers for at least two antigens to allow in frame translation of a fusion polypeptide comprising the at least two antigens.

[0126] The polycistronic nucleotide sequence may further comprise a nucleotide sequence that promotes localization of the nucleotide sequence or encodes a peptide that promotes localization of the peptide (e.g., a signal peptide (2)). In some embodiments, the nucleotide sequence promotes localization to cells comprising a human leukocyte antigen molecule (HLA). In some embodiments, the HLA is an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule. In some embodiments, the nucleotide sequence promotes localization to cells comprising a major histocompatibility complex (MHC) molecule. In some embodiments, the MHC molecule is an MHC Class I molecule or an MHC Class II molecule. Localization patterns for nucleotides and peptides of the present technology may be assessed using predictive localization software, including but not limited to TargetP, WoLF PSORT, DeepLoc, CELLO, YLoc, BaCelLo, and LocTree3.

[0127] In some embodiments, the compositions of the present technology comprise features that promote binding to and / or co-localization with an HLA (e.g., HLA-A, HLA-B, or HLA- C), an APC (e.g., a peptide or nucleotide sequence that targets or binds to an MHC Class II molecule), and / or a T-cell receptor (TCR). In some embodiments, the compositions comprise a carbohydrate that binds a carbohydrate receptor on an APC. In some embodiments, the carbohydrate is a mannose carbohydrate. The mannose carbohydrate may be selected from the group consisting of D-mannose, mannose-6-phosphate (M6P), a mannans, a mannose polymer, and a mannose receptor ligand.

[0128] In some embodiments, the compositions of the present technology comprise a hydrophobic tail. The hydrophobic tail may act as a targeting moiety, promoting binding to APCs. In some embodiments, the hydrophobic tail comprises a fatty acid, a phospholipid, a cholesterol, a retinoid, a steroid, an alkyl chain, or a nonpolar amino acid side chain.

[0129] Peptides

[0130] In some embodiments, the LNP of the present disclosure may comprise at least one polypeptide. In some embodiments, the polypeptides encode for antigens or epitopes. The present technology comprises compositions having one or more cancer antigens that are a protein or peptide antigen. In some embodiments, the composition comprises two different peptide antigens. Any one of the protein or peptide antigens may promote an immune response or immunological memory. In some embodiments, each of the protein or peptide antigens promote an immune response or immunological memory. In some embodiments, the peptides are delivered using nanoparticles that can target DCs.

[0131] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi -molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain.

[0132] "Amino acids" are molecules containing an amine group, a carboxylic acid group, and a side-chain that is specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, andnitrogenandhavethegenericformulaH2N-CHR- COOH, wherein R represents a side chain group. The various a-amino acids differ in the sidechain moiety that is attached to the a-carbon. The "amino acids" of the present technology include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User's Guide, G. A. Grant, editor, W.H. Freeman & Co., New York(1992),theteachingsofwhichareincorporated hereinby reference, includingthetext and table set forth at pages 11 through 24. As set forth above, the term "amino acid" also includes stereoisomers andmodifi cations of naturally occurring protein aminoacids, nonprotein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. Modified and unusual amino acids are described generally inSynthetic Peptides: A User's Guide, supra; Hruby et al., Biochem. J. 268:249-262 (1990); and Toniolo, 7n / . J. Peptide Protein Res. 35:287-300 (1990); the teachings of all of which are incorporated herein by reference. Amino acids, including stereoisomers and modifications of naturally occurring amino acids, protein amino acids, non-protein amino acids, post- translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs, or structures designed to mimic amino acids (peptide mimetics), and the like, including all of the foregoing, are sometimes referred to herein as "residues."

[0133] Natural amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), Lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Vai or V).

[0134] Unnatural amino acids include, but are not limited to, azetidinecarboxylic acid, 2- aminoadipic acid, 3-aminoadipic acid, beta-alanine, naphthylalanine (“naph”), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2- aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid, tertiary-butylglycine (“tBuG”), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N- ethylglycine, N-ethylasparagine, homoproline (“hPro” or “homoP”), hydroxylysine, allohydroxylysine, 3 -hydroxyproline (“3Hyp”), 4-hydroxyproline (“4Hyp”), isodesmosine, alloisoleucine, N-methylalanine (“MeAla” or “Nime”), N-alkylglycine (“NAG”) including N- methylglycine, N-methylisoleucine, N-alkylpentylglycine (“NAPG”) including N- methylpentylglycine. N-methylvaline, naphthylalanine, norvaline (“Norval”), norleucine (“Norleu”), octylglycine (“OctG”), ornithine (“Orn”), pentylglycine (“pG” or “PGly”), pipecolic acid, thioproline (“ThioP” or “tPro”), homoLysine (“hLys”), and homoArginine (“hArg”).

[0135] The term “amino acid analog” refers to a natural or unnatural amino acid where one or more of the C-terminal carboxy group, the N-terminal amino group and side-chain bioactive group has been chemically blocked, reversibly or irreversibly, or otherwise modified to another bioactive group. For example, aspartic acid-(beta-methyl ester) is an amino acid analog of aspartic acid; N- ethylglycine is an amino acid analog of glycine; or alanine carboxamide is an amino acid analog of alanine. Other amino acid analogs include methionine sulfoxide, methionine sulfone, S- (carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.

[0136] As used herein, a “conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A) and Glycine (G); 2) Aspartic acid (D) and Glutamic acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V); 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W); 7) Serine (S) and Threonine (T); and 8) Cysteine (C) and Methionine (M). Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (or basic) (histidine (H), lysine (K), and arginine (R)); polar negative (or acidic) (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi-conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.

[0137] In some embodiments, unless otherwise specified, a conservative or semi -conservative amino acid substitution may also encompass non-naturally occurring amino acid residues that have similar chemical properties to the natural residue. These non-natural residues are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include, but are not limited to, peptidomimetics and other reversed or inverted forms of amino acid moieties. Embodiments herein may, in some embodiments, be limited to natural amino acids, nonnatural amino acids, and / or amino acid analogs.

[0138] Non-conservative substitutions may involve the exchange of a member of one class for a member from another class.

[0139] The term "peptide" as used herein includes any structure comprised of two or more amino acids, including chemical modifications and derivatives of amino acids. The amino acids forming all or a part of a peptide may be naturally occurring amino acids, stereoisomers and modifications of such amino acids, non-protein amino acids, post translationally modified amino acids, enzymatically modified amino acids, constructs or structures designed to mimic amino acids, and the like, so that the term "peptide" includes pseudopeptides and peptidomimetics, including structures which have a non-peptidic backbone. The term "peptide" also includes dimers or multimers of peptides. A "manufactured" peptide includes a peptide produced by chemical synthesis, recombinant DNA technology, biochemical, or enzymatic fragmentation of larger molecules, combinations of the foregoing or, in general, made by any other method. The term "peptide" includes peptides containing a variable number of amino acid residues, optionally with non amino acid residue groups at the N- and C-termini, such groups including acyl, acetyl, alkenyl, alkyl, N-alkyl, amine, or amide groups, among others.

[0140] By employing chemical synthesis, a useful means of production, it is possible to introduce various amino acids which do not naturally occur along the chain, modify the N or C -terminus, and the like, thereby providing for improved stability and formulation, resistance to protease degradation, and the like.

[0141] The polypeptides of the present disclosure may also comprise a linker sequence. Peptide linker sequences may be used to connect or separate different antigens, enhance peptide packaging into various compositions, or to facilitate molecular manipulations. The peptide linker sequence may be nonimmunogenic or comprises low immunogenicity. In some embodiments the linker sequence (4) may comprise GGSGGGSGG (SEQ ID NO: 93) or sequences about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 93. In some embodiments, the peptide linker sequence comprises a specific cleavage site linker (e.g., a Furin cleavage site; 2A linker peptides). The specific cleavage site linker may comprise a protease recognition site that is selectively cleaved by certain proteases. In some embodiments, the peptide linker sequence comprises a targeting or tagging linker that permits the localization, detection, or purification of peptides. The peptide linker sequence may comprise a flexible linker (e.g., a glycine and serine rich linker) or a rigid linker (e.g., a glycine rich linker or an alpha-helical structure linker). The peptide linker sequence may disrupt an active site in a cancer antigen. In some embodiments, the peptide linker sequence may prevent tertiary structure formation of the antigen and / or may prevent function of the antigen.

[0142] In some embodiments, the compositions of the present technology may further comprise a signal peptide. Nonlimiting examples of signal peptides include peptides that promote localization to cellular compartments (e.g., endoplasmic reticulum, mitochondria, nucleus, or peroxisomes), signal peptides that promote peptide secretion, and signal peptides that promote cleavage.

[0143] In some embodiments, the signal peptide promotes localization and / or binding to cells comprising an MHC Class I molecule. In some embodiments, the signal peptide promotes localization and / or binding to cells comprising an MHC Class IT molecule. In some embodiments, the cells comprising an MHC Class II molecule are APC cells. In some embodiments, the APC cells are DCs. In some embodiments, the signal peptide promotes localization and / or binding to cells comprising an HLA. In some embodiments, the HLA is an HLA-A molecule, an HLA-B molecule, or an HLA-C molecule. Peptide binding for peptides of the present technology may be assessed using predictive binding software, including but not limited to NetMHC, NetMHCpan, NetMHCllpan, Immune Epitope Database and Analysis Resource (IEDB), SYFPEITHI, Stabilized Matrix Method (SMM), ProPred-I, ProPred, and NetCTLpan.

[0144] In some embodiments, the peptide is about 5 to about 50 amino acids, about 8 to about 50 amino acids, about 15 to about 40 amino acids, about 50 to about 100 amino acids, about 100 to about 200 amino acids, about 200 to about 500 amino acids, about 500 to about 1000 amino acids, about 1000 to about 2500 amino acids, about 2500 to about 5000 amino acids, about 5000 to about 10000 amino acids, about 10000 to about 100000 amino acids, or more. In some embodiments, the peptide is less than about 30 amino acids. In some embodiments, a longer polypeptide is generated as a fusion protein in which each of the peptides is linked via a linker to the next peptide antigen. In these polypeptides each of the peptides in the compositions are less than about 30 amino acids. In some embodiments, the compositions comprise a peptide that is less than about 25 amino acids. In some embodiments, each of the peptides in the compositions are less than about 25 amino acids. In some embodiments, the compositions comprise a peptide that is less than about 20 amino acids. In some embodiments, each of the peptides in the compositions are less than about 20 amino acids. In some embodiments, the compositions comprise a peptide that is less than about 15 amino acids. In some embodiments, each of the peptides in the compositions are less than about 15 amino acids. In some embodiments, the compositions comprise a peptide that is less than about 10 amino acids. In some embodiments, each of the peptides in the compositions are less than about 10 amino acids.

[0145] In some embodiments, the compositions comprise one or more peptides of the present technology and one or more polynucleotide sequences of the present technology. In some embodiments, the compositions of the present technology comprise two or more peptides of the present technology and one or more nucleotide sequences of the present technology. In some embodiments the compositions of the present technology comprise two or more nucleotide sequences of the present technology and one or more peptides of the present technology. In some embodiments, the compositions comprise two or more nucleotide sequences of the present technology and two or more peptides of the present technology.

[0146] Antigens

[0147] In some embodiments, the polynucleotide or polypeptide of the present disclosure encodes and antigen. An antigen is a molecule, moiety, foreign particulate matter, or an allergen, such as pollen, that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. In some embodiments, the antigen is a cancer antigen, or a cancer epitope. "Cancer" as used herein may refer to one or more of bone cancer, testicular cancer, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, squamous cell head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, etc.), ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma. "Cancer" may also refer to an individual cancer cell, multiple cancer cells, or a tumor. Cancer may also comprise metastasized tumors and pre-cancers.

[0148] "Cancer antigen" as used herein may refer to any antigen expressed by or associated with a cancer. This may include antigens found on both noncancer and cancer cells but are upregulated by cancer cells, antigens only found on cancer cells, or antigens modified by a cancer.

[0149] The present technology comprises compositions which provide cancer antigens and promote immunological surveillance and memory. The compositions elicit an immune response, thereby triggering the recognition, response, and or elimination of the cancer antigens and cells comprising the cancer antigens. In some embodiments, the compositions of the present technology induce an immune response in a subject to the cancer antigens. In some embodiments, the compositions of the present technology may be administered to a subject to treat, prevent, and / or reduce / slow the growth or progression of a cancer.

[0150] The cancer antigens of the present technology may be comprised of protein and / or peptide antigens, glycoprotein antigens, or lipid antigens. In some embodiments, the composition comprises one or more nucleotide sequences which encode cancer antigens. In some embodiments, the composition comprises one or more peptide antigens and one or more polynucleotide encoding an antigen.

[0151] The cancer antigens of the present technology may be an antigen associated with more than one cancer. In some embodiments, the subject has more than one cancer, is at risk for developing more than one cancers, or has symptoms of more than one cancer.

[0152] In some embodiments, the composition comprises one or more nucleotide sequence and or one or more peptide sequence. In some embodiments, the composition comprises two or more nucleotide sequences. In some embodiments, the composition comprises a nucleotide sequence encoding a cancer antigen protein or peptide thereof. The cancer antigen may be a neoantigen. The cancer antigen may comprise a product of a mutated oncogene, a product of a mutated tumor suppressor gene, a cancer antigen produced by a cancer, an oncofetal antigen, an altered cell surface glycolipid and / or glycoprotein, a lineage restricted antigen, an idiotypic antigen, a post-translationally altered antigen, or a product of other mutated genes (e g., over expressed and / or aberrantly expressed cellular proteins).

[0153] The cancer antigen may comprise a product of a mutated oncogene, a product of a mutated tumor suppressor gene, a cancer antigen produced by a cancer, an oncofetal antigen, an altered cell surface glycolipid and / or glycoprotein, a lineage restricted antigen, an idiotypic antigen, a post-translationally altered antigen, or a product of other mutated genes (e.g., over expressed and / or aberrantly expressed cellular proteins).

[0154] Cancer antigens of the present disclosure may be any antigen that have increased frequency in a cancer. Cancer antigens of the present disclosure may be selected based on genetic sequencing of a subject and their cancer. As such, the compositions described herein may be used as a personalized therapeutic. Cancer antigens of the present disclosure may be selected based on population-based sequencing and frequency data. Population based data may be obtained from public or private databases including but not limited to National Cancer Institute databases (e.g. The Cancer Genome Atlas, Mutagene, COSMIC, LocusLink, OMIM). Population based data may be based on the general population, or on a population of subjects selected based on a particular distinguishing factor. Examples of populations of subject with distinguishing factors include, but are not limited to, subject with one or more genetic markers (e.g. a population of subjects with BRACA1 / BRAC2 mutations), a population based on age, a population based on environmental exposures or a population based on lifestyle factors (e.g. tobacco use).

[0155] In some embodiments, the cancer antigen is a cancer antigen produced by a cancer (e.g., an oncogenic cancer). In some embodiments, the cancer is selected from the group consisting of a Human Papillomacancer (HPV), a Hepatitis cancer (e.g., Hepatitis B (HBV) andHepatitis C (HCV)), a Human Immunodeficiency Cancer (HIV), an Epstein-Barr Cancer (EBV), a Human T-cell Lymphotropic Cancer (HTLV-1), a Herpescancer (e.g., Kaposi's Sarcoma-associated Herpescancer (KSHV), Human Herpescancer 8 (HHV-8)), a Simian Cancer 40 (SV40), an Adenocancer, or a Polyomacancer (e.g., Merkel Cell Polyomacancer (MCV)).

[0156] In some embodiments, the cancer is selected from the group consisting of bone cancer, testicular cancer, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, squamous cell head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, etc.), ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma.

[0157] In some embodiments, the composition comprises a nucleotide sequence encoding a protein and or a peptide thereof selected from the group of Tumor Associated Antigens (overexpressed in cancer) including but not limited to Carcinoembryonic Antigen (CEA), Prostate-specific Antigen (PSA), Prostate-Specific Membrane Antigen (PSMA), p53 (TP53), Wilm's Tumor 1 (WT1), CA-125, Alpha-fetoprotein (AFP), Cancer antigen 125, cancer antigen 15-3, carbohydrate antigen 19-9, carcinoembryonic antigen, human chronic gonadotrophin, Human Epidermal Growth Factor Receptor 2 (HER2), Melanoma-associated Antigen (MAGE), MUC1, NY-ESO-1, Glycoprotein 100 (GP100), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), human papilloma cancer E6 protein, human papilloma cancer E7 protein, CEA, Immature laminin receptor, TAG-72, BING-4, Calcium-activated chloride channel 2, cyclin-Bl, 9D7, Ep-CAM, EphA3, telomerase, Mesothelin, SAP-1, Survivin, a BAGE family antigen, a CAGE family antigen, a GAGE family antigen, a MAGE family antigen, a SAGE family antigen, a XAGE family antigen, NY-ESO-l / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, Gpl00 / pmell7, Tyrosinase, TRP-1 / -2, P.polypeptide, MC1R, 13- catenin, BRCA1, BRCA2, CDK4, CML66, Fibronectin, MART-2, Ras, TGF-1311, orMUCl.

[0158] The composition may comprise a nucleotide sequence encoding a protein and or a peptide thereof of the cancer antigens disclosed in (1) Zarour HM, Deleo A, Finn OJ, et al. Categories of Tumor Antigens. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland- Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003; (2) Vogel WH . Diagnostic evaluation, classification and staging. Yarbro CH, Wujcki D, Holmes Gobel B, (eds.). Cancer Nursing: Principles and Practice. 8th ed. Burlington, MA: Jones and Bartlett Learning; 2018: 7: 169-203; (3) Fischbach FT, Fischbach MA Fischbach's A Manual of Laboratory and Diagnostic Tests. 10th ed. Wolters Kluwer; 2018, the entire contents of each of which are incorporated by reference herein.

[0159] In some embodiments, the composition comprises a first nucleotide sequence encoding a first antigen and a second nucleotide sequence encoding a second antigen, the first and second antigens each independently selected from the group consisting of a Carcinoembryonic Antigen (CEA) peptide, a Prostate-specific Antigen (PSA) peptide, a Prostate-Specific Membrane Antigen (PSMA) peptide, a p53 (TP53) peptide, a Wilm's Tumor 1 (WT1) peptide, a CA-125 peptide, a Alpha-fetoprotein (AFP) peptide, a Cancer antigen 125 peptide, a cancer antigen 15-3 peptide, a carbohydrate antigen 19-9 peptide, a carcinoembryonic antigen peptide, a human chronic gonadotrophin peptide, a Human Epidermal Growth Factor Receptor 2 (HER2) peptide, a Melanoma-associated Antigen (MAGE) peptide, a MUC1 peptide, a NY-ESO-1 peptide, a Glycoprotein 100 (GP100) peptide, a alphafetoprotein (AFP) peptide, a carci noembryonic antigen (CEA) peptide, a human papilloma cancer E6 protein peptide, a human papilloma cancer E7 protein peptide, a CEA peptide, a Immature laminin receptor peptide, a TAG-72 peptide, a BING-4 peptide, a Calcium-activated chloride channel 2 peptide, a cyclin-B 1 peptide, a 9D7 peptide, a Ep- CAM peptide, a EphA3 peptide, a telomerase peptide, a Mesothelin peptide, a SAP-1 peptide, a Survivin peptide, aB AGE family antigen peptide, a CAGE family antigen peptide, a GAGE family antigen peptide, a MAGE family antigen peptide, a SAGE family antigen peptide, a XAGE family antigen peptide, a NY-ES 0-1 / LAGE- 1 peptide, a PRAME peptide, a SSX-2 peptide, a Melan-A / MART-1 peptide, a Gpl00 / pmell7 peptide, a Tyrosinase peptide, a TRP-1 / -2 peptide, a P. polypeptide peptide, a MC1R peptide, a 13- catenin peptide, a BRCA1 peptide, a BRCA2 peptide, a CDK4 peptide, a CML66 peptide, a Fibronectin peptide, a MART-2 peptide, a Ras peptide, a TGF-13II peptide, a or MUC1 peptide.

[0160] In some embodiments, the antigen is selected from those in any of Tables 1-14, and combinations thereof.

[0161] In some embodiments, a polynucleotide described herein may encode at least two antigens separated by a linker. In some embodiments, the antigens are selected from the group consisting of DNMT3A, TP53, PIK3CA, EGFR, NRAS, IDH2, FLT3, nucleophosmin, and KRAS, and combinations thereof. In some embodiments the antigen is selected from the group consisting of mutations in DNMT3A, TP53, PIK3CA, EGFR, NRAS, IDH2, FLT3, nucleophosmin, and KRAS, and combinations thereof.

[0162] In some embodiments, a polynucleotide described herein may encode at least two antigens separated by a linker. In some embodiments, the antigens are selected from the group consisting of DNMT3A R882C, TP53 R158L, PIK3CA Q546K, TP53 C238Y, EGFR 747-751 A, TP53 E286K, TP53 V157F, PIK3CA E542K, PIK3CA E545K, NRAS Q61K, PIK3CA Q546K, IDH2 R140Q, TP53 R175H, DNMT3A R882H, TP53 G266E, FLT3 D835Y, TP53 H214R, nucleophosmin NPM1 W288Cfs*30, KRAS G12D, TP53 R248Q, KRAS G12V, TP53 R248W, KRas G12C, EGFR T790M, KRas G13D and combinations thereof. In some embodiments, the polynucleotides comprise any of those in Tables 11-14. Tables 1-10 and 15 and 16 provide additional proteins and mutation containing epitopes for use as antigens in the compositions. Exemplary polynucleotides and peptides are shown in Tables 11-14. Exemplary polynucleotides include SEQ ID NO: 18-67; SEQ ID NO: 1-67; SEQ ID NOs: 1-4 and 8-67; SEQ ID NOs: 2-7 and 9-12 and 17-67; SEQ ID NOs: 2-5 and 8-12 and 17-67; SEQ ID NO: 99. Exemplary peptides comprise SEQ ID NOs: 68-92 and SEQ ID NO: 100. Exemplary polynucleotides can be encoded by SEQ ID NO: 95, 96, 97 and 98.

[0163] Pharmaceutical Compositions

[0164] In some embodiments, the composition of the present disclosure may comprise an additional carrier, excipient or diluent. The composition may comprise proteolipids (e.g., protamine), carrier proteins, and / or small molecules. As used herein, the term “carrier” refers to a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and / or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the above categories, may be found in the U.S. Pharmacopeia National Formulary, 1857-1859, (1990).

[0165] Some examples of the materials which can serve as pharmaceutically acceptable carriers are 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; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; 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 nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions, according to the desires of the formulator.

[0166] Examples of pharmaceutically acceptable antioxidants include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and metal-chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

[0167] In another embodiment, the present formulation may also comprise other suitable agents such as a stabilizing delivery vehicle, carrier, support or complex-forming species. The coordinate administration methods and combinatorial formulations of the instant invention may optionally incorporate effective carriers, processing agents, or delivery vehicles, to provide improved formulations for delivery of the NLP.

[0168] The composition may additionally include a biologically acceptable buffer to maintain a pH close to neutral (7.0-7.3). Such buffers preferably used are typically phosphates, carboxylates, and bicarbonates. More preferred buffering agents are sodium phosphate, potassium phosphate, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, and potassium bicarbonate. The buffer may comprise about 0.0001-5% (w / v) of the formulation, more preferably about 0.001-1% (w / v). Other excipients, if desired, may be included as part of the final formulation.

[0169] Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g. , aluminum hydroxide); and preservative.

[0170] In some embodiments, the compositions of the present technology comprise adjuvants. Adjuvants may stimulate or enhance an immune response upon administration of the composition. Nonlimiting examples of adjuvants include aluminum salts, oil-in- water emulsions (e.g., MF59 or AS03), pathogen mimics (e.g., CpG oligonucleotides, monophosphoryl lipid A), squalene, virosomes, and liposomes). In some embodiments, the composition is sterilized (e.g., sterilization by filtration)

[0171] As used herein, a "composition" or a "pharmaceutical composition" refers to a mixture of the active ingredient with other chemical components, such as pharmaceutically acceptable carriers and / or excipients. Tn some embodiments, the compositions comprise one or more peptides of the present technology (e.g., antigen peptides) or nucleotide sequences encoding peptides of the present technology in an amount of about 0.05 % w / v or w / w of the composition, about 0.1% w / v or w / w of the composition, about 1% w / v or w / w of the composition; about 10% w / v or w / w of the composition; about 20% w / v or w / w of the composition; about 30% w / v or w / w of the composition; about 40% w / v or w / w of the composition; about 50% w / v or w / w of the composition; about 60% w / v or w / w of the composition; about 70% w / v or w / w of the composition; about 80% w / v or w / w of the composition; about 90% w / v or w / w of the composition; about 95% w / v or w / w of the composition; or about 99% w / v or w / w of the composition.

[0172] In some embodiments, the composition comprises a lyophilized drug product (lyophilized powder or lyophilized cake of the peptides or nucleotide sequences). In some embodiments, the lyophilized drug product is reconstituted (e.g., using water or saline) prior to administration.

[0173] In some embodiments, the reconstituted composition provides a solution having a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.

[0174] In some embodiments, the reconstituted composition provides a solution having a concentration of a peptide of the present technology or a nucleotide sequence of the present technology that is about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml about 13 mg / ml, about 14 mg / ml, about 15mg / ml, about 20 mg / ml, about 25 mg / ml, about 30 mg / ml, or about 40 mg / ml, about 50mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, or about 100 mg / ml.

[0175] Cell compositions The present technology comprises cell compositions that are immunogenic to antigens and promote immunological surveillance and memory. The cell composition may degenerated such that the cells are immunogenic to specific antigens (e.g., cancer antigens). Additionally, the cell compositions may stimulate an immune reaction leading to the production of an immune response. Nonlimiting examples of immune responses include the production of antibodies or activation of lymphatic cells (e.g., T-cells). In some embodiments, the cell compositions of the present technology induce an immune response in a subject to one or more antigens. In some embodiments, the cell compositions of the present technology may be administered to a subject to treat, prevent, and / or reduce / slow the growth of a cancer and / or other conditions associated with cancer.

[0176] The cell composition may stimulate an immune response to a protein and / or peptide of the present technology. In some embodiments, the cell composition is stimulating an immune response to a nucleotide sequence of the present technology (e.g., a nucleotide sequence encoding a peptide of the present technology). In some embodiments, the cell composition is stimulating an immune response to one or more peptides of the present technology and one or more nucleotide sequences of the present technology (e.g., a nucleotide sequence encoding a cancer peptide). Tn some embodiments, the cell composition is stimulating an immune response to two or more peptides of the present technology and one or more nucleotide sequences of the present technology. In some embodiments the cell composition is stimulating an immune response to two or more nucleotide sequences of the present technology and one or more peptides of the present technology. In some embodiments, the cell composition is stimulating an immune response to two or more nucleotide sequences of the present technology and two or more peptides of the present technology.

[0177] The cell compositions of the present technology may be generated from a subject sample (e.g., a biological sample). The subject sample may be a circulatory fluid sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), a lymphoid tissue sample (e.g., lymphnode, spleen, ortonsil tissue), amucosal sample (e.g., mucosal tissue), a bone marrow sample, a cerebrospinal fluid (CSF) sample, or a synovial fluid sample. Tn some embodiments, the cell composition is a lymphatic cell composition. The cell composition may comprise one or more of T-cells, B cells, Natural Killer (NK) cells, Dendritic Cells (DCs), macrophages, or granulocytes. The cell composition may be ex-vivo cells or differentiated cells.

[0178] In some embodiments, the cell composition is a dendritic cell composition. The DC composition may comprise one or more subsets of DC. The DC may be isolated from a subject or may be differentiated. In some embodiments, the DC may be contacted ex-vivo with a composition described herein. Cell compositions may further comprise T lymphocytes.

[0179] In some embodiments, the cell composition is a T-cell composition. T-cells may comprise helper T-cells (Th cells) (e.g., CD4+ cells), cytotoxic T-cells (e.g., CD8+ cells), regulatory T-cells (Tregs), memory T-cells, and follicular helper T-cells (Tfh cells). Nonlimiting examples of Th cells include Thl cells, Th2 cells, Th 17 cells.

[0180] In some embodiments, the cell composition comprises one or more CD4+ cell subsets. In some embodiments, the one or more CD4+ cell subsets are selected from the group consisting of Thl cells, Th2 cells, and Th 17 cells.

[0181] In some embodiments, the Thl (CD4+) T-cells produce cytokines, including LFNy, and generate a CD8+ T-cell (e.g., cytotoxic T-cell) response. In some embodiments, Th2 (CD4+) T-cells produce cytokines, including IL4, and generate a high affinity antibody immune response, including class switching to IgG and IgA.

[0182] In some embodiments, the cell composition is reactive to one or more cytokines and / or chemokines (e.g., activation, differentiation, signaling, and / or function of an immune cell is stimulated in the presence of the cytokines or chemokines). Nonlimiting examples of cytokines and chemokines include interleukins (Ils) (e.g., IL-2, IL-4, IL-6, IL-8, IL-10, or IL- 12), tumor necrosis factors (TNFs) (e.g., TNFa or TNF13), interferons (INFs) (e.g., INFy, INF a, orINF13), chemokines (e.g., CXCL8, CXCL10, or CCL2), colony-stimulating factors (CSFs) (e.g., granulocyte-macrophage CSF or granulocyte CSF), or Transforming Growth F actor-b eta (e . g . , TGF13 ) . Tn some embodiments, the DC cell composition is present in a composition or an infusion.

[0183] Methods

[0184] Another aspect of the present disclosure provides methods of using the compositions provided herein. In one embodiment, a method of preventing or treating cancer in a subject in need is provided. In some embodiments, the method comprises administering a nanoparticle comprising a ligand for targeting dendritic cells (DC) and a polynucleotide, wherein the polynucleotide encodes at least two cancer antigens separated by a linker, to the subject. In other embodiments, the method comprises administering a nanoparticle containing multiple separate RNA molecules encoding different sets of antigens or co-administration of combinations of nanoparticles containing such different RNA molecules.

[0185] As used herein, "treatment,” “therapy” and / or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder or condition. As used herein, the terms “prevent,” “prevention,” and preventing” refer to reducing the likelihood of a particular condition or disease state (e.g., cancer) from occurring in a subject not presently experiencing or afflicted with the condition or disease state. The terms encompass delaying, reducing and stopping the development of cancer, including the progression of pre-cancer to cancer. Precancer is a condition where abnormal cells have developed in tissue and could potentially become cancer if left untreated. The terms do not necessarily indicate complete or absolute prevention. “Prevention,” encompasses any administration or application of a therapeutic or technique to reduce the likelihood of a disease developing (e.g., in a mammal, including a human). Such a likelihood may be assessed for a population or for an individual. The compositions provided herein may be prophylactic, e.g., to prevent or ameliorate the effects of a future disease.

[0186] Treating cancer in a subject includes the reducing, repressing, delaying or preventing cancer growth, reduction of tumor volume, and / or preventing, repressing, delaying or reducing metastasis of the tumor. Treating cancer in a subject also includes the reduction of the number of tumor cells within the subject. The term "treatment" can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; and (d) reducing or ameliorating at least one symptom of cancer. In some embodiments, the optimum effective amount can be readily determined by one skilled in the art using routine experimentation.

[0187] As used herein, the term "administering" an agent, such as a composition described herein to an animal or cell, is intended to refer to dispensing, delivering or applying the substance to the intended target. The terms "administering" or "administer" include delivery of therapies (e.g., compositions) of the present technology to a subject either by local or systemic administration. Administration may be parenteral. In terms of the therapeutic composition, the term "administering" is intended to refer to contacting or dispensing, delivering or applying the therapeutic agent to a subject by any suitable route for delivery of the therapeutic agent to the desired location in the animal, including delivery by either the parenteral or oral route, intertumoral, intramuscular injection, subcutaneous / intradermal injection, intravenous injection, intrathecal administration, buccal administration, or transdermal delivery. In some embodiments, the administration is subcutaneous. In a preferred embodiment the therapeutic composition is administered subcutaneously.

[0188] A “subject in need thereof’ as utilized herein may refer to a subject in need of treatment for a disease or disorder associated with cancer or a genetic disease which carries an increased risk of cancer. A subject in need thereof may include a subject having a cancer that is characterized by gross abnormality visible by X-ray, computerized tomography (CT), or magnetic resonance imaging (MRI). The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. The term “subject” does not denote a particular age or sex. In some embodiments, the subject may include a subject with precancerous cells or a subject with a genetic predisposition to cancer.

[0189] In some embodiments, the composition described herein may be administered with other treatments, such as those considered standard of care including chemotherapy, radiation, bone marrow transplant, immunotherapy, and surgery.

[0190] As used herein the term “effective amount” or “therapeutically effective amount” refers to the amount or dose of the compound that provides the desired effect. In some embodiments, the effective amount is the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment.

[0191] An effective amount can be readily determined by those of skill in the art, including an attending diagnostician, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

[0192] In some embodiments, a method of treating cancer in a subject with a cancer-associated genetic mutation is provided, the method comprising administering a nanoparticle comprising a ligand for targeting dendritic cells (DC) and a polynucleotide, wherein the polynucleotide encodes at least two tumor antigens separated by a linker to the subject. In some embodiments, the subject in need is diagnosed with a genetic condition, wherein the genetic condition has an increased risk or predisposition for the development of cancer.

[0193] The term “mutation” or “genetic mutation” as used herein indicates any modification of a nucleic acid and / or polypeptide which results in an altered nucleic acid or polypeptide (i.e., relative to the wild-type nucleic acid or polypeptide sequence). Mutations include, for example, point mutations, substitutions, deletions, or insertions of single or multiple residues in a polynucleotide (or the encoded polypeptide), which includes alterations arising within a proteinencoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A genetic alteration may be a mutation of any type. For instance, the mutation may constitute a point mutation, a frame-shift mutation, an insertion, or a deletion of part or all of a gene.

[0194] A subject with cancer-associated genetic mutation or a genetic condition has an increased risk or predisposition for the development of cancer. In some embodiments, subjects may have hereditary cancer or family cancer syndrome. Such cancers are caused by inherited genetic variants in certain cancer-related genes. In some embodiments, a subject has a novel or spontaneous mutation that is associated with an increased risk for cancer. In some embodiments, the subject has a genetic disease or condition associated with an increased risk of cancer. Examples of cancer-associated genetic mutation include, but are not limited to, breast, ovarian, pancreatic and prostate cancer associated with the BRCA1 and BRCA2 genes, lynch syndrome associated with the EPCAM, MLH1, MSH2, MSH6, and PMS2 genes, Li-Fraumeni syndrome associated with the TP53 gene, hereditary diffuse gastric cancer associated with the CDH1 gene, Multiple endocrine neoplasia type 1 and 2 (MEN) associated with the RET gene, Familial adenomatous polyposis (FAP) associated with the APC gene and Von Hippel-Lindau disease associated with the VHL gene, PTEN Hamartoma tumor syndrome and Cowden syndrome associated with the PTEN gene, MUTYH associated polyposis (MAP) associated with the MUTYH gene, Juvenile Polyposis syndrome (JPS) associated with the BMPR1A and SMALM genes, PALB2 gene, Tuberous sclerosis (TS) associated with the TSC1 and TSC2 genes, Birt-Hogg-Dube syndrome (BHDS) associated with the FLCN gene, RBI gene, Familial atypical multiple mole melanoma syndrome (FAMMM), Hereditary kidney cancer syndromes associated with the MET gene, CHEK2 gene.

[0195] In some embodiments, subjects with colon cancer may be administered a polynucleotide and or peptide encoding an antigen selected from Table 1.

[0196] In some embodiments, subjects with lung cancer may be administered a polynucleotide and or peptide encoding an antigen selected from Table 2.

[0197] In some embodiments, subjects with pancreatic cancer may be administered a polynucleotide and or peptide encoding an antigen selected from Table 3 or Table 9.

[0198] In some embodiments, subjects with Diffuse large B-cell lymphoma (DLBCL)cancer may be administered a polynucleotide and or peptide encoding an antigen selected from Table 4.

[0199] In some embodiments, subjects with Acute myeloid leukemia (AML) cancer may be administered a polynucleotide and or peptide encoding an antigen selected from Table 5.

[0200] In some embodiments, subjects with melanoma cancer may be administered a polynucleotide and or peptide encoding an antigen selected from Table 6.

[0201] In some embodiments, subjects with bladder cancer may be administered a polynucleotide and or peptide encoding an antigen selected from Table 7.

[0202] In some embodiments, subjects with glioblastoma cancer may be administered a polynucleotide and or peptide encoding an antigen selected from Table 8.

[0203] In some embodiments, subjects with cancer may be administered a polynucleotide and or peptide encoding an antigen selected from any one of Tables 1-10. In some embodiments, subjects may be administered a DC targeted nanoparticle with antigens specific for their cancer or disease. By way of example and not limitation, subjects with a BRCA1 mutation may be administered a polynucleotide or peptides encoding BRCA1 mutations such as K654Sfs*47, El 1 lGfs*3, E720*, K223Rfs*l 1, El 112*, Y655Vfs*18, E572*, K339Rfs*2, Q94*, and R1516Kfs*6. Subjects with a BRAC2 mutation may be administered a polynucleotide or peptides encoding BRCA2 mutations such as T3033Lfs*29, K1691Nfs*15, N1784Kfs*3, I605Yfs*9, N1784Tfs*7, K3416Nfs*l l, E510*, N863Kfs*18, N986Ifs*5, and S599*. The BRCA antigens are further expanded to include but are not limited to those in Table 15.

[0204] Subjects with Lynch syndrome may be administered a polynucleotide or peptide encoding mutant epitopes from multiple genes that have somatic mutations in Lynch syndrome patients such as the following sequences: SPRRSPAL, GLMTLSKMIK, MTLSKMIKK, SPSQPKKMSV, FLLALWECSL, TPQDSRQVL, FVMSDTTYK, MSDTTYKIY, AMTTSSSQK, RLSSCVPVA, SAMTTSSSQK, VPVALMSAM, FPITPPVW,

[0205] GPRMQLCTQL, and TQLARFFPI. The antigens are further expanded to include but are not limited to those in Table 16 and the sequence identifiers can be found in Table 16.

[0206] In some embodiments the method further comprises determining the mutations specific to a subject, for example by sequencing a biopsy sample from a subject. In some embodiments, the subject can be administered a targeted nanoparticle comprising antigens specific for their mutations. In some embodiment, the subject may also be administered antigens to additional mutations in addition to their specific mutations.

[0207] In some embodiments, the method comprises isolating or differentiating DC from a subject and contacting the DC ex-vivo to the nanoparticles described herein. In some embodiments, the nanoparticles may comprise antigens specific to the subject, or not-subject specific cancer antigens. In some embodiments the contacted DC may be exposed to T- lymphocytes ex-vivo, the exposed DC or T-lymphocytes may also administered back to the subject.

[0208] In some embodiments, a method of inducing a dendritic cell mediated anti-tumor immune response is provided, the method comprising administering a nanoparticle comprising a ligand for targeting dendritic cells and a polynucleotide, wherein the polynucleotide encodes at least two cancer antigens separated by a linker to the subject. A dendritic cell mediated immune response refers to the process where dendritic cells, acting as professional antigen-presenting cells, capture, process, and present antigens to T cells, initiating an adaptive immune response against a pathogen or foreign substance, an anti-tumor immune response is initiated when the antigen is a tumor antigen. A dendritic cell mediated immune response may comprise proliferation, cytokine production and immunologic memory.

[0209] In some embodiments a method of inducing an immune response to more than one antigen is provided, the method comprising administering a nanoparticle comprising a ligand for targeting dendritic cells (DC) and a polynucleotide, wherein the polynucleotide encodes at least two antigens separated by a linker. In some embodiments, an immune response to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, or more and any amount in-between, antigens is provided. In some embodiments, the polynucleotide encodes for the more than one antigen, and an immune response is generated to the more than one antigen. In some embodiments, the immune response is a dendritic cell mediated immune response.

[0210] The present technology comprises compositions having nucleotide sequences that encode cancer antigens. Any one of the nucleotide sequences may promote a response by an immune cell, an immune response, or immunological memory against the cancer antigen which it encodes (e.g., generation of a CD4+ T-cell memory response, a CD8+ T-cell memory response, central memory T-cells (TCM), effector memory T-cells (TEM), crossreactivity of memory T-cells, or the maintenance of memory T-cells). In some embodiments, each of the nucleotide sequences promote an immune response or immunological memory against one or more of the cancer antigens to which the nucleotide sequence corresponds to.

[0211] In some embodiments, a method for targeting and delivering an agent to dendritic cells is provided. In some embodiments, the method comprises exposing DC to a nanoparticle comprising a DC targeting ligand, wherein the nanoparticle comprises a agent to be delivered. In some embodiments, the agent is cargo. A cargo molecule may comprise any molecule which is to be transported or delivered by the DC. By way of example and not limitation nanoparticle cargo may comprise RNA, DNA, active pharmaceutical ingredients, adjuvants, proteins, therapeutic cargo, gene editing cargo such as Cas9 and gRNA, delivery of vaccines including mRNA and / or adjuvants. Cargo may be organ, tissue or cell type specific.

[0212] Quantifying Lymphocyte Cell Populations Tn some embodiments, the methods of generating the compositions and / or the cell compositions of the present technology comprise a step of quantifying a lymphocyte cell (e.g., T-cell, B cell, NK cells, DC, macrophage, or granulocyte) population in a sample from a subject that has is in remission for a cancer and / or has successfully cleared a cancer (e.g., elimination or reduction of active cancer cell and / or tumor, establishment of immune memory against a cancer antigen) or an oncogenic cancer (e.g., reduction of oncogenic viral load, resolution of a symptom associated with infection of the oncogenic cancer). Quantifying the lymphocyte cell population may comprise methods including, but not limited to, flow cytometry, immunohistochemistry, immunofluorescence, protein quantification and / or detection methods (e.g., assessing levels of lymphocyte cell makers), or gene expression quantification methods (e.g., assessing expression levels of transcripts associated with lymphocyte cells).

[0213] In some embodiments, the lymphocyte cell population is a T-cell population. T cells may comprise helper T-cells (Th cells) (e.g., CD4+ cells), cytotoxic T-cells (e.g., CD8+ cells), regulatory T-cells (Tregs), memory T-cells, and follicular helper T-cells (Tfh cells). Nonlimiting examples of Th cells include Thl cells, Th2 cells, Thl7 cells.

[0214] In some embodiments, the T-cell composition comprises one or more CD4+ cell subsets. In some embodiments, the one or more CD4+ cell subsets are selected from the group consisting of Thl cells, Th2 cells, and Th 17 cells.

[0215] The sample from the subject may be a circulatory fluid sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), a lymphoid tissue sample (e.g., lymph node, spleen, or tonsil tissue), a mucosal sample (e.g., mucosal tissue), a bone marrow sample, a cerebrospinal fluid (CSF) sample, or a synovial fluid sample.

[0216] In some embodiments, the subject that is in remission for a cancer or has successfully cleared a cancer or an oncogenic cancer.

[0217] Exposing the Sample to a Cancer Antigen

[0218] In some embodiments, the methods of generating the compositions and / or cell compositions of the present technology comprise a step of exposing a sample to one or more antigens from the cancer. Exposing the sample may comprise direct exposure (e.g., inoculating the sample with a protein antigen, peptide antigen, lipid antigen, glycoprotein antigen, or a nucleotide sequence that produces an antigen), or indirect exposure. Indirect exposure may comprise exposing the sample to the one or more antigens using an antigen- presenting cell (APC). Nonlimiting examples of APCs comprise DCs (e.g., follicular DCs), macrophages, B cells, monocytes, and Langerhans cells.

[0219] In some embodiments, the methods comprise quantifying the lymphocyte population before, during, and / or after exposure to the one or more cancer antigens.

[0220] In some embodiments, the lymphocyte population is quantified about 1 minute, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours,

[0221] 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 15, or 20 weeks before and / or after exposure to the one or more cancer antigens. In some embodiments, the lymphocyte population is quantified at least 1 minute, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 2 days,

[0222] 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 15, or 20 weeks before and / or after exposure to the one or more cancer antigens. In some embodiments, the lymphocyte population is quantified at least about 1 minute, 30 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 15, or 20 weeks before and / or after exposure to the one or more cancer antigens.

[0223] In some embodiments, quantifying the lymphocyte cell population comprises a normalization step.

[0224] In some embodiments, the methods comprise a step of calculating a difference in the lymphocyte cell population quantity between two different time points. In some embodiments, the two different time points comprise a time point before exposure to the one or more cancer antigens and a time point after exposure to the one or more cancer antigens. In some embodiments, the two different time points comprise a time point before exposure to the one or more cancer antigens and a time point during exposure to the one or more cancer antigens. In some embodiments, the two different time points comprise a time point during exposure to the one or more cancer antigens and a time point after exposure to the one or more cancer antigens.

[0225] In some embodiments, calculating the difference in the lymphocyte cell population between two different time points further comprises comparing the difference to a threshold value. The threshold value may be useful in determining whether lymphocyte cell populations change in response to the one or more cancer antigens.

[0226] In some embodiments, the one or more cancer antigens or a nucleotide sequence encoding the one or more cancer antigens is included in a composition if the difference in the lymphocyte cell population exceeds the threshold value

[0227] Expansion of Lymphocyte Cells

[0228] In some embodiments, the methods of generating cell compositions of the present technology comprise a step of exposing a second sample to one or more antigens from the cancer. In some embodiments, the one or more cancer antigens or a nucleotide sequence encoding the one or more cancer antigens are used to expose a second sample from a second subject if the difference in the lymphocyte cell population exceeds the threshold value. The second sample may comprise a biological sample. In some embodiments, the second sample is a circulatory fluid sample (e.g., a peripheral blood mononuclear cell (PBMC) sample), a lymphoid tissue sample (e.g., lymph node, spleen, or tonsil tissue), a mucosal sample (e.g., mucosal tissue), a bone marrow sample, a cerebrospinal fluid (CSF) sample, or a synovial fluid sample.

[0229] The second subject may comprise a subject that has never been exposed to the cancer (i.e., a naive subject), a subject that has never been infected a cancer oncogenic virus, a subject that is infected with an oncogenic virus, or a subject that has symptoms associated with infection with the oncogenic virus.

[0230] In some embodiments, the second subject is in the early stages of cancer or infection with an oncogenic virus.

[0231] In some embodiments, the second subject has cancer. The second subject may have Stage Om Stage I, Stage II, Stage Ill, or Stage IV cancer.

[0232] In some embodiments, the lymphocyte cell population is expanded in the second sample after exposure to the one or more cancer antigens or the nucleotide sequence encoding the one or more cancer antigens. Nonlimiting examples of methods that may be used to expand the lymphocyte cell population include cytokine stimulation, cell culture methods (e.g., addition of nutrients or growth factors to promote proliferation; co- culture systems); or bioreactors, antibody stimulation. In some embodiments, the expanded lymphocyte cell population is isolated and / or may be screened for reactivity to a cytokine and / or chemokine (e.g., IFNy, IL4, IL2, IL6, IL7, IL15, GM-CSF, SCF, TGF13, CXCL12, CCL19) formulation for administration (e.g., in a vaccine composition).

[0233] Adoptive T cell manufacturing

[0234] In another embodiment, we use our DC targeted nanoparticles in an ex vivo T cell manufacturing process to expand antigen specific T cells for adoptive T cell therapy. The manufacturing involves the use of autologous dendritic cells and mRNA to neoantigens found in a patient’s liquid biopsy. Specifically, monocytes are isolated by exposure to polystyrene and expanded / matured as per the art. The use of DC targeted nanoparticles is more efficient than untargeted nanoparticles at delivering the RNA or DNA into the Dendritic cells and less toxic to the cells. It can deliver RNA with a comparable efficiency to nucleoporation with significant workflow advantages.

[0235] In another embodiment, ex vivo expanded SMART-T cells are infused back into the patient. The same patient specific neoantigen mRNA enclosed in LNP is formulated for subcutaneous injection. The patient can now be vaccinated to restimulate the SMART-T cells infused and in circulation.

[0236] In some embodiments, if there is a specific cancer within the patient’s family history, then descendants of the patient may be vaccinated prophylactically to prevent the potential cancer in the future.

[0237] Additional definitions

[0238] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps.

[0239] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0240] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more” of the grammatical object of the article. For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0241] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth.

[0242] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0243] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise.

[0244] In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0245] The terms "bind," "binding," "complex," and "complexing," refer to all types of physical and chemical binding, reactions, complexing, attraction, chelating and the like.

[0246] The term "conserved epitope" refers to conserved proteins with epitopes identified by the methods of the present technology that are conserved across the multiple strains of the cancer.

[0247] The term "pathogen" refers to a bacterium, cancer, or other microorganism that may cause disease. Other microorganisms may include fungi, mold, parasites, viruses and prions.

[0248] Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0249] 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., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0250] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0251] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0252] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0253] EXAMPLES

[0254] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0255] Example 1:

[0256] Profiling Thl and Th2 T-cells

[0257] T-cells reactive to antigens were plated (IxlO5per well) on enzyme-linked immunosorbent spot (ELISpot) plates comprising IFNy and IL4. After 24-hour incubation, the plates were washed and incubated with a second antibody against IFNy which enzymatically catalyzes a first color and anti IL-4 which enzymatically catalyzes a second color. After drying, the first color spots and the second color spots were counted. Each spot represented a single T-cell secreting the analyte cytokine, where Thl cells secreted IFNy and Th2 cells secrete IL-4. These assays were performed on T-cells at day 14 and day 21 post stimulation. T-cell immunological memory was then measured as a percentage of CD3+CD62L+CD197+ T-cell populations (FIG. 1A and IB).

[0258] Assessing Vaccine Compositions in a Primate Model

[0259] To assess vaccine compositions in a primate model, Macaque monkeys were injected intramuscularly on day 0 and day 21 in 3 groups including 6 animals each. Group 1 was administered a vaccine generated from sequences of a SARS-CoV-2 alpha strain with 200 ug of mRNA conserved antigen+ 100 ug of mRNA for Spike vaccine for two injections. Group 2 was administered a vaccine generated from sequences of a SARS-CoV-2 alpha strain with 200 ug of mRNA conserved antigen alone. Group 3 was administered a vaccine generated from the sequences of a SARS-CoV-2 alpha strain with lOOug of mRNA for Spike antigen alone. The mRNA antigen vaccines were comprised of 6 designated conserved antigens. Blood was drawn on days 14 and 35 to test for T cell gamma interferon production in response to the injected antigens. Antigen specific T cell frequency was determined by pulsing with antigen specific peptides and screened using the ELISpot assay for INFy. The results showed that T cell spots that equate to each T cells' TFNy release, in a Th-1 response to each target antigen multivalent target. At day 35 post challenge, half of the animals (N=3) in each Group were challenged with SARS-Cov-2 alpha strain or with Omicron XBB strain. The animals were then followed for 30 days for pulmonary symptoms, weight loss and, starting on Day 42 and every 7 days thereafter, Nasal swab sampling and antigen testing for viral peptides was used to screen for active SARS-CoV-2 cancer.

[0260] After 30 days, none of the animals vaccinated with the SARS-CoV-2 alpha strain and challenged with the SARS-CoV-2 alpha had weight loss, pulmonary infection, or presence of viral peptides. The identical results are observed by challenge with the XBB strain for animals in Group 1 and Group 2. However, 2 of 3 of the animals in Group 3 who only received the vaccine to the SARS-CoV-2 alpha Spike as the vaccination had weight loss, pulmonary infection, and presence of viral peptides (Table 18). This suggests that the conserved proteins are protective across viral strains.

[0261] Table 18: Animals Dosed with Alpha Strain Vaccine

[0262] Development of a DC targeted nanoparticle may enhance the delivery of RNA encoding vaccine antigens to DCs. The lipids will be used to encapsulate and deliver an RNA encoding green fluorescent protein (GFP) in vitro. DCs will be analyzed 24 hours after nanoparticle delivery vis flow cytometry to assess GFP expression. DCs will either be exposed to nanoparticles carrying GFP RNA as a control or nanoparticles carrying a neoantigen RNA encoding cancer peptides (FIG. 9A and FIG. 9B). Nanoparticles will be designed to target DCs using mannose, CD180, CD209, or HLA-DR-targeting ligands. Nanoparticles with targeting ligands were more efficient in RNA delivery, as measured by GFP levels (FIG. 10).

[0263] Macaque monkeys were administered nanoparticles comprising 6 SARS-CoV- 2 cancer antigens, via vaccine. Controls comprised nanoparticles having no targeting ligand and / or no cancer antigen load. DC targeted lipid nanoparticles resulted in greater protection of Macaques when challenged with Omicron XBB at lower concentrations of RNA than the protection achieved with non -targeted lipid nanoparticles (Table 19).

[0264] Table 19: Infection Rates in Animals Administered Lipid Nanoparticle Compositions

[0265] Example 2: Cancer Genomics

[0266] The following example demonstrates the ability to detect all cancer mutations / rearrangements, referred to as neoantigens, present in a patient from a single blood draw and to receive results within a week, allowing for fully personalized cancer treatment. In developing this treatment model, typical mutations of a cancer genome were first identified and then adapted for use with the disclosed process of growing targeted T cells by the introduction of autologous dendritic cells (“DCs”). Initial efforts included conducting an analysis on the most common mutations found in cancer in order to model the types of targets for a typical cancer treatment. The analysis included using The Cancer Genome Atlas (TCGA), which is a curated collection of genome sequencing, next generation sequencing, and RNA sequencing of various types of tumors. TCGA draws from well over 10,000 patients and is, therefore, representative of neoantigens in cancer patient populations. Using a process of elimination, the most common oncogenic mutations were identified. The oncogenic mutations are significant as they are most likely founder antigens (i.e., the first mutation that occurs in the cell as the cells become cancerous) and therefore should be common to all cancer cells. Additionally, these mutations tend to be critical for the growth of cancer cells, and, if the treatment eliminates these mutations from the body, then the cancer may lose its propagation potential. While each type of cancer has a set of mutations that are commonly associated with that cancer, there are some mutations common to all cancer types. To ensure that every cancer is represented, and not just a common mutation in a common form of cancer, each cancer type was analyzed independently.

[0267] This analysis provided gene frequency, site frequency, identification of individual mutation frequency, and eliminated mutations not believed to be oncogenic. All data was drawn from TCGA (www.genome.gov / Funded-Programs-Projects / Cancer-Genome-Atlas). The database also provided information on whether a mutation is a hotspot or believed to be oncogenic. Data for each cancer type, gene, and mutation was accessed by using the filter sets to specify cancer type, frequency of mutated genes, and frequency of mutation sites. The data was aggregated manually in Excel and further analysis was done to select mutations that could be functionally significant with regards to oncogenic potential. Site frequencies, not sample number, was used to rank all the mutations found using the algorithm outlined here

[0268] From this analysis, the most common genes and mutations for colon cancer, lung cancer, pancreatic cancer, diffuse large B cell lymphoma (DLBCL), acute myeloid leukemia (AML), melanoma, bladder cancer, and glioblastoma were determined (Tables 1-8).

[0269] Table 1 shows the genes and corresponding mutations associated with colon cancer, with the most represented determined to be KRAS G12, KRAS G13, and BRAF V600E. All genes were hotspots

[0270] Table 2 shows the genes and corresponding mutations associated with lung cancer, with the most represented determined to be KRAS G12 and EGFR E760_A750del L858R. All genes are hotspots

[0271] Table 3 shows the genes and corresponding mutations associated with pancreatic cancer, with the most represented determined to be KRAS G12. All genes are hotspots

[0272]

[0273] Table 4 shows the genes and corresponding mutations associated with DLBCL, with the most represented determined to be MYD88, L256P, and EZH2 Y641. 1266 patient samples, all mutation types are missense

[0274]

[0275] Table 5 shows the genes and corresponding mutations associated with AML, with the most represented determined to be FLT3 D835. All genes are hotspots

[0276] Table 6 shows the genes and corresponding mutations associated with melanoma, with the most represented determined to be BRAF V600E and NRAS Q61. From 955 patient samples

[0277]

[0278] Table 7 shows the genes and corresponding mutations associated with bladder cancer, with the most represented determined to be FGFR3 S249C, FGFR3 Y373C, and PIK3CA E545K. All genes are hotspots, data from 1429 patient samples

[0279]

[0280] Table 8 shows the genes and corresponding mutations associated with glioblastoma, with the most represented determined to be IDH1 R132H, EGFR A289V, and EGFR G598V. Data from 585 patient samples, all mutations are missense

[0281]

[0282] Table 9 shows the genes and corresponding mutations associated with pancreatic cancer.

[0283] Pancreatic cancers are known to have fewer mutations than other cancer types, but as shown there are still numerous detected mutations.

[0284] The results shown in Tables 1-9 indicate that two genes, TP53 and KRAS, were present in all cancers. KRAS G12D was the most common mutation across all cancers (Table 10)

[0285] Table 10: The Gene and Mutation Frequency Found in All Cancers

[0286] Table 11. mRNA polycistronic antigen with signal sequence

[0287]

[0288]

[0289]

[0290]

[0291]

[0292] Table 12, mRNA polycistronic antigen with no signal sequence

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] Table 13. polycistronic DNA polynucleotide with signal sequence

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306] Table 14. Polycistronic DNA polynucleotide with no signal sequence

[0307]

[0308]

[0309]

[0310]

[0311]

[0312] Example 3: BRCA related breast cancer

[0313] Women with BRCA 1 or BRCA 2 abnormalities are at increased risk for developing breast and / or ovarian cancer. Discovering one has the abnormality leads to wrenching decisions regarding prophylactic mastectomies and / or oophorectomies. A vaccine targeting the neoantigens associated with those abnormalities might prevent the cancer from developing. Table 15 lists the mutations.

[0314] Table 15. BRACA mutations and antigens

[0315]

[0316]

[0317]

[0318] The BRCA mutations include but are not limited to the mutations above that are pathogenic.

[0319] For BRCA 2 (Chromosome 13) the targetable neoantigens are those in the following positions: 32332280, 32333271, 323338762, 3240266, 32340800, 32345938,32357930,32398507

[0320] For BRCA 1 (Chromosome 17), the targetable neoantigens are those in positions: 43051116, 43051118, 43057078, 43071072, 43071239, 43091619, 43092821,43094366, 43094800, 43124016, 43124029.

[0321] A vaccine to prevent cancer from developing in people with BRCA 1 mutations. A vaccine composed of a polycistronic mRNA coding for 2 or more of the 11 mutations referenced above is created according to methods described herein. An intramuscular, intradermal or subcutaneous injection of a dose of mRNA is administered to cohorts of volunteers in standard studies to determine the best initial dosing and timing of boosts for the vaccine. In a preferred clinical trial design, women with BRCA mutations who opted out of bilateral mastectomy would be enrolled at age 40 to 50 (as this the age of peak incidence of breast cancer in these patients). An exemplary dose is 0.5 ml of a vaccine containing 50gg of mRNA vaccine. An exemplary schedule would be on day 1, 14 and then day 60 as the initial series. Once dosing is established, people with the BRCA1 mutation are administered the vaccine and followed by drawing blood and testing T cells for reactivity with the neoantigens over time. Boosters are administered if the activity of the T cells falls.

[0322] Similarly, a vaccine to prevent people with BRCA 2 mutations from developing breast and / or ovarian cancer can be generated and the methods are performed as in Example 1, except the mRNA construct is for the BRCA 2 mutations.

[0323] Example 4 Lynch Syndrome

[0324] Lynch Syndrome patients have a high rate of mis-match repair mutations that frequently lead to malignancy. A vaccine to create an immune response to known mutations might prevent or substantially delay onset of cancer. People with the syndrome have stages of malignant transformation of colonic polyps that can be characterized as normal, pre-cancer, advanced precancer and early-stage cancer.

[0325] Table 16 is a compilation of the neoantigens found in these stages. A strategy for vaccine development may be targeted at the “next stage” (ie, if a patient has pre-cancer, then a vaccine that addresses advanced pre-cancer may prevent progression), or, alternatively, a vaccine targeting all of the neoantigens might be more efficacious especially since some cells with more advanced stage may be missed.

[0326] Table 16. Lynch Mutations

[0327] In a preferred embodiment, a vaccine targeting all of the mutations identified in Table 16 which occur in cancers associated with Lynch syndrome is constructed. Based upon the 335% cumulative percentage in all 57 of these mutations which occur in cancers associated with Lynch syndrome, the vaccine should prime immunosurveillance to 3 mutations per patient. This immune response rate is predicted to be sufficient to prevent the development of cancer in these patients.

[0328] Using the methods described herein, we construct an mRNA vaccine for all 57 neoantigens. The mRNA may be a single chain, or several. The mRNA is encapsulated in dendritic cell targeted nanoparticle and constitutes the vaccine.

[0329] The vaccine is tested by administering intramuscularly, intradermally, or subcutaneously, with an exemplary route being intramuscularly, on day 0 (the day of injection), day 14, and then at 6 months. Prior to each injection, PBMCs are withdrawn from the participant, and tested for activity of the T cells to the targets. Finding dose and schedule is a standard practice, and different dosing and scheduling may be required to initiate and maintain immune cell reactivity to the target neoantigens. An exemplary starting dose would be 0.5ml of vaccine containing 50pg of mRNA coding for the neoantigens.

[0330] Once an optimal dose and schedule is determined, people with Lynch syndrome will be treated with the vaccine and followed longitudinally for the development of colonic polyps and the progression, regression or stability of stage compared to pre-vaccination. In a preferred clinical trial design, pateints with Lynch syndrome would be enrolled at age 40 to 60 (as this the age of peak incidence of colon cancer occurs in these patients. 40 to 80% of these patients will get colon cancer by age 70 so this is a high-risk population in which we will have optimal chance of demonstrating prevention with our vaccine). They will be followed longitudinally for the development of colonic polyps and the progression, regression or stability of stage compared to pre-vaccination.

Claims

CLAIMSWhat is claimed:

1. A composition comprising a nanoparticle and a polynucleotide, wherein the nanoparticle comprises a ligand for targeting dendritic cells (DC), and wherein the polynucleotide encodes at least two antigens separated by a linker.

2. The composition of claim 1, wherein the ligand for targeting DC is selected from the group consisting of mannose, CDla, CDlc (BOCA1), CD1 lb (ITGAM), CD11c (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD180 (RP105), CD205 (DEC- 205), CD206 (MRC1), CD209 (DC-SIGN), FcsRl, HLA-DR, TLR2, TLR3, TLR4 , TLR7 / 8, and TLR9 and combinations thereof.

3. The composition of claim 1 or 2, wherein the polynucleotide encodes 3 to 750 antigens each of which are separated by a linker.

4. The composition of claim 3, wherein the polynucleotide encodes 3 to 50 antigens each of which are separated by a linker.

5. The composition of any one of the preceding claims, wherein the antigens are between about 8 amino acids and 50 amino acids in length.

6. The composition of any one of the preceding claims, wherein the antigens are between about 15 and about 40 amino acids in length.

7. The composition of any one of the preceding claims, wherein the antigens are cancer antigens.

8. The composition of claim 7, wherein the cancer antigens are selected from the cancer antigens in Tables 1-14, SEQ ID NO: 100-157 or are encoded by at least one of SEQ ID NO: 95- 99.

9. The composition of claim 7, wherein the cancer antigens are selected from the group consisting of KRAS, TP53, PIK3CA, EGFR, NRAS, IDH2, FLT3, nucleophosmin, BRCA1, BRCA2, and DNMT3A, and combinations thereof.

10. The composition of claim 7, wherein the cancer antigens are selected from the group consisting of TP53, and KRAS.

11. The composition of claim 7, wherein the cancer antigens are selected from the group consisting of DNMT3A R882C, TP53 R158L, PIK3CA Q546K, TP53 C238Y, EGFR 747-751 A, TP53 E286K, TP53 V157F, PIK3CA E542K, PIK3CA E545K, NRAS Q61K, PIK3CA Q546K, IDH2 R140Q, TP53 R175H, DNMT3A R882H, TP53 G266E, FLT3 D835Y, TP53 H214R, nucleophosmin NPM1 W288Cfs*30, KRAS G12D, TP53 R248Q, KRAS G12V, TP53 R248W, KRas G12C, EGFR T790M, KRas G13D.

12. The composition of claim 7, wherein the cancer antigens are selected from SEQ ID NOs: 18-67.

13. The composition of any one of the preceding claims, wherein the polynucleotide is DNA or RNA.

14. The composition of claim 13, wherein the DNA is in a construct and wherein the construct comprises a promoter.

15. The composition of any one of the preceding claims, wherein the polynucleotide comprises modifications.

16. The composition of claim 15, wherein the polynucleotide is RNA, and wherein the RNA comprises modifications selected from the group consisting of a 5’ cap, a poly A tail, untranslated regions, circularization, 2’0-methyl modified bases and combinations thereof.

17. A composition comprising a lipid nanoparticle and a plurality of cancer antigen peptides, wherein the lipid nanoparticle comprises a ligand for targeting dendritic cells (DC).

18. The composition of claim 17, wherein the plurality of cancer antigens is selected from the cancer antigens in Tables 1-14, SEQ ID NO: 100-157 or are encoded by at least one of SEQ ID NO: 95-99.

19. The composition of claim 17, wherein the plurality of cancer antigens is selected from the group consisting of DNMT3A, TP53, PIK3CA, EGFR, NRAS, IDH2, FLT3, nucleophosmin, and KRAS, and combinations thereof.

20. The composition of claim 17, wherein the cancer antigens are selected from SEQ ID NOs: 68-92.

21. The composition of any one of claims 17-20, wherein at least two cancer antigens are part of a fusion peptide with a linker separating the cancer antigens.

22. The composition of any one of claims 17-21, wherein the ligand for targeting DC is selected from the group consisting of mannose, CDla, CDlc (BOCA1), CD1 lb (ITGAM), CDl lc (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD180 (RP105), CD205 (DEC -205), CD206 (MRC1), CD209 (DC-SIGN), FcsRl, HLA-DR, TLR2, TLR3, TLR4 , TLR7 / 8, and TLR9 and combinations thereof.

23. The composition of any one of claims 17-22, wherein the plurality of cancer antigen peptides includes 3 to 750 peptides each of which are separated by a linker.

24. The composition of any one of claims 17-23, wherein the plurality of cancer antigen peptides includes 3 to 50 peptides each of which are separated by a linker.

25. The composition of any one of claims 17-24, wherein each peptide in the plurality of cancer antigen peptides is between about 8 and 50 amino acids in length.

26. The composition of any one of claims 17-25, wherein each peptide in the plurality of cancer antigen peptides is between about 15 and 40 amino acids in length.

27. A pharmaceutical composition comprising the composition of any one of the preceding claims and a pharmaceutically acceptable carrier.

28. A method of preventing or treating cancer or slowing the rate at which a pre-cancer develops into a cancer in a subject, the method comprising administering the composition of any one of claims 1-26, the pharmaceutical composition of claim 27 or combinations thereof to the subject.

29. A method of inducing an anti-tumor immune response in a subject in need thereof, the method comprising administering the composition of any one of claims 1-26, the pharmaceutical composition of claim 27 or combinations thereof to the subject.

30. A method of inducing a dendritic cell mediated anti-tumor immune response the method comprising administering the composition of any one of claims 1-26, the pharmaceutical composition of claim 27 or combinations thereof to the subject.

31. A method of inducing an immune response to more than one antigen, the method comprising administering the composition of any one of claims 1-26, the pharmaceutical composition of claim 27 or combinations thereof to the subject.

32. The method of any one of claims 28-31, wherein subject in need is diagnosed with a genetic condition, wherein the genetic condition is associated with an increased risk or predisposition for the development of cancer.

33. A method of preventing or treating cancer in a subject with a cancer-associated genetic mutation the method comprising administering the composition of any one of claims 1-26, the pharmaceutical composition of claim 27 or combinations thereof to the subject.

34. The method of claim 32 or 33, wherein the genetic condition is selected from the group consisting of Lynch syndrome, Von Hippel -Lindau disease (VHL), Li-Fraumeni syndrome (LFS), Hereditary Breast and Ovarian Cancer (HBOC) syndrome, Peutz-Jeghers syndrome (PJS), Familial adenomatous polyposis (FAP), Hereditary leiomyomatosis and renal cell cancer (HLRCC), DICER1 syndrome, Gorlin syndrome, Cowden Syndrome, Hereditary Leukemia and Hematologic Malignancies Syndromes, Multiple endocrine neoplasias (MEN), PTEN Hamartoma tumor syndrome, MUTYH associated polyposis, juvenile polyposis syndrome, PALB2 gene, Birt-Hogg Dube syndrome, ataxia-telangiectasis, Familial GIST syndrome, and Carney Complex.

35. The method of claim 34, wherein the subject has or has been diagnosed with having Lynch syndrome and the at least two antigens comprise at least two of SEQ ID NO: 101-157.

36. The method of any one of claims 28-35, wherein the ligand for targeting DC is selected from the group consisting of mannose, CDla, CDlc (BOCA1), CD1 lb (ITGAM), CD11c (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD180 (RP105), CD205 (DEC-205), CD206 (MRC1), CD209 (DC-SIGN), FcsRl, HLA-DR, TLR2, TLR3, TLR4 , TLR7 / 8, and TLR9 and combinations thereof.

37. The method of any one of claims 28-36, wherein the antigens are selected from the antigens in Tables 1-14, SEQ ID NO: 100-157 or are encoded by at least one of SEQ ID NO: 95- 99.

38. The method of any one of claims 28-37, wherein the antigens are selected from the group consisting of DNMT3A, TP53, PIK3CA, EGFR, NRAS, IDH2, FLT3, nucleophosmin, and KRAS, and combinations thereof.

39. The method of any one of claims 28-38, wherein the subject in need is diagnosed with a cancer selected from the group consisting of bone cancer, testicular cancer, gastric cancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neck cancer, squamous cell head and neck cancer, thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, etc.), ovarian cancer, breast cancer, prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma.

40. The method of claim 39, wherein the subject in need is diagnosed with cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Tables 1-10.

41. The method of claim 39, wherein the subject in need is diagnosed with colon cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 1.

42. The method of claim 39, wherein the subject in need is diagnosed with lung cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 2.

43. The method of claim 39, wherein the subject in need is diagnosed with pancreatic cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 3 or 9.

44. The method of claim 39, wherein the subject in need is diagnosed with Diffuse large B- cell lymphoma (DLBCL)cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 4.

45. The method of claim 39, wherein the subject in need is diagnosed with Acute myeloid leukemia (AML) cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 5.

46. The method of claim 39, wherein the subject in need is diagnosed with melanoma cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 6.

47. The method of claim 39, wherein the subject in need is diagnosed with bladder cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 7.

48. The method of claim 39, wherein the subject in need is diagnosed with glioblastoma cancer and the nanoparticle comprises a polynucleotide and or peptide encoding an antigen selected from Table 8.

49. The method of any one of claims 28-48, wherein the nanoparticle is administered intramuscularly, intravenously, subcutaneously, or intratum orally.

50. A method of delivering cargo to a dendritic cell, the method comprising contacting a dendritic cell with a nanoparticle comprising a dendritic cell targeting ligand and the cargo.

51. The method of claim 50, wherein the dendritic cell targeting ligand is selected from the group consisting of mannose, CDla, CDlc (BOCA1), CDl lb (ITGAM), CDl lc (ITGAX), CD40, CD49d, CD80, CD83, CD85, CD86, CD172a (SIRPa), CD180 (RP105), CD205 (DEC- 205), CD206 (MRC1), CD209 (DC-SIGN), FcsRl, HLA-DR, TLR2, TLR3, TLR4 , TLR7 / 8, and TLR9 and combinations thereof.

52. The method of any one of claims 50 or 51, wherein the cargo comprises DNA, RNA, protein, gRNA, and enzymes.

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