Combinatorial cancer vaccine

Abstract

A combinatorial cancer vaccine for cancer therapy includes a lipid nanoparticle that includes a plurality of pH sensitive protonatable or ionizable lipids, at least one of a TLR7 agonist, TLR 8 agonist or a TLR9 agonist complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids, optionally a nucleic acid encoding a cancer antigen or neoantigen complexed or conjugated with and / or encapsulated by the pH sensitive protonatable or ionizable lipids, and optionally a stabilizing amount of at least one stabilizing polymer, polyethylene glycol or polysaccharide, or structural lipid that is conjugated to and / or complexed with the pH sensitive protonatable or ionizable lipids.

Description

COMBINATORIAL CANCER VACCINERELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application 63 / 625,377, filed January 26, 2024, the subject matter of which is incorporated herein by reference in its entirety.GOVERNMENT FUNDING

[0002] This invention was made with government support under CA235152 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 23, 2025, is named CWR-033302WO ORD st.26 and is 36,864 bytes in size.BACKGROUND

[0004] Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis. It is the fourth leading cause of cancer-related deaths in the United States, with a five-year survival rate of less than 10%. Of the current treatment options for PDAC, surgery is considered the only curative option; however, reoccurrence is common affecting greater than 50% of patients who have undergone pancreatic resection. Additionally, chemotherapy and radiation have limited efficacy and often result in significant side effects.

[0005] Cancer vaccines have emerged as a promising approach to prevent and treat cancer. The goal of cancer vaccines is to stimulate the immune system to recognize and attack cancer cells, either by targeting tumor-specific antigens or by activating immune cells, such as T-cells or natural killer cells. Several cancer vaccines have been developed and tested in clinical trials for PDAC, including GVAX, a granulocyte-macrophage colonystimulating factor (GM-CSF)-allogeneic vaccine made from whole, irradiated tumor cells, and CRS-207, a vaccine made from a modified version of the Listeria bacteria that expresses the tumor-associated antigen mesothelin.

[0006] However, despite promising results in preclinical studies and early-stage clinical trials, the efficacy of these cancer vaccines in improving survival in PDAC patients has been limited. This may be due to the heterogenous and immunosuppressive microenvironment of pancreatic cancer, which inhibits the activation and function of immune cells. Therefore, there is a need for new and more effective cancer vaccines that can overcome these challenges and improve outcomes for PDAC patients.SUMMARY

[0007] Embodiments described herein relate to a combinatorial cancer vaccine that includes a plurality of lipid nanoparticles and their use in treating or preventing cancer in a subject in need thereof. The lipid nanoparticles c n include a plurality of pH protonatable or ionizable lipids that can complex or conjugate with and / or encapsulate at least one TLR agonist and cancer antigen or neoantigen. The lipid nanoparticles upon administration to the subject can be readily internalized by endosome / lysosomes of immune cells of the subject and stimulate a combinatorial innate and adaptive immune therapeutic or prophylactic anti- cancer immune response. The vaccine’s unique composition and mechanism of action can address the limitations of current cancer vaccines and improve the outcomes of subjects with cancer.

[0008] In some embodiments, the combinatorial cancer vaccine can include at least one lipid nanoparticle, preferably a plurality of lipid nanoparticles. The lipid nanoparticle can include: a) a plurality of pH sensitive protonatable or ionizable lipids having the structure of formula (i):wherein R1is an alkylamino group, a hydroxylalkyl group, or a group containing at least one aromatic group;R2and R3are independently an aliphatic group or a hydrophobic group;R4and R5are independently H, an alkyl group, an alkenyl group, an acyl group, or an aromatic group, or each R4or R5independently includes a polymer which isoptionally linked to a targeting group or linked to at least one of a cancer antigen or neoantigen; a, b, c, and d are independently an integer from 1 to 10; and pharmaceutically acceptable salts thereof; b) at least one of a TLR7 agonist, TLR8 agonist, or a TLR9 agonist complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids; c) optionally a nucleic acid encoding a cancer antigen or neoantigen complexed or conjugated with and / or encapsulated by the pH sensitive protonatable or ionizable lipids; and d) optionally a stabilizing amount of at least one stabilizing polymer, polyethylene glycol, or polysaccharide, or structural lipid that is conjugated to and / or complexed with the pH sensitive protonatable or ionizable lipids; and wherein at least one R4or Rsincludes a polymer linked to the cancer antigen or neoantigen and / or the lipid nanoparticle includes the nucleic acid encoding the cancer antigen or neoantigen.

[0009] In some embodiments, the TLR9 agonist can include lefitolimod, tilsotolimod, or a cytidine-phosphate-guanosine (CpG) oligonucleotide.

[0010] In some embodiments, the TLR7 agonist or TLR8 agonist can include 4-amino- 2-(ethoxymethyl)-a,a-dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol (R848), imiquimod, gardiquimod, their fatty acid or lipid derivatives, or mixtures thereof.

[0011] In some embodiments, the lipid nanoparticles can include CpG ODN and R848 complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids.

[0012] In some embodiments, the neoantigen can include a peptide encoded by a mutation of a gene selected from ABL1, BRAF, CDKN1A, EPHA3, FGFR4, IKZF1, MCL1, NKX2-1, PMS2, RNF43, TET2, ACVR1B, BRCA1, CDKN1B, EPHB1, FH, INPP4B, MDM2, NOTCH1, POLDI, ROS1, TGFBR2, AKT1, BRCA2, CDKN2A, EPHB4, FLCN, IRF2, MDM4, NOTCH2, POLE, RPTOR, TIP ARP, AKT2, BRD4, CDKN2B, ERBB2, FLT1, IRF4, MED12, NOTCH3, PPARG, SDHA, TNFAIP3, AKT3, BRIP1, CDKN2C, ERBB3, FLT3, IRS2, MEF2B, NPM1, PPP2R1A, SDHB, TNFRSF14, ALK, BTG1, CEBPA, ERBB4, FOXL2, JAK1, MEN1, NRAS, PPP2R2A, SDHC, TP53, ALOX12B, BTG2, CHEK1, ERCC4, FUBP1, JAK2, MERTK, NT5C2, PRDM1, SDHD, TSC1, AMER1, BTK, CHEK2, ERG, GABRA6, JAK3, MET, NTRK1, PRKAR1A, SETD2, TSC2,APC, Cl lorf30, CIC, ERRFI1, GATA3, JUN, MITF, NTRK2, PRKC1, SF3B1, TYR03, AR, CALR, CREBBP, ESRI, GATA4, KDM5A, MKNK1, NTRK3, PTCHI, SGK1, U2AF1, ARAF, CARD11, CRKL, EZH2, GATA6, KDM5C, MLH1, P2RY8, PTEN, SMAD2, VEGFA, ARFRP1, CASP8, CSF1R, FAM46C, GID4, (C17orf39), KDM6A, MPL, PALB2, PTPN11, SMAD4, VHL, ARID1A, CBFB, CSF3R, FANCA, GNA11, KDR, MRE11 A, PARK2, PTPRO, SMARCA4, WHSCI , ASXL1 , CBL, CTCF, FANCC, GNA13, KEAP1, MSH2, PARP1, QKI, SMARCB1, WHSC1L1, ATM, CCND1, CTNNA1, FANCG, GNAQ, KEL, MSH3, PARP2, RAC1, SMO, WT1, ATR, CCND2, CTNNB1, FANCL, GNAS, KIT, MSH6, PARP3, RAD21, SNCAIP, XPO1, ATRX, CCND3, CUL3, FAS, GRM3, KLHL6, MST1R, PAX5, RAD51, SOCS1, XRCC2, AURKA, CCNE1, CUL4A, FBXW7, GSK3B, KMT2A, (MLL), MTAP, PBRM1, RAD51B, SOX2, ZNF217, AURKB, CD22, CXCR4, FGF10, H3F3A, KMT2D, (MLL2), MTOR, PDCD1, RAD51C, SOX9, ZNF703, AXIN1, CD274, CYP17A1, FGF12, HDAC1, KRAS, MUTYH, PDCD1LG2, RAD51 D, SPEN, AXL, CD70, DAXX, FGF14, HGF, LTK, MYC, PDGFRA, RAD52, SPOP, BAP1, CD79A, DDR1, FGF19, HNF1A, LYN, MYCL, PDGFRB, RAD54L, SRC, BARD1, CD79B, DDR2, FGF23, HRAS, MAF, MYCN, PDK1, RAFI, STAG2, BCL2, CDC73, DIS3, FGF3, HSD3BI, MAP2KI, MYD88, PIK3C2B, RARA, STAT3, BCL2L1, CDH1, DNMT3A, FGF4, ID3, MAP2K2, NBN, P1K3C2G, RBI, STK11, BCL2L2, CDK12, DOT IL, FGF6, IDH1, MAP2K4, NF1, PIK3CA, RBM10, SUFU, BCL6, CDK4, EED, FGFR1, IDH2, MAP3K1, NF2, P1K3CB, REL, SYK, BCOR, CDK6, EGFR, FGFR2, IGF1R, MAP3K13, NFE2L2, PIK3R1, RET, TBX3, BCORL1, CDK8, EP300, FGFR3, IKBKE, MAPK1, NFKBIA, PIM1, RICTOR, TEK, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, MYB, NUTM1, RSPO2, SDC4, SLC34A2, TERC, TERT, or TMPRSS2.

[0013] For example, the neoantigen can include at least one of KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12S, KRAS G12V, KRAS G13D, or KRAS G13C.

[0014] In some embodiments, the targeting group can include at least one of immune cell or cancer cell targeting peptide.

[0015] In some embodiments, R1can include at least one of:- (CH2)eNR5R7.5- (CH2)fN(CH2)gNR9R10R8; or- (CH2)|N - {(CH2)jN}k- (CH2),NR13R14R11R12-(CH2)mOR15; where R6, R7, R8, R9, R10, R11, R12, R13, R14, and R15are each independently hydrogen, an alkyl group, a hydrophobic group, a nitrogen containing substituent, or an oxygen containing substituent; and e, f, g, i, j, k, 1, and m are an integer from 1 to 10.

[0016] For example, R1can include at least one of CH2CH2NH2, CH2CH2OH, CH2CH2OCH2CH2OH, CH2CH2OCH2CH2NH2, CH2CH2NHCH2CH2NHCH2CH2NH2, orCH2CH2NHCH2CH2CH2CH2NHCH2CH2CH2NH2.

[0017] In some embodiments, R2and R3are each independently a saturated alkyl with long or branched chains, and or a fatty acid hydrophobic group derived from oleic acid or linoleic acid.

[0018] In other embodiments, at least one of R4or R5includes the polymer, such as polyethylene glycol (PEG), linked to the cancer antigen or neoantigen.

[0019] In some embodiments, the pH sensitive protonatable or ionizable lipids are selected frompolyethylene glycol (PEG) modified lipids thereof, dextran modified lipids thereof, or combinations thereof.

[0020] In some embodiments, the plurality of pH sensitive protonatable or ionizable lipids include a plurality of ECO and / or ECLn lipids and a plurality of ECLn and / or ECO modified with PEG linked to the cancer antigen or neoantigen.

[0021] Other embodiments describe herein relate to a vaccine that includes a plurality of lipid nanoparticles as described herein or the pharmaceutical composition described herein.

[0022] Other embodiments described herein relate to a method of treating or preventing cancer in a subject in need thereof. The method can include administering to the subject a therapeutically effective or prophylactic amount of the combinatorial cancer vaccine as described herein.

[0023] In some embodiments, the method can further include administering at least one immune checkpoint inhibitor in combination with the vaccine. The immune checkpoint inhibitor can include an inhibitor for any immune checkpoint molecules selected from the group consisting of PD-1, CTLA-4, TIM-3, BTLA, LAG-3, A2aR, KIR, VISTA, TIGIT, PD- L1 PD-L2, CD80, CD86, GAL-9, HVEM, CD160, MHC class II, B7-H3, B7-H4, B7-H5. B7-H6, and B7-H7, or a combination of two or more inhibitors thereof.

[0024] In some embodiments, the immune checkpoint inhibitor is selected from an antibody against the immune checkpoint molecule, an antigen-binding fragment of the antibody, or a combination thereof.

[0025] For example, the immune checkpoint inhibitor can be selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, avelumab, atezolizumab, and durvalumab.

[0026] In some embodiments, the cancer that is treated can be selected from colorectal cancer, breast cancer, lung cancer, melanoma, hepatoma, head and neck cancers, squamous cell carcinomas of the lung, ovarian cancer, uterine cancer, prostate cancer, gastric carcinoma, cervical cancer, esophageal carcinoma, bladder cancer, kidney cancer, braincancer, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular malignant melanoma, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin’s lymphoma, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, adrenal gland cancer, sarcoma of soft tissue, urethra cancer, penis cancer, chronic or acute leukemias solid tumors of childhood, lymphocytic lymphoma, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, medulloblastoma pilomatrixomas, endometrial cancer, multiple myeloma, or T-cell lymphoma.BRTEF DESCRIPTION OF THE DRAWINGS

[0027] Fig. 1 illustrates a schematic showing the combinatorial cancer vaccine and its administration to a subject in accordance with an embodiment.

[0028] Fig. 2 illustrates the preparation strategy of G12D-(CpG / R848)-ECO lipid nanoparticle (ELNP) of the combinatorial cancer vaccine.

[0029] Fig. 3 illustrates gel electrophoresis showing the formation of peptide- (CpG / R848)-ELNP particles. 1, naked CpG ODN; 2, RGD-CpG-ELNP; 3, ECO / CpG / R848; 4. RGD-CpG / R848-ELNP.

[0030] Fig. 4 illustrates long-term survival of the mice bearing orthotopic KPC1242 PDAC tumors after treatment with PBS, cocktail vaccine, ELNP nanovaccine.

[0031] Figs. 5(A-B) illustrate presentative MT218-MR1 images of the mice bearing orthotopic KPC pancreatic cancer treated with saline control and nanovaccine (A) and the untreated naive mice and complete responding (CR) mice challenged by subcutaneous injection of the KPC cancer cells (B). T, tumor; P, pancreas.

[0032] Fig. 6 illustrates survival curve of the mice with complete response to the nanovaccine treatment (n= 9) and the untreated naive mice after subcutaneous injection of the cancer cells.

[0033] Figs. 7(A-D) illustrate (A) Agarose gel encapsulation with 3 nanoparticle formulations: NP1 : CpG / ECO-PEG-RGD, NP2: CpG-R848 / ECO and NP3: CpG-R848 / ECO-PEG-RGD. (B-D) size distribution of particles measured by DLS. (E) Free CpG in solution vs nanoparticle formulations.

[0034] Fig. 8 illustrates size and zeta-potentials of ECO / CpG-ODN nanoparticle formulations with different targeting moieties.

[0035] Fig. 9 illustrates axial slices of mice administered the vaccine over time. Mouse 1 shows a non-responder who developed a tumor similar to that of the i.p. saline controls. Mouse 2 had a major pathological response and the MRI demonstrates the associated size reduction. Mouse 3 shows cancer vaccine mouse with strong signal enhancement at days 10 and 27 but no signs of tumor at day 45. Mice 2 and 3 that are responders have higher signal (characterized by contrast-to-noise ratio) at day 27. Comparatively, mouse 3 with vaccine therapy has with less signal enhancement at 10 min within the tumor than responder mice 2 and 3.

[0036] Fig. 10 illustrates axial slices of mice administered the vaccine and an immune checkpoint inhibitor over time. Mouse 1 shows cancer vaccine + VISTA mAb mouse with strong signal enhancement at days 10 and 27 but no signs of tumor at day 45. Mice 2 and 3 show tumors that have a partial response to therapy with less signal enhancement at 10 min within the tumor than responder mouse 1.

[0037] Fig. 11 illustrates tumor growth from 3D MRMI scans over time in immunotherapy treated groups.

[0038] Fig. 12 illustrates survival proportions of groups treated with vaccine combination alone IP and PBS treated groups

[0039] Figs. 13(A-C) illustrate physical characterization of nanovaccine particlesA. encapsulation of 2 batches of nanovaccine in lanes 2 and 3 versus free CpG in lane 1.B. Nanovaccine size distribution as quantified by DLS by intensity weighted measurement.C. Charge distribution of nanovaccine via zeta-potential.

[0040] Figs. 14(A-C) illustrate A. MRMI of mice over time, left is saline controls before and after contrast administration, middle is batch vaccine and right is a nanovaccine (combinatorial cancer vaccine). Nanovaccine shows clear hinderance of tumor growth and reduction in signal at day 45 compared to control and soluble vaccine. B. Quantification of CNR at pre-contrast and post-contrast 15-minute timepoint. C. Tumor growth based on 2D and 3D imaging of tumors with MRMI.

[0041] Figs. 15(A-C) illustrate evaluation of nanovaccine efficacy in C57BL / 6 mice bearing orthotopic pancreatic tumors (red dashed lines). T1 -weighted contrast-enhanced MRI was performed at days 14 and 45 post-implantation. A. Nanovaccine administration (days 5 and 12) resulted in visible tumor suppression by day 14, with complete tumor regression observed at day 45. Control mice exhibited progressive tumor growth, with extensive abdominal cavity involvement by day 45. B. Final tumor and metastatic burden showed a trend toward significance between nanovaccine-treated and control groups. C. In survival studies, nanovaccine treatment extended median survival to 88 days compared to 66 days in controls, with 50% overall survival in the treatment group.

[0042] Figs. 16(A-C) illustrate evaluation of vaccine memory response in C57BL / 6 mice using subcutaneous pancreatic tumor rechallenge (white arrows, red dashed lines). Following initial orthotopic tumor treatment (day 75), mice received subcutaneous tumor implants. T2-weighted MRI monitoring occurred biweekly post-implantation. A. Nanovaccine-treated mice showed reduced tumor burden 30 days post-rechallenge, while controls exhibited progressive growth with peritoneal invasion by day 60 in surviving control mice. B. Terminal tumor and pancreas mass demonstrated highly significant differences between treatment groups. C. Survival analysis established 100% disease-free survival in nanovaccine-treated mice versus median survival of 50 days in controls.

[0043] Fig. 17 illustrates plots and graph showing therapeutic outcomes of mice administered the vaccine.DETAILED DESCRIPTION

[0044] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Commonly understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th Ed., Springer- Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994. The definitionsprovided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present invention.

[0045] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like. "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.For example, the phrase "optionally substituted lower alkyl" means that the lower alkyl group can or cannot be substituted and that the description includes both unsubstituted lower alkyl and lower alkyl where there is substitution.

[0046] The terms “approximately” and “about,” as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” may refer to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0047] The term "alkenyl group" is defined herein as a C2-C20 alkyl group possessing at least one C=C double bond.

[0048] The term "alkyl group" as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 25 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A "lower alkyl" group is an alkyl group containing from one to six carbon atoms.

[0049] The term "acyl" group as used herein is represented by the formula C(O)R, where R is an organic group such as, for example, an alkyl or aromatic group as defined herein.

[0050] The term "alkylene group" as used herein is a group having two or more CH2 groups linked to one another. The alkylene group can be represented by the formula (CH2)a, where a is an integer of from 2 to 25.

[0051] The term "aromatic group" as used herein is any group containing an aromatic group including, but not limited to, benzene, naphthalene, etc. The term "aromatic" also includes "heteroaryl group," which is defined as an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy.

[0052] The term “antigen” is used to describe a compound, composition, or chemical that induces an immune response, e.g., cytotoxic T lymphocyte (CTL) response, a B cell response (for example, production of antibodies that specifically bind the epitope), an NK cell response or any combinations thereof, when administered to an immunocompetent subject. Thus, an immunogenic or antigenic composition is a composition capable of eliciting an immune response in an immunocompetent subject.

[0053] The term “cancer” refers to the physiological condition in subjects in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or certain morphological features. Often cancers can be in the form of a tumor or mass, but may exist alone within the subject, or may circulate in the blood stream as independent cells, such as leukemic or lymphoma cells. The term cancer includes all types of cancers and metastases, including hematological malignancy, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer e.g., triple negative breast cancer, hormone receptor positive breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, and various types of head and neck cancers.

[0054] The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive as referred to herein.

[0055] The term “checkpoint inhibitor” (the term “immune checkpoint inhibitor” may be used interchangeably herein) refers to a therapeutic agent that targets at least one immune checkpoint protein to alter the regulation of an immune response, e.g., down-modulating or inhibiting an immune response. Immune checkpoint proteins are known in the art and include, without limitation, cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), V-domain Ig suppressor of T cell activation (VISTA), B7-H2, 137-13, B7-H4, B7- 116, 2B4, ICOS, IVEM, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM- 4. LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIG1T, LAG-3, BTLA, IDO, 0X40, and A2aR.

[0056] The term “delivering” means providing an entity to a destination. For example, delivering a therapeutic and / or prophylactic to a subject may involve administering a lipid nanoparticle composition including the cancer antigen or neoantigen and TLR agonists to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). Administration of the lipid nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the lipid nanoparticle composition.

[0057] The phrase "nitrogen containing substituent" is defined herein as any amino group. The term "amino group" is defined herein as a primary, secondary, or tertiary amino group. In the alternative, the nitrogen containing substituent can be a quaternary ammonium group. The nitrogen containing substituent can be an aromatic or cycloaliphatic group, where the nitrogen atom is either part of the ring or directly or indirectly attached by one or more atoms (i.e., pendant) to the ring. The nitrogen containing substituent can be an alkylamino group having the formula RNH2, where R is a branched or straight alkyl group, and the amino group can be substituted or unsubstituted.

[0058] The term “encapsulation,” or its grammatical equivalent, refers to the process of confining a nucleic acid or other molecule within a nanoparticle.

[0059] The term “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides (e.g., heavy chain or light chain ofantibody) into an intact protein (e.g., antibody) and / or post-translational modification of a polypeptide or fully assembled protein e.g., antibody). In this application, the terms “expression” and “production,” and their grammatical equivalents, are used interchangeably.

[0060] The terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein.

[0061] The term “impurities” refers to substances inside a confined amount of liquid, gas, or solid, which differ from the chemical composition of the target material or compound. Impurities are also referred to as contaminants.

[0062] The term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0063] The term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0064] The term “isolated” refers to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and / or entities should not include excipients (e.g., buffer, solvent, water, etc.).

[0065] The terms “local distribution,” “local delivery,” or grammatical equivalent, refer to tissue specific delivery or distribution. Typically, local distribution or delivery requires apeptide or protein (e.g., enzyme) encoded by mRNAs be translated and expressed intracellularly or with limited secretion that avoids entering the patient's circulation system.

[0066] The term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one peptide, polypeptide or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA 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, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5' to 3' direction unless otherwise indicated. In some embodiments, an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C 5 -bromouridine, C5 -fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2- thiocytidine, pseudouridine, and 5-methylcytidine); 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., phosphorothioates and 5'-N-phosphoramidite linkages).

[0067] The term “mRNA integrity” refers to the quality of mRNA. In particular, mRNA integrity refers to the percentage of mRNA that is not degraded. mRNA integrity may be determined using methods well known in the art, for example, by RNA agarose gel electrophoresis (e.g., Ausubel et al., John Weley <&. Sons, Inc., 1997, Current Protocols in Molecular Biology) or capillary electrophoresis. In some embodiments, mRNA integrity can be quantified and expressed as a percent. For example, capillary electrophoresis and similar methods can be uses to separate degraded mRNA from mRNA that is not degraded and then the percent integrity, i.e., percent of mRNA not degraded relative to total mRNA, can be calculated based on the relative areas from the resulting chromatogram.

[0068] The term “N / P ratio” refers to a molar ratio of positively charged molecular units in the cationic lipids in a lipid nanoparticle relative to negatively charged molecular units in the nucleic acids encapsulated within that lipid nanoparticle. As such, N / P ratio istypically calculated as the ratio of moles of amine groups in cationic lipids in a lipid nanoparticle relative to moles of phosphate groups in nucleic acids encapsulated within that lipid nanoparticle.

[0069] The term “nucleic acid,” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain 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 a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double- stranded DNA and / or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences that encode proteins and / or RNA may include introns. 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, backbone modifications, etc. 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, deoxy thymidine, deoxy guanosine, and deoxycytidine); nucleoside analogs e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5 -fluorouridine, C5-iodouridine, C5 -propynyl-uridine, C5 -propynyl-cytidine, C5 -methylcytidine, 2-aminoadenosine, 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 / ormodified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some embodiments, the present invention is specifically directed to “unmodified nucleic acids,” meaning nucleic acids e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery. In some embodiments, the nucleotides T and U are used interchangeably in sequence descriptions.

[0070] The term “pharmaceutically acceptable” as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0071] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulf onate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(CI-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counter ions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quatemization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quarternized alkylated amino salt.

[0072] The term “potency,” or grammatical equivalents, refers to level of protein(s) or peptide(s) of the cancer antigen or neoantigen or nucleic acid encoding the cancer antigen or neoantigen along with TLR agonists and the resulting biological effect.

[0073] The term “salt” refers to an ionic compound that does or may result from a neutralization reaction between an acid and a base.

[0074] The terms “systemic distribution,” “systemic delivery,” or grammatical equivalent, refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body’s circulation system, e.g., blood stream. Compared to the definition of “local distribution or delivery.”

[0075] The term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0076] The term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0077] The term “substantially free” refers to a state in which relatively little or no amount of a substance to be removed e.g., prematurely aborted RNA sequences) are present. For example, “substantially free of prematurely aborted RNA sequences” means the prematurely aborted RNA sequences are present at a level less than approximately 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (w / w) of the impurity. Alternatively, “substantially free of prematurely aborted RNA sequences” meansthe prematurely aborted RNA sequences are present at a level less than about 100 ng, 90 ng, 80 ng, 70 ng, 60 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 1 ng, 500 pg, 100 pg, 50 pg, 10 pg, or less.

[0078] The term “target tissues” refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease- associated pathology, symptom, or feature.

[0079] The term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and / or reduce incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0080] The phrase “pharmaceutically acceptable excipient,” as used herein, refers to any ingredient other than the compounds described herein (for example, a vehicle capable of suspending, complexing, or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: anti- adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin D, vitamin E (alpha-tocopherol), vitamin C, vitamin K, xylitol, and other species disclosed herein.

[0081] In the present specification, the structural formula of the compound represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers,such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like, it being understood that not all isomers may have the same level of activity. In addition, a crystal polymorphism may be present for the compounds represented by the formula. It is noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof is included in the scope of the present disclosure.

[0082] A “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell. In certain aspects, the stabilized lipid nanoparticles described herein are free of phospholipids, i.e., does not have the phospholipid component used in the traditional lipid nanoparticle compositions.

[0083] The term “polypeptide” or “polypeptide of interest” refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically.

[0084] As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

[0085] The term “therapeutic agent” or “prophylactic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. Therapeutic agents are also referred to as “actives” or “active agents.” Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0086] Embodiments described herein relate to a combinatorial cancer vaccine (or nanovaccine or vaccine adjuvant) that includes a plurality of lipid nanoparticles and their use in treating or preventing cancer in a subject in need thereof. The lipid nanoparticles include a plurality of pH protonatable or ionizable lipids that can complex or conjugate with and / or encapsulate at least one TLR agonist and cancer antigen or neoantigen. The lipid nanoparticles upon administration to the subject can be readily internalized byendosome / lysosomes of immune cells of the subject and stimulate a combinatorial innate and adaptive immune therapeutic or prophylactic anti-cancer immune response. The vaccine’s unique composition and mechanism of action can address the limitations of current cancer vaccines and improve the outcomes of subjects with cancer.

[0087] In some embodiments, the pH sensitive protonatable or ionizable lipids of the lipid nanoparticle can include: a protonable amino head group, which can complex with the negatively charged molecules, such as nucleic acid TLR agonists or other nucleic acids, e.g., mRNA; fatty acid or lipid tails, which can participate in hydrophobic condensation; two cysteine residues, which are capable of forming disulfide bridges via autooxidation; optionally, cancer antigens or neoantigens that can be readily internalized by endosome / lysosomes of immune cells and trigger an immune response; and / or optionally a targeting group that targets and / or binds to protein or other biological target molecule in a subject.

[0088] The protonable amino head group can complex with negatively charged molecules, such as nucleic acid TLR agonists or other nucleic acids (e.g., mRNA) to form stabilized lipid nanoparticles for delivery of TLR agonists or nucleic acids to cells. The amines in the head groups contribute to the essential pH-sensitive characteristic of the carrier system, which is important for improving endosomal escape. Greater protonation of the amino head groups can occur in the relatively acidic environment (pH = 5-6) of the endosome and lysosome compartments after cellular uptake. This enhances electrostatic interactions between the cationic carriers and the anionic phospholipids of endosomal / lysosomal membranes, promoting the bilayer destabilization and nanoparticle charge neutralization events required for efficient cytosolic release of their TLR agonist or nucleic acid payload. By affecting the number of amines, and thus overall pKa, of the cationic carrier, the choice of head group can ultimately determine the degree to which such protonation can occur. The pH-sensitive property of the lipid nanoparticles is essential so that the nanoparticles do not affect the integrity of the outer cell membrane and cause cell death, but instead are able to selectively fuse with and destabilize the endosomal and lysosomal membranes.

[0089] The cysteine residues can form disulfide bridges via autooxidation and react with functional groups of other compounds, such as those containing thiol groups. Once the TLR agonists or nucleic acids are complexed with the lipids, the thiol groups can producedisulfide (S— S) bonds or bridges by autooxidation to form oligomers and polymers or crosslinking. The disulfide bonds can stabilize the lipid nanoparticles and help achieve release of the TLR agonists or nucleic acids once the nanoparticle is in the cell.

[0090] The fatty acid or lipid tails groups can participate in hydrophobic condensation and help form compact, stable nanoparticles with the TLR agonists or nucleic acids and introduce amphiphilic properties to facilitate pH sensitive escape of nanoparticles from endosomal and lysosomal compartments. This is particularly useful when the nanoparticles are used in vivo.

[0091] In general, the transfection efficiency of lipid nanoparticles has been shown to decrease with increasing alkyl chain length and saturation of the lipid tail groups. When saturated, shorter aliphatic chains (C12 and C14) favor higher rates of inter-membrane lipid mixing and reportedly allow for better transfection efficiencies in vitro, as compared to in vivo, whereas the opposite is true for longer chains (C16 and C18). Typically, saturated fatty acids greater than 14 carbons in length are not favorable for nucleic acid transfections due to their elevated phase transition temperature and overall less fluidity than those that are unsaturated. However, it has been discovered that there exists a limit, at which point an increase in unsaturation and lipid fluidity is inversely correlated to transfection efficiency, primarily because some degree of rigidity is required for particle stability, as evidenced by the widespread use of cholesterol in lipid nanoparticle formulations.

[0092] Advantageously, the lipid nanoparticles formed using the plurality of pH sensitive protonatable or ionizable lipids have improved stability when administered systemically to a subject, protect condensed TLR agonists or nucleic acids from degradation, and promote endosomal escape and cytosolic release upon cellular uptake.

[0093] In some embodiments, the lipid nanoparticles of the vaccine can include: a) a plurality of pH sensitive protonatable or ionizable lipids having the structure of formula (I):wherein R1is an alkylamino group, a hydroxylalkyl group, or a group containing at least one aromatic group;R2and R3are independently an aliphatic group or a hydrophobic group;R4and R5are independently H, an alkyl group, an alkenyl group, an acyl group, or an aromatic group, or each R4or Rsindependently includes a polymer which is optionally linked to a targeting group or linked to at least one of a cancer antigen or neoantigen; a, b, c, and d are independently an integer from 1 to 10; and pharmaceutically acceptable salts thereof; b) at least one of a TLR7 agonist, TLR8 agonist, or a TLR9 agonist complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids; c) optionally a nucleic acid encoding a cancer antigen or neoantigen complexed or conjugated with and / or encapsulated by the pH sensitive protonatable or ionizable lipids; and d) optionally a stabilizing amount of at least one stabilizing polymer, polyethylene glycol or polysaccharide, or structural lipid that is conjugated to and / or complexed with the pH sensitive protonatable or ionizable lipids; and wherein at least one R4or Rsincludes a polymer linked to the cancer antigen or neoantigen and / or the lipid nanoparticle includes the nucleic acid encoding the cancer antigen or neoantigen.

[0094] In some embodiments, R1can include at least one of:- (CH2)eNR6R7.- (CH2)fN(CH2)gNR9R10R8; or- (CH2)|N - {(CH2)jN}k- (CH2),NR13R14R11R12-(CH2)mOR15where R6, R7, R8, R9, R10, R11, R12, R13, R14, and R15are independently hydrogen, an alkyl group, a hydrophobic group, a nitrogen containing substituent, or an oxygen containing substituent; and e, f, g, i, j, k, 1, and m are an integer from 1 to 10.

[0095] For example, R1can include at least one of CH2NH2, CH2CH2NH2, CH2CH2CH2NH2, CH2CH2OH, CH2CH2OCH2CH2OH, CH2CH2OCH2CH2NH2, CH2CH2CH2CH2NH2, CH2CH2CH2CH2CH2NH2, CH2NHCH2CH2CH2NH2, CH2CH2NHCH2CH2CH2NH2, CH2CH2CH2NHCH2CH2CH2CH2NHCH2CH2CH2NH2, CH2CH2NHCH2CH2CH2CH2NH2, CH2CH2NHCH2CH2CH2NHCH2CH2CH2HN2, or CH2CH2NH(CH2CH2NH)dCH2CH2NH2, where d is from 0 to 10.

[0096] In some embodiments, R1can be CH2CH2NH2 or CH2CH2NHCH2CH2CH2NHCH2CH2CH2HN2. In other embodiments, R1is CH2CH2NH2.

[0097] In other embodiments, R2and R3are independently an aliphatic group or a hydrophobic group derived from fatty acid, such as oleic acid or linoleic acid, and are the same or different. The additional double bond in linoleic acid introduces an extra kink into the hydrocarbon backbone, giving the compound a broader conical shape than oleic acid and increasing its fluidity. When incorporated into a nanoparticle structure, the extra degree of unsaturation elevates the propensity to form the hexagonal phase during an impending membrane fusion event of cellular uptake.

[0098] In some embodiments, R2and R3are each independently a saturated alkyl with long or branched chains, and or a fatty acid hydrophobic group derived from oleic acid or linoleic acid.

[0099] In other embodiments, R2and R3are the same or different.

[0100] In some embodiments, at least one of R4or R5includes a polymer which is optionally linked to a targeting group or linked to at least one of a cancer antigen or neoantigen. The cancer antigen or neoantigen can include a peptide and the targeting group can be for example, an antibody, antibody fragment, nanobody, peptide saccharide, organic compound, etc.

[0101] In some embodiments, the polymer of at least one of R4or R5is a stabilizing polymer, such as polyethylene glycol (PEG) or a stabilizing polysaccharide.

[0102] In some embodiments, for at least some of the pH sensitive protonatable or ionizable lipids R4and R5are each H and for other of the pH sensitive protonatable orionizable lipids at least one of R4or R5includes a polymer each optionally linked to a targeting group or linked to at least one of a cancer antigen or neoantigen.

[0103] In some embodiments, the pH sensitive protonatable or ionizable lipids can be selected from:polymer modified lipids thereof, e.g., polyethylene glycol (PEG) modified lipids thereof, dextran modified lipids thereof, or combinations thereof each optionally linked to a targeting group or linked to at least one of a cancer antigen or neoantigen.

[0104] In some embodiments, at least one of R4or Rsof the plurality of pH sensitive protonatable or ionizable lipids can include a polymer linked to at least one of cancer antigen or neoantigen. The term “cancer antigen” as used herein can be used interchangeably herein with the terms “tumor-specific antigen”, “tumor-associated antigen”, “cancer-associated antigen” or “cancer-specific antigen”. Tumor-associated or cancer-associated antigens include a molecule, e.g., protein, present on tumor or cancer cells and on normal cells, or on many normal cells, but at much lower concentration on normal cells than on tumor cells. In contrast, tumor- specific or cancer-specific antigens generally include a molecule, e.g., protein which is present on tumor or cancer cells but absent from normal cells. The tumor-associated or cancer-associated antigen can be an antigen associated with a cancer cell, e.g., a breast cancer cell, a B cell lymphoma, a pancreatic cancer, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell, a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, aneuroblastoma cell, a glioma, a glioblastoma, a colorectal cancer cell, etc. It will also be understood that in some cases, a tumor-associated antigen may also be expressed by a non- cancerous cell.

[0105] The term “neoantigen” refers to a tumor-specific antigen, i.e., an antigen found on tumor cells but not on non-tumor cells. Neoantigens may arise from one or more tumorspecific alterations in a native protein or from non-native proteins, such as viral proteins. Alterations in a native protein that give rise to a neoantigen may be the result of one or more mutations, including for example point mutations, rearrangements, insertions, deletions, or frameshift mutations in the gene encoding the protein or a proximal non-coding region, and / or one or more post-translational modifications, such as glycosylation, lipidation, phosphorylation, acetylation, ubiquitination, or sumoylation. In some cases, post- translational modifications may be the result of an underlying mutation. Mutations giving rise to a neoantigen sometimes result in altered protein expression, for example overexpression, underexpression, or differently timed expression.

[0106] Cancer antigens or neoantigens can include antigens from any tumor or cancer, including, but not limited to, melanomas, squamous cell carcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers, prostatic cancers, ovarian cancers, bladder cancers, skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast cell tumors, leukemias, lymphomas, primary hepatic cancers, lung cancers, pancreatic cancers, gastrointestinal cancers (including colorectal cancers), renal cell carcinomas, hematopoietic neoplasias and metastatic cancers thereof.

[0107] Examples of cancer antigens include carcinoembryonic antigen (CEA) and epitopes thereof such as CAP-1, CAP-1-6D (GenBank Accession No. M29540), MART-1 (Kawakami et al, J. Exp. Med. 180:347-352, 1994), MAGE-1 (U.S. Pat. No. 5,750,395), MAGE-3, GAGE (U.S. Pat. No. 5,648,226), GP-100 (Kawakami et al Proc. Nat’l Acad. Sci. USA 91 :6458-6462, 1992), MUC-1, MUC-2, point mutated Ras oncoprotein, normal and point mutated p53 oncoproteins (Hollstein et al Nucleic Acids Res. 22:3551-3555, 1994), PSMA (Israeli et al Cancer Res. 53:227-230, 1993), tyrosinase (Kwon et al PNAS 84:7473- 7477, 1987), TRP-1 (gp75) (Cohen et al Nucleic Acid Res. 18:2807-2808, 1990; U.S. Pat. No. 5,840,839), NY-ESO-1 (Chen et al PAS 94:1914-1918, 1997), TRP-2 (Jackson et al EMBOJ, 11:527-535, 1992), TAG72, KSA, CA-125, PSA, HER-2 / neu / c-erb / B2, (U.S. Pat.No. 5,550,214), EGFR, hTERT, p73, B-RAF, adenomatous polyposis coli (APC), Myc, von Hippel-Lindau protein (VHL), Rb-1, Rb-2, androgen receptor (AR), Smad4, MDR1, Flt-3, BRCA-1, BRCA-2, Bcr-Abl, pax3-fkhr, ews-fli-1, Brachyury, HERV-H, HERV-K, TWIST, Mesothelin, NGEP, modifications of such antigens and tissue specific antigens, splice variants of such antigens, and / or epitope agonists of such antigens. Other cancer antigens are known in the art. Other cancer antigens may also be identified, isolated and cloned by methods known in the art such as those disclosed in U.S. Pat. No. 4,514,506. Cancer antigens may also include one or more growth factors and splice variants of each.

[0108] In some embodiments, the neoantigen can include a peptide encoded by a mutation of gene selected from ABL1 , BRAF, CDKN1A, EPHA3, FGFR4, IKZF1, MCL1, NKX2-1, PMS2, RNF43, TET2, ACVR1B, BRCA1, CDKN1B, EPHB1, FH, INPP4B, MDM2, NOTCH1, POLDI, ROS1, TGFBR2, AKT1, BRCA2, CDKN2A, EPHB4, FLCN, IRF2, MDM4, NOTCH2, POLE, RPTOR, TIP ARP, AKT2, BRD4, CDKN2B, ERBB2, FLT1, 1RF4, MED 12, NOTCH3, PPARG, SDHA, TNFAIP3, AKT3, BRIP1, CDKN2C, ERBB3, FLT3, IRS2, MEF2B, NPM1, PPP2R1A, SDHB, TNFRSF14, ALK, BTG1 , CEBPA, ERBB4, FOXL2, JAK1, MEN1, NRAS, PPP2R2A, SDHC, TP53, ALOX12B, BTG2, CHEK1, ERCC4, FUBP1, JAK2, MERTK, NT5C2, PRDM1, SDHD, TSC1, AMER1, BTK, CHEK2, ERG, GABRA6, JAK3, MET, NTRK1, PRKAR1A, SETD2, TSC2, APC, Cl lorf30, CIC, ERRFI1, GATA3, JUN, MITF, NTRK2, PRKC1, SF3B1, TYRO3, AR, CALR, CREBBP, ESRI, GATA4, KDM5A, MKNK1, NTRK3, PTCHI, SGK1 , U2AF1, ARAF, CARD11, CRKL, EZH2, GATA6, KDM5C, MLH1, P2RY8, PTEN, SMAD2, VEGFA, ARFRP1, CASP8, CSF1R, FAM46C, GID4, (C17orf39), KDM6A, MPL, PALB2, PTPN11, SMAD4, VHL, ARID1A, CBFB, CSF3R, FANCA, GNA11, KDR, MRE11A, PARK2, PTPRO, SMARCA4, WHSCI, ASXL1, CBL, CTCF, FANCC, GNA13, KEAP1, MSH2, PARP1, QKI, SMARCB1, WHSC1L1, ATM, CCND1, CTNNA1, FANCG, GNAQ, KEL, MSH3, PARP2, RAC1, SMO, WT1, ATR, CCND2, CTNNB1, FANCL, GNAS, KIT, MSH6, PARP3, RAD21, SNCAIP, XPO1, ATRX, CCND3, CUL3, FAS, GRM3, KLHL6, MST1R, PAX5, RAD51, SOCS1, XRCC2, AURKA, CCNE1, CUL4A, FBXW7, GSK3B, KMT2A, (MLL), MTAP, PBRM1, RAD51B, SOX2, ZNF217, AURKB, CD22, CXCR4, FGF10, H3F3A, KMT2D, (MLL2), MTOR, PDCD1, RAD51C, SOX9, ZNF703, AXIN1, CD274, CYP17A1, FGF12, HDAC1, KRAS, MUTYH, PDCD1LG2, RAD51 D, SPEN, AXL, CD70, DAXX, FGF14, HGF, LTK, MYC, PDGFRA, RAD52,SPOP, BAP1, CD79A, DDR1, FGF19, HNF1A, LYN, MYCL, PDGFRB, RAD54L, SRC, BARD1, CD79B, DDR2, FGF23, HRAS, MAF, MYCN, PDK1, RAFI, STAG2, BCL2, CDC73, DIS3, FGF3, HSD3BI, MAP2KI, MYD88, PIK3C2B, RARA, STAT3, BCL2L1, CDH1, DNMT3A, FGF4, ID3, MAP2K2, NBN, P1K3C2G, RBI, STK11, BCL2L2, CDK12, DOT IL, FGF6, IDH1, MAP2K4, NF1, PIK3CA, RBM10, SUFU, BCL6, CDK4, EED, FGFR1 , IDH2, MAP3K1 , NF2, P1 K3CB, REL, SYK, BCOR, CDK6, EGFR, FGFR2, IGF1R, MAP3K13, NFE2L2, PIK3R1, RET, TBX3, BCORL1, CDK8, EP300, FGFR3, IKBKE, MAPK1, NFKBIA, PIM1, RICTOR, TEK, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, MYB, NUTM1, RSPO2, SDC4, SLC34A2, TERC, TERT, or TMPRSS2.

[0109] For example, a neoantigen peptide encoded by a mutation of KRAS gene associated with cancer, such as pancreatic cancer, can include at least one of KRAS G12A (KLVVVGAAGVGKSALTI (SEQ ID NO: 1)), KRAS G12C (KLVVVGACGVGKSALTI (SEQ ID NO: 2)), KRAS G12D (KLVVVGADGVGKSALTI (SEQ ID NO: 3)), KRAS G12R (KLVVVGARGVGKSALT1 (SEQ ID NO: 4)), KRAS G12S (KLVVVGASGVGKSALTI (SEQ ID NO: 5)), KRAS G12V (KLVVVGAVGVGKSALTI (SEQ ID NO: 6)), KRAS G13D (KLVVVGAGDVGKSALTI (SEQ ID NO: 7)), or KRAS G13C (KLVVVGAGCVGKSALTI (SEQ ID NO: 8)).

[0110] Other neoantigen peptides encoded by a mutated gene associated with colon cancer, lung cancer, pancreatic cancer, diffuse large B cell lymphoma (DBCL), acute myeloid leukemia (AML), melanoma, bladder cancer, and glioblastoma are described in Tables 1-8 of U.S. Patent Application Publication No. 2024 / 0000935 Al, U.S. Patent Application Publication No. 2025 / 0009888, and U.S. Patent Application Publication No. 2024 / 0374697, which are herein incorporated by reference in their entirety.

[0111] In other embodiments, at least one of R4or R5of the plurality of pH sensitive protonatable or ionizable lipids can include a polymer linked to a targeting group.

[0112] The targeting group can be configured to specifically bind to and / or target molecules of a cell, tissue, and / or disease site of interest in a subject and be useful in the delivery of lipid nanoparticles to cell, tissue, and / or disease site, such as cancer or tumor site.

[0113] In certain embodiments, the targeting group specifically binds a cell surface molecule of a target cell. As used herein, a targeting group "specifically binds" to a target molecule if it binds to or associates with the target molecule with an affinity or Ka (that is, an equilibrium association constant of a particular binding interaction with units of 1 / M) of, forexample, greater than or equal to about 105M1. In certain embodiments, the first molecule binds to the second molecule with a Ka greater than or equal to about 106M1, 107M1, 108M’1, 109M1, IO10M1, 1011M1, 1012M1, or 1013M’1. "High affinity" binding refers to binding with a Ka of at least 107M’1, at least 108M1, at least 109M’1, at least IO10M'1, at least 1011M1, at least 1012M1, at least 1013M1, or greater. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 10"5M to 10"13M, or less). In certain aspects, specific binding means binding to the target molecule with a KD of less than or equal to about 10’5M, less than or equal to about 106M, less than or equal to about 107M, less than or equal to about 108M, or less than or equal to about 10‘9M, IO10M, 10’11M, or 1012M or less. The binding affinity of the first molecule for the target can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0114] In some embodiments, the targeting group can include, but is not limited to, synthetic compounds, natural compounds or products, macromolecular entities, bioengineered molecules (e.g., polypeptides, lipids, polynucleotides, antibodies, antibody fragments), and small entities (e.g., small molecules, neurotransmitters, substrates, ligands, hormones and elemental compounds).

[0115] In one example, the targeting group can comprise an antibody, such as a monoclonal antibody, a polyclonal antibody, or a humanized antibody, including without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelized antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent targeting moieties including without limitation: monospecific or bispecific antibodies, such as disulfide Fv fragments, scFv tandems ((scFv)2 fragments), diabodies, tribodies or tetrabodies, which typically are covalently linked or otherwise stabilized (i.e., leucine zipper or helix stabilized) scFv fragments; and receptor molecules, which naturally interact with a desired target molecule.

[0116] The targeting group need not originate from a biological source. The targeting group may, for example, be screened from a combinatorial library of synthetic peptides. Onesuch method is described in U.S. Pat. No. 5,948,635, incorporated herein by reference, which describes the production of phagemid libraries having random amino acid insertions in the pill gene of Ml 3. This phage may be clonally amplified by affinity selection.

[0117] In certain embodiments, a targeting group as described herein may comprise a homing peptide, which selectively directs the lipid nanoparticles of the vaccine to a targeted cell. Homing peptides for a targeted cell can be identified using various methods well known in the art. Many laboratories have identified the homing peptides that are selective for cells of the vasculature of brain, kidney, lung, skin, pancreas, intestine, uterus, adrenal gland, retina, muscle, prostate, or tumors. See, for example, Samoylova et al., 1999, Muscle Nerve, 22:460; Pasqualini et al., 1996 Nature, 380:364; Koivunen et al., 1995, Biotechnology, 13:265; Pasqualini et al., 1995, J. Cell Biol., 130: 1189; Pasqualini et al., 1996, Mole. Psych., 1:421, 423; Rajotte et al., 1998, J. Clin. Invest., 102:430; Rajotte et al., 1999, J. Biol. Chem., 274:11593. See, also, U.S. Pat. Nos. 5,622,6999; 6,068,829; 6,174,687; 6,180,084; 6,232,287; 6,296,832; 6,303,573; and 6,306,365.

[0118] In other embodiments, the targeting group may comprise a ligand molecule, including, for example, ligands which naturally recognize a specific desired receptor of a target cell. Such ligand molecules include ligands that have been modified to increase their specificity of interaction with a target receptor, ligands that have been modified to interact with a desired receptor not naturally recognized by the ligand, and fragments of such ligands.

[0119] In still other embodiments, the targeting group may comprise an aptamer. Aptamers are oligonucleotides that are selected to bind specifically to a desired molecular structure of the target cell. Aptamers typically are the products of an affinity selection process similar to the affinity selection of phage display (also known as in vitro molecular evolution). The process involves performing several tandem iterations of affinity separation, e.g., using a solid support to which the diseased immunogen is bound, followed by polymerase chain reaction (PCR) to amplify nucleic acids that bound to the immunogens. Each round of affinity separation thus enriches the nucleic acid population for molecules that successfully bind the desired immunogen. In this manner, a random pool of nucleic acids may be "educated" to yield aptamers that specifically bind target molecules. Aptamers typically are RNA, but may be DNA or analogs or derivatives thereof, such as, without limitation, peptide nucleic acids (PNAs) and phosphorothioate nucleic acids.

[0120] In yet other embodiments, the targeting group may be a peptidomimetic. By employing, for example, scanning mutagenesis to map the amino acid residues of a protein, which is involved in binding other proteins, peptidomimetic compounds can be generated that mimic those residues, which facilitate the interaction. Such mimetics may then be used as a targeting moiety to deliver the nanobubble to a target cell. For instance, non-hydrolyzable peptide analogs of such resides can be generated using benzodiazepine (<?.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al., 1986, J Med Chem 29:295; and Ewenson et al., in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al., 1985, Tetrahedron Lett 26:647; and Sato et al., 1986, J Chem Soc Perkin Trans 1: 1231), and P-aminoalcohols (Gordon et al., 1985, Biochem Biophys Res Cummun 126:419; and Dann et al., 1986, Biochem Biophys Res Commun 134:71).

[0121] In some embodiments, the targeting group can bind to or specifically bind to a cancer antigen. The cancer cell antigen can include at least one of 5T4, a2pi integrin, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET (Hepatocyte Growth Factor Receptor), C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, collagen receptor, Cripto protein, CS1, delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvIll, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), extracellular domain B of fibronectin (EDB-FN), extracellular domain A of fibronectin (EDB-FN, fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), glycoprotein non- metastatic B (GPNMB), guanylate cyclase 2 C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Integrin alpha, lysosomal-associated membrane protein 1 (LAMP-1), Lewis Y, LIV-1, leucine rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16),sodium-dependent phosphate transport protein 2B (NaPi2b), Nectin-4, NMB, N0TCH3, p- cadherin (p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), protein tyrosine phosphatase mu (PTPmu) solute carrier family 44 member 4 (SLC44A4), SLIT like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T cell immunoglobulin and mucin protein- 1 (TIM-1), or trophoblast cellsurface antigen (TROP-2).

[0122] Non-limiting examples of antibodies that specifically bind to tumor antigens which may be used as a targeting moiety include Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritumumab, Radretumab, Ramucirumab, Rilotumumab, Robatumumab, Seribantumab, Tarextumab, Teprotumumab, Tovetumab, Vantictumab, Vesencumab, Votumumab, Zalutumumab, Flanvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab, Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Girentuximab, Indatuximab, Isatuximab, Margetuximab, Rituximab, Siltuximab, Ublituximab, Ecromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontictuzumab, Cantuzumab, Cantuzumab, Citatuzumab, Clivatuzumab, Dacetuzumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab, Tucotuzumab, Vandortuzumab, Vanucizumab, Veltuzumab, Vorsetuzumab, Sotituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or a tumor antigen-binding variant thereof. As used herein,"variant" is meant the antibody specifically binds to the particular antigen (e.g., HER2 for trastuzumab) but has fewer or more amino acids than the parental antibody (e.g., is a fragment e.g., scFv) of the parental antibody), has one or more amino acid substitutions relative to the parental antibody, or a combination thereof.

[0123] In some embodiments, the targeting moiety can include a targeting peptide that can specifically bind to EDB-FN. Targeting peptides that specifically bind EDB-FN can include linear peptides having the amino acid sequences of TVRTSAD (SEQ ID NO: 9), NWGDRIL (SEQ ID NO: 10), NWGKPIK (SEQ ID NO: 11), SGVKSAF (SEQ ID NO: 12), GVKSYNE (SEQ ID NO: 13), IGKTNTL (SEQ ID NO: 14), IGNSNTL (SEQ ID NO: 15), IGNTIPV (SEQ ID NO: 16), and LYANSPF (SEQ ID NO: 17), cyclic peptides having the amino acid sequences of CTVRTSADC (SEQ ID NO: 18), CNWGDRILC (SEQ ID NO: 19), CNWGKPIKC (SEQ ID NO: 20), CSGVKSAFC (SEQ ID NO: 21), CGVKSYNEC (SEQ ID NO: 22), CIGKTNTLC (SEQ ID NO: 23), CIGNSNTLC (SEQ ID NO: 24), CIGNTIPVC (SEQ ID NO: 25), or CLYANSPFC (SEQ ID NO: 26), linear peptides with cysteine linkers, or retro-inverso peptides having a retro-inverso amino acid sequence of the linear peptides thereof.

[0124] In other embodiments, the targeting moiety can include a targeting peptide that specifically binds to EDA-FN. Targeting peptides that specifically bind EDA-FN can include linear peptides having the amino acid sequences of WNYPFRL (SEQ ID NO: 27), SNTSYVN (SEQ ID NO: 28), SFSYTSG (SEQ ID NO: 29), WSPAPMS (SEQ ID NO: 30), TREHPAQ (SEQ ID NO: 31), or ARIIDNA (SEQ ID NO: 32), cyclic peptides having the amino acid sequences of CWNYPFRLC (SEQ ID NO: 33), CSNTSYVNC (SEQ ID NO: 34), CSFSYTSGC (SEQ ID NO: 35), CWSPAPMSC (SEQ ID NO: 36), CTREHPAQC (SEQ ID NO: 37), or CARIIDNAC (SEQ ID NO: 38), linear peptides with cysteine linkers, or retro- inverso peptides having a retro-inverso amino acid sequence of the linear peptides thereof.

[0125] The cancer antigen or neoantigen peptides as well as the target group peptides can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a solidphase Merrifield-type synthesis, can be used for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production and the like. A summary of the many techniques available can be found in Steward et al., "Solid Phase Peptide Synthesis", W. H. Freeman Co., San Francisco, 1969; Bodanszky, et al., "Peptide Synthesis", John Wiley& Sons, Second Edition, 1976; J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969; Fields et al., int. J. Peptide Protein Res., 35:161-214, 1990; and U.S. Pat. No. 4,244,946 for solid phase peptide synthesis, and Schroder et al., "The Peptides", Vol. 1, Academic Press (New York), 1965 for classical solution synthesis, each of which is incorporated herein by reference. Appropriate protective groups usable in such synthesis are described in the above texts and in J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973, which is incorporated herein by reference.

[0126] The polymer of at least one of R4 or R5 linked to the cancer antigen, neoantigen, and / or targeting group can include a homo- or hetero- bifunctional polymer with two reactive groups or linkers separated by a polyamine, a polyalkylene, a polyamino acid, a polyethylene glycol (PEG), or a polysaccharide. The reactive moieties in the bifunctional polymer, which may be the same or, different, can be capable of reaction with the thiol group of formula (1) and a functional group of the cancer antigen, neoantigen, and / or targeting group, such as carboxylic groups and amino groups. An example of a reactive group capable of reaction with the thiol group of formula (I) is a maleimide group or derivative thereof. An example of a reactive groups capable of reaction with the amine group of a peptide of the cancer antigen, neoantigen, and / or targeting group is a succinimide group or derivative thereof. It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, Ill.), can be employed as a reactive group.

[0127] Other types of binding chemistries are also available. For example, methods for conjugating polysaccharides to peptides are exemplified by, but not limited to coupling via alpha- or epsilon-amino groups to NaIO4-activated oligosaccharide (Bocher et al., J.Immunol. Methods 27, 191-202 (1997)), using squaric acid diester (1,2-diethoxycyclobutene- 3, 4-dione) as a coupling reagent (Tietze et al. Bioconjug Chem. 2:148-153 (1991)), coupling via a peptide binder wherein the polysaccharide has a reducing terminal and is free of carboxyl groups (U.S. Pat. No. 5,342,770), and coupling with a synthetic peptide carrier derived from human heat shock protein hsp65 (U.S. Pat. No. 5,736,146). Further methods for conjugating polysaccharides, proteins, and lipids to peptides are described by U.S. Pat. No. 7,666,624.

[0128] In some embodiments, the polymer includes polyethylene glycol (PEG) having a molecular weight from 500 to 10,000, 500 to 9,000, 500 to 8,000, 500 to 7,000, or 2,000 to 5,000. In certain aspects, the cancer antigen, neoantigen, and / or targeting group is first reacted with the reactive group or linker in a manner such that the cancer antigen, neoantigen, and / or targeting group is covalently attached to the linker. For example, the linker can possess one or more groups that can react with an amino group present on a peptide of the cancer antigen, neoantigen, and / or targeting group. The linker also possesses additional groups that react with and form covalent bonds with the polymer (e.g., PDG) described herein. For example, the linker can possess maleimide groups that readily react with the thiol groups. The selection of functional groups present on the linker can vary depending upon the functional groups present on the cancer antigen, neoantigen, and / or targeting group.

[0129] In some embodiments, the linker can include an acid labile bond, such as formed by incorporation of a hydrazone into the linker, that is hydrolysable in an endolysomal environment following uptake to cells, such as cancer cells. For example, the linker can be covalently linked to the pH sensitive protonatable or ionizable lipid of the lipid nanoparticle by at least one of a covalent hydrolysable ester, covalent hydrolysable amide, covalent photodegradable urethane, or covalent hydrolysable acrylate-thiol linkage. Following cellular uptake of the lipid nanoparticle, within the endosomes, the increasingly acidic environment can cleave the acid labile linkage to promote shedding of a polymer linker, such as PEG, and expose the core of the compound / nucleic complex nanoparticle.

[0130] The cancer antigen, neoantigen, and / or targeting group can be linked to the polymer prior or during the formation of nanoparticles. Depending upon the selection of the cancer antigen, neoantigen, or targeting group, the cancer antigen, neoantigen, or targeting group polymer can be covalently bound to the polymer via a linker, such as a maleimide (MAL)-lysine linker.

[0131] The TLR7, TLR8 agonist, and / or a TLR9 agonist complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids can include, for example, any TLR7 agonist, TLR8 agonist, and / or a TLR9 agonist. The term “agonist,” as used herein, refers to a compound that can combine with a receptor (e.g., a TLR) to produce a cellular activity. An agonist may be a ligand that directly binds to the receptor. Alternatively, an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise results in themodification of another compound so that the other compound directly binds to the receptor. An agonist may be referred to as an agonist of a particular TLR e.g., a TLR7 agonist) or a particular combination of TLRs (e.g., a TLR 7 / 8 agonist — an agonist of both TLR7 and TLR8).

[0132] In some embodiments, TLR7 agonists include isolated, naturally-occurring TLR7 agonists; and synthetic TLR7 agonists. TLR7 agonists isolated from a naturally- occurring source of TLR7 agonist are generally purified, e.g., the purified TLR7 agonist is at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure. Synthetic TLR7 agonists are prepared by standard means, and are generally at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure.

[0133] TLR7 agonists can include imidazoquinoline compounds; guanosine analogs; pyrimidinone compounds, such as bropirimine and bropirimine analogs; and the like. Imidazoquinoline compounds that function as TLR7 ligands include, but are not limited to, imiquimod, (also known as Aldara, R-837, S-26308), and R-848 (also known as resiquimod, S-28463; having the chemical structure: 4-amino-2-ethoxymethyl-a, a-dimethyl-lH- imidazol[4,5-c]quinoli-ne-l-ethanol). Imidazoquinoline agents can include imidazoquinoline amines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2 bridged imidazoquinoline amines. These compounds have been described in U.S. Pat. Nos. 4,689,338, 4,929,624, 5,238,944, 5,266,575, 5,268,376, 5,346,905, 5,352,784, 5,389,640, 5,395,937, 5,494,916, 5,482,936, 5,525,612, 6,039,969 and 6,110,929. Particular species of imidazoquinoline agents that can be used include R-848 (S-28463); 4-amino-2ethoxymethyl- a, a-dimethyl-lH-imidazo[4,5-c]quinoline-s-i-ethanol; and l-(2-methylpropyl)-lH- imidazo[4,5-c]quinolin-4-amine (R-837 or Imiquimod). Other compounds that can be used include 4-amino-2-(ethoxymethyl)-a, a-dimethyl-6,7,8,9-tetrahydro-lH-imidazo[4,5- c]quinoline-l -ethanol hydrate (see, e.g., BM-003 in Gorden et al. (2005).

[0134] In some embodiments, TLR7 agonists include those having a 2- aminopyridine fused to a five membered nitrogen-containing heterocyclic ring. Such compounds include, for example, imidazoquinoline amines including but not limited to substituted imidazoquinoline amines such as, for example, amide substituted imidazoquinoline amines, sulfonamide substituted imidazoquinoline amines, urea substituted imidazoquinoline amines, aryl ethersubstituted imidazoquinoline amines, heterocyclic ether substituted imidazoquinoline amines, amido ether substituted imidazoquinoline amines, sulfonamido ether substituted imidazoquinoline amines, urea substituted imidazoquinoline ethers, thioether substituted imidazoquinoline amines, and 6-, 7-, 8-, or 9-aryl or heteroaryl substituted imidazoquinoline amines; tetrahydroimidazoquinoline amines including but not limited to amide substituted tetrahydroimidazoquinoline amines, sulfonamide substituted tetrahydroimidazoquinoline amines, urea substituted tetrahydroimidazoquinoline amines, aryl ether substituted tetrahydroimidazoquinoline amines, heterocyclic ether substituted tetrahydroimidazoquinoline amines, amido ether substituted tetrahydroimidazoquinoline amines, sulfonamido ether substituted tetrahydroimidazoquinoline amines, urea substituted tetrahydroimidazoquinoline ethers, and thioether substituted tetrahydroimidazoquinoline amines; imidazopyridine amines including but not limited to amide substituted imidazopyridine amines, sulfonamido substituted imidazopyridine amines, urea substituted imidazopyridine amines, aryl ether substituted imidazopyridine amines, heterocyclic ether substituted imidazopyridine amines, amido ether substituted imidazopyridine amines, sulfonamido ether substituted imidazopyridine amines, urea substituted imidazopyridine ethers, and thioether substituted imidazopyridine amines; 1,2-bridged imidazoquinoline amines; 6,7-fused cycloalkylimidazopyridine amines; imidazonaphthyridine amines; tetrahydroimidazonaphthyridine amines; oxazoloquinoline amines; thiazoloquinoline amines; oxazolopyridine amines; thiazolopyridine amines; oxazolonaphthyridine amines; thiazolonaphthyridine amines; and IH-imidazo dimers fused to pyridine amines, quinoline amines, tetrahydroquinoline amines, naphthyridine amines, and tetrahydronaphthyridine amines.

[0135] In other embodiments, TLR7 agonists include a substituted imidazoquinoline amine, a tetrahydroimidazoquinoline amine, an imidazopyridine amine, a 1 ,2-bridged imidazoquinoline amine, a 6,7-fused cycloalkylimidazopyridine amine, an imidazonaphthyridine amine, a tetrahydroimidazonaphthyridine amine, an oxazoloquinoline amine, a thiazoloquinoline amine, an oxazolopyridine amine, a thiazolopyridine amine, an oxazolonaphthyridine amine, and a thiazolonaphthyridine amine.

[0136] As used herein, a substituted imidazoquinoline amine refers to an amide substituted imidazoquinoline amine, a sulfonamide substituted imidazoquinoline amine, a urea substituted imidazoquinoline amine, an aryl ether substituted imidazoquinoline amine, aheterocyclic ether substituted imidazoquinoline amine, an amido ether substituted imidazoquinoline amine, a sulfonamido ether substituted imidazoquinoline amine, a urea substituted imidazoquinoline ether, a thioether substituted imidazoquinoline amines, or a 6-, 7-, 8-, or 9-aryl or heteroaryl substituted imidazoquinoline amine.

[0137] Guanosine analogs that function as TLR7 agonists include certain C8- substituted and N7,C8-disubstituted guanine ribonucleotides and deoxyribonucleotides, including, but not limited to, Loxoribine (7-allyl-8-oxoguanosine), 7-thia-8-oxo-guanosine (TOG), 7-deazaguanosine, and 7-deazadeoxyguanosine (Lee et al., 2003). Bropirimine (PNU- 54461), a 5-halo-6-phenyl-pyrimidinone, and bropirimine analogs are described in the literature and are also suitable for use. See, e.g., Vroegop et al. (1999). Additional examples of C8-substituted guanosines include 8-mercaptoguanosine, 8-bromoguanosine, 8- methylguanosine, 8-oxo-7,8-dihydroguanosine, C8-arylamino-2'-deoxyguanosine, C8- propynyl-guanosine, C8- and N7-substituted guanine ribonucleosides such as 7-allyl-8- oxoguanosine (loxoribine) and 7-methyl-8-oxoguanosine, 8-aminoguanosine, 8-hydroxy-2'- deoxyguanosine, and 8-hydroxyguanosine.

[0138] In some embodiments, a TLR7 agonist is a selective TLR7 agonist, e.g., the agonist modulates cellular activity through TLR7, but does not modulate cellular activity through TLR8. TLR7-selective agonists include those shown in U.S. Patent Publication 2004 / 0171086. Such TLR7 selective agonist compounds include Nl-{4-[4-amino-2-(2- methoxyethyl)-6,7,8,9-tetrahydro-lH-imidazo[4,5-c]quinolin-l-yl]butyl]-4-fluoro-l- benzenesulfonamide, Nl-[4-(4-amino-2-(2-methoxyethyl)-lH-imidazo[4,5-c]quinolin-l- yl)butyl]-4-fluoro-l -benzenesulfonamide, N-[4-(4-amino-2-propyl-lH-imidazo[4,5- c]quinolin-l-yl)butyl]methanesulfonamide, N-{ 3-[4-amino-2-(2-methoxyethyl)-lH- imidazo[4,5-c]quinolin-l-yl]-2,2-dimethylpropyl] benzamide, N-(2-{2-[4-amino-2-(2- methoxyethyl)-lH-imidazo[4,5-c]quinolin-l-yl]ethoxy)ethyl)-N-methylmethanesulfonamide, N-(2-{2-[4-amino-2-(2-methoxyethyl)-6,7,8,9-tetrahydro-lH-imidazo[4,5-c]quinolin-l- yl]ethoxy]ethyl)benzamide, N-[4-(4-amino-2-methyl-lH-imidazo[4,5-c]quinolin-l- yl)butyl]cyclopentanecarboxamide, 1 -[4-( 1 , 1 -dioxidoisothiazolidin-2-yl)butyl]-2-(2- methoxyethyl)-lH-imidazo[4,5-c]quinolin-4-amine, 2-methyl-l-[5-methylsulfonyl)pentyl- 6,7,8,9-tetrahydro-lH-imidazo[4,5-c]quinolin-4-amine, N-(2-[4-amino-2-(ethoxymethyl)-6,7- dimethyl-lH-imidazo[4,5-c]pyridin-l-yl]-l,l-dimethylethyl]-N-cyclohexylurea, N-[2-(4- amino-2-ethyl- 1 H-imidazo[4,5-c]quinolin- 1 -y 1)- 1 , 1 -dimethylethyl]benzamide, N-[3-(4-amino-2-butyl-lH-imidazo[4,5-c]quinolin-l-yl)-2,2-dimethylpropyl]methanesulfonamide, 1- [6-(methanesulfonyl)hexyl]-6,7-dimethyl-2-propyl-lH-imidazo[4,5-c]pyridin-4-amine, 6-(6- amino-2-propyl-lH-imidazo[4,5-c]quinolin-l-yl)-N-methoxy-N-methylhexamide, 1 -[2,2- dimethyl-3-(methylsulfonyl)propyl]-2-(ethoxymethyl)-lH-imidazo[4,5-c]quinolin-4-amine, N-[4-(4-amino-2-methyl-lH-imidazo[4,5-c]quinolin-l-yl)butyl]-N-methyl-N-phenylurea, 1- {3-[4-amino-l-(2-methylpropyl)-lH-imidazo[4,5-c]quinolin-8-yl]phenyl {ethanone, 7-(4- amino-2-propyl-lH-imidazo[4,5-c]quinolin-l-yl)-2-methylheptan-2-ol, N-methyl-4-(4- amino-2-ethyl-lH-imidazo[4,5-c]quinolin-l-yl)butane-l -sulfonamide, N-(4-methoxybenzyl)- 4-(4-amino-2-ethyl-lH-imidazo[4,5-c]quinolin-l-yl)butane-l -sulfonamide, N-{2-[4-amino-3- (ethoxymethyl)-6,7-dimethyl- 1 H-imidazo [4,5 -c]pyridin- 1 -yl] - 1 , 1 - dimethylethyl } methanesulfonamide, 2-ethoxymethyl- 1 -(3 -methoxypropyl)-7-(5 - hydroxymethylpyridin-3-yl)-lH-imidazo[4,5-c]quinolin-4-amine, l-[(2,2-dimethyl-l,3- dioxolan-4-yl)methyl]-2-(ethoxymethyl)-7-(pyridin-3-yl)-lH-imidazo[4,5-c]quinolin-4- amine, 4- 13 -(4-amino-6,7 -dimethyl-2-propyl- 1 H-imithizo| 4,5-c Ipyridin- 1 -y 1 [propane- 1 - sulfonyl]-benzoic acid ethyl ester, 2-butyl-l-{2-[2-(methylsulfonyl)ethoxy]ethyl}-lH- imidazo[4,5-c]quinolin-4-amine, N-(2-{4-amino-2-ethoxymethyl-7-[6- (methanesulfony lamino)hexy loxy ] - 1 H-imidazo [4,5-c] quinolin- 1 -y 1 } - 1 , 1 - dimethylethyl)methanesulfonamide, N-(6-{ [4-amino-2-ethoxymethyl- 1 -(2- methanesulfonylamino-2-methylpropyl)-lH-imidazo[4,5-c]quinolin-7- yl]oxy}hexyl)acetamide, l-[4-(l,l-dioxidoisothiazolidin-2-yl)butyl]-2-ethoxymethyl-7- (pyridin- 3 -yl) - 1 H-imidazo [4,5 -c]quinolin-4- amine , 1 - [4- ( 1 , 1 -dioxidoisothiazolidin-2- yl)butyl]-2-ethoxymethyl-7-(pyridin-4-yl)-lH-imidazo[4,5-c]quinolin-4-amine, l-[4-( 1 , 1 - dioxidoisothiazolidin-2-yl)butyl]-2-ethoxymethyl-7-phenyl-lH-imidazo[4,5-c]quinolin-4- amine, 2-(ethoxymethyl)- 1 - { [ 1 -(methylsulf onyl)piperidin-4-yl]methyl } -7-(pyridin-3 -yl)- 1 H- imidazo[4,5-c]quinolin-4-amine, 2-(ethoxymethyl)-l-[(l-isobutyrylpiperidin-4-yl)methyl]-7- (pyridin-3-yl)-lH-imidazo[4,5-c]quinolin-4-amine, 2-(ethoxymethyl)-l-{ [l-(morpholic-4- ylcarbonyl)piperidin-4-yl]methyl}-7-(pyridin-3-yl)-lH-imidazo[4,5-c]quinolin-4-amine, Cyclopropanecarboxylic acid [3-(4-amino-2-propyl-lH-imidazo[4,5-c]quinolin-l- yl)propoxy] amide, Isopropylcarbamic acid 4-amino-2-(2-methoxyethyl)-l -propyl- 1H- imidazo[4,5-c]quinolin-7-yl ester, Ethyl 4-(4-amino-2-propyl-lH-imidazo[4,5-c]quinolin-l- yl)butyrate, 1 - [4-amino-2-ethyl-7-(pyridin-3 -yl)- 1 H-imidazo [4,5-c]quinolin- 1 -yl] -2- methylpropan-2-ol, l-(4-amino-2-ethyl-7-[5-{hydroxymethyl)pyridin-3-yl]-lH-imidazo[4,5-c]quinolin- 1 -yl } -2-methylpropan-2-ol, 1 -(3-[4-amino-2-(2-methoxyethyl)-8-(pyridin-3-yl)- lH-imidazo[4,5-c]quinolin-l-yl]propyl]pyrolidin-2-one, N-(2-{4-amino-2-ethoxymethyl-7- [6-(methanesulfonylamino)hexyloxy] - 1 H-imidazo [4,5 -c]quinolin- l-yl}-l,l- dimethylethyl) acetamide, l-{3-[4-amino-7-(3-hydroxymethylphenyl)-2-(2-methoxyethyl)- lH-imidazo[4,5-c]quinolin-l-yl]propyl}pyrrolidin-2-one, N-(4-[4-amino-2-ethoxymethyl-7- (pyridin-3-yl)-lH-imidazo[4,5-c]quinolin-l-yl]butyl)-N'-propylurea, N-{4-[4-amino-2- ethoxymethyl-7-(pyridin-3-yl)-lH-imidazo[4,5-c]quinolin-l-yl]butyl}butyramide, 5-(4- amino-2-propyl-lH-imidazo[4,5-c]quinolin-l-yl)-4,4-dimethylpentan-2-one, 1- cyclohexylmethyl-2-ethoxymethyl-7-(5-hydroxymethylpyridin-3-yl)-lH-imidazo[4,5- c]quinolin-4-amine, N,N-dimethyl-5-(4-amino-2-ethoxymethyl-lH-imidazo[4,5-c]quinolin-l- yl)pentane-l -sulfonamide, N-{3-[(4-amino-2-ethoxymethyl-lH-imidazo[4,5-c]quinolin-l- yl)amino]propyl } methanesulfonamide, and / or N,N-dimethyl-4-(4-amino-2-ethoxymethyl- lH-imidazo[4,5-c]quinolin-l-yl)butane-l-sulfonamide. Additional suitable TLR7 selective agonists include, but are not limited to, 2-(ethoxymethyl)-l-(2-methylpropyl)-lH- imidazo[4,5-c]quinolin-4-amine (U.S. Pat. No. 5,389,640); 2-methyl-l-[2-(3-pyridin-3- ylpropoxy)ethyl]-lH-imidazo[4,5-c]quinolin-4-amine (WO 02 / 46193); N-(2-{2-[4-amino-2- (2-methoxyethyl)-lH-imidazo[4,5-c]quinolin-l-yl]ethoxy}ethyl)-N- methylcyclohexanecarboxamide (U.S. Patent Publication 2004 / 0171086); l-[2- (benzyloxy)ethyl]-2-methyl-lH-imidazo[4,5-c]quinolin-4-amine (WO 02 / 46189); N-{8-[4- amino-2-(2-methyoxyethyl)-lH-imidazo[4,5-c]quinolin-l-yl]octyl}-N-phenylurea (U.S. Patent Publication 2004 / 0171086 (IRMS)); 2-butyl-l-[5-(methylsulfonyl)pentyl]-lH- imidazo[4,5-c]quinolin-4-amine (WO 02 / 46192); N-{3-[4-amino-2-(2-methoxyethyl)-lH- imidazo[4,5-c]quinolin-l-yl]propyl}-4-methylbenzenesulfonamide (U.S. Pat. No. 6,331,539); and N-[4-(4-amino-2-ethyl-lH-imidazo[4,5-c]quinolin-l-yl)butyl]cyclohexanecar-boxamide (U.S. Patent Publication 2004 / 0171086 (IRM8)). Other TLR7-selective agonists include N- [4-(4-amino-2-ethyl-lH-imidazo[4,5-c]quinolin-l-yl)butyl-]methanesulfon-amide (Gorden et al., 2005).

[0139] TLR8 agonists include isolated, naturally-occurring TLR8 agonists; and synthetic TLR8 agonists. TLR8 agonists isolated from a naturally-occurring source of TLR8 agonist are generally purified, e.g., the purified TLR8 agonist is at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure. Synthetic TLR8 agonists are prepared by standard methods,and are generally at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure.

[0140] In some embodiments, TLR8 agonists can include compounds, such as R-848, and derivatives and analogs thereof. TLR8 agonists can include compounds having a 2- aminopyridine fused to a five membered nitrogen-containing heterocyclic ring. Such compounds include, for example, imidazoquinoline amines including but not limited to substituted imidazoquinoline amines such as, for example, amide substituted imidazoquinoline amines, sulfonamide substituted imidazoquinoline amines, urea substituted imidazoquinoline amines, aryl ether substituted imidazoquinoline amines, heterocyclic ether substituted imidazoquinoline amines, amido ether substituted imidazoquinoline amines, sulfonamido ether substituted imidazoquinoline amines, urea substituted imidazoquinoline ethers, thioether substituted imidazoquinoline amines, and 6-, 7-, 8-, or 9-aryl or heteroaryl substituted imidazoquinoline amines; tetrahydroimidazoquinoline amines including but not limited to amide substituted tetrahydroimidazoquinoline amines, sulfonamide substituted tetrahydroimidazoquinoline amines, urea substituted tetrahydroimidazoquinoline amines, aryl ether substituted tetrahydroimidazoquinoline amines, heterocyclic ether substituted tetrahydroimidazoquinoline amines, amido ether substituted tetrahydroimidazoquinoline amines, sulfonamido ether substituted tetrahydroimidazoquinoline amines, urea substituted tetrahydroimidazoquinoline ethers, and thioether substituted tetrahydroimidazoquinoline amines; imidazopyridine amines including but not limited to amide substituted imidazopyridine amines, sulfonamide substituted imidazopyridine amines, urea substituted imidazopyridine amines, aryl ether substituted imidazopyridine amines, heterocyclic ether substituted imidazopyridine amines, amido ether substituted imidazopyridine amines, sulfonamido ether substituted imidazopyridine amines, urea substituted imidazopyridine ethers, and thioether substituted imidazopyridine amines; 1,2-bridged imidazoquinoline amines; 6,7-fused cycloalky limidazopyridine amines; imidazonaphthyridine amines; tetrahydroimidazonaphthyridine amines; oxazoloquinoline amines; thiazoloquinoline amines; oxazolopyridine amines; thiazolopyridine amines; oxazolonaphthyridine amines; thiazolonaphthyridine amines; and IH-imidazo dimers fused to pyridine amines, quinoline amines, tetrahydroquinoline amines, naphthyridine amines, or tetrahydronaphthyridine amines.

[0141] In one particular embodiment, the TLR8 agonist is an amide substituted imidazoquinoline amine. In an alternative embodiment, the TLR8 agonist is a sulfonamide substituted imidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a urea substituted imidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is an aryl ether substituted imidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a heterocyclic ether substituted imidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is an amido ether substituted imidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a sulfonamido ether substituted imidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a urea substituted imidazoquinoline ether. In another alternative embodiment, the TLR8 agonist is a thioether substituted imidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a 6-, 7-, 8-, or 9-aryl or heteroaryl substituted imidazoquinoline amine.

[0142] In another alternative embodiment, the TLR8 agonist is an amide substituted tetrahydroimidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a sulfonamide substituted tetrahydroimidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a urea substituted tetrahydroimidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is an aryl ether substituted tetrahydroimidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a heterocyclic ether substituted tetrahydroimidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is an amido ether substituted tetrahydroimidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a sulfonamido ether substituted tetrahydroimidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a urea substituted tetrahydroimidazoquinoline ether. In another alternative embodiment, the TLR8 agonist is a thioether substituted tetrahydroimidazoquinoline amine.

[0143] In another alternative embodiment, the TLR8 agonist is an amide substituted imidazopyridine amine. In another alternative embodiment, the TLR8 agonist is a sulfonamide substituted imidazopyridine amine. In another alternative embodiment, the TLR8 agonist is a urea substituted imidazopyridine amine. In another alternative embodiment, the TLR8 agonist is an aryl ether substituted imidazopyridine amine. In another alternative embodiment, the TLR8 agonist is a heterocyclic ether substituted imidazopyridine amine. In another alternative embodiment, the TLR8 agonist is an amido ether substitutedimidazopyridine amine. In another alternative embodiment, the TLR8 agonist is a sulfonamido ether substituted imidazopyridine amine. In another alternative embodiment, the TLR8 agonist is a urea substituted imidazopyridine ether. In another alternative embodiment, the TLR8 agonist is a thioether substituted imidazopyridine amine.

[0144] In another alternative embodiment, the TLR8 agonist is a 1 ,2-bridged imidazoquinoline amine. In another alternative embodiment, the TLR8 agonist is a 6,7-fused cycloalkylimidazopyridine amine.

[0145] In another alternative embodiment, the TLR8 agonist is an imidazonaphthyridine amine. In another alternative embodiment, the TLR8 agonist is a tetrahydroimidazonaphthyridine amine. In another alternative embodiment, the TLR8 agonist is an oxazoloquinoline amine. In another alternative embodiment, the TLR8 agonist is a thiazoloquinoline amine. In another alternative embodiment, the TLR8 agonist is an oxazolopyridine amine. In another alternative embodiment, the TLR8 agonist is a thiazolopyridine amine. In another alternative embodiment, the TLR8 agonist is an oxazolonaphthyridine amine. In another alternative embodiment, the TLR8 agonist is a thiazolonaphthyridine amine.

[0146] In yet another alternative embodiment, the TLR8 agonist is a IH-imidazo dimer fused to a pyridine amine, quinoline amine, tetrahydroquinoline amine, naphthyridine amine, or a tetrahydronaphthyridine amine.

[0147] In some embodiments, the TLR8 agonist is a selective TLR8 agonist, e.g., the agonist modulates cellular activity through TLR8, but does not modulate cellular activity through TLR7. TLR8-selective agonists include those in U.S. Patent Publication 2004 / 0171086. Such TLR8 selective agonist compounds include, but are not limited to, the compounds shown in U.S. Patent Publication No. 2004 / 0171086 that include N-{4-[4-amino- 2-(2-methoxyethyl)-lH-imidazo[4,5-c]quinolin-l-yl]butyl]quinolin-3-carboxamide, N-{4-[4- amino-2-(2-methoxyethyl)-lH-imidazo[4,5-c]quinolin-l-yl]butyl}quinoxoline-2- carboxamide, and N-[4-(4-amino-2-propyl-lH-imidazo[4,5-c]quinolin-l- yl)butyl]morpholine-4-carboxamide.

[0148] Other suitable TLR8-selective agonists include, but are not limited to, 2- propylthiazolo[4,5-c]quinolin-4-amine (U.S. Pat. No. 6,110,929); N.sup.l-[2-(4-amino-2- butyl-lH-imidazo[4,5-c][l,5]naphthridin-l-yl)ethyl]-2-amino-4-methylpentanamide (U.S. Pat. No. 6,194,425); N'-[4-(4-amino-lH-imidazo[4,5-c]quinolin-l-yl)butyl]-2-phenoxy-benzamide (U.S. Pat. No. 6,451,810); N'-[2-(4-amino-2-butyl-lH-imidazo[4,5-c]quinolin-l- yl)ethyl]-l-propa-nesulfonamide (U.S. Pat. No. 6,331,539); N-{2-[2-(4-amino-2-ethyl-lH- imidazo[4,5-c]quinolin-l-yl)ethyoxy]ethyl}-N'-phenylurea (U.S. Patent Publication 2004 / 0171086); l-{4-[3,5-dichlorophenyl)thio]butyl}-2-ethyl-lH-imidazo[4,5-c]quinolin-4- amine (U.S. Patent Publication 2004 / 0171086); N-{2-[4-amino-2-(ethoxymethyl)-lH- imidazo[4,5-c]quinolin-l -yl]ethyl }-N'-(3-cyanophenyl)urea (WO 00 / 76518 and U.S. Patent Publication No. 2004 / 0171086); and 4-amino-a,a-dimethyl-2-methoxyethyl-lH-imidazo[4,5- c]quinoli-ne- 1 -ethanol (U.S. Pat. No. 5,389,640). Included for use as TLR8-selective agonists are the compounds in U.S. Patent Publication No. 2004 / 0171086. Another TLR8-selective agonist is the compound 2-propylthiazolo-4,5-c]quinolin-4-amine.

[0149] In some embodiments, the TLR agonist is an agonist of both TLR7 and TLR8, i.e., a TLR 7 / 8 agonist. The TLR7 / 8 agonist can include, for example, 4-amino-2- (ethoxymethyl)-a,a-dimethyl- lH-imidazo[4,5-c]quinoline- 1-ethanol (R848), imiquimod, gardiquimod, their lipid derivatives, or mixtures thereof.

[0150] In some embodiments, the TLR7 agonist, TLR8 agonist, and / or TLR7 / 8 agonist can be conjugated to a fatty acid, such as oleic acid, or lipid to increase the stability of the TLR7 agonist, TLR8 agonist, and / or TLR7 / 8 agonist and / or promote complexing with the lipids during formation of the lipid nanoparticles.

[0151] In some embodiments, the TLR9 agonists can include isolated, naturally- occurring TLR9 agonists; and synthetic TLR9 agonists. TLR9 agonists isolated from a naturally -occurring source of TLR9 agonist are generally purified, e.g., the purified TLR9 agonist is at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure. Synthetic TLR9 agonists are prepared by standard methods, and are generally at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure.

[0152] Examples of TLR9 agonists include nucleic acids having a CpG oligonucleotide motif that can enhance or induce an immune stimulation mediated by TLR9. CpG nucleotides can be isolated from endogenous sources or synthesized in vivo or in vitro. Examples of sources of endogenous CpG oligonucleotides include, but are not limited to, microorganisms, bacteria, fungi, protozoa, viruses, molds, or parasites. Alternatively, endogenous CpG oligonucleotides are isolated from mammalian benign or malignantneoplastic tumors. Synthetic CpG oligonucleotides can be synthesized in vivo following transfection or transformation of template DNA into a host organism. Alternatively, synthetic CpG oligonucleotides can be synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods (Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 ( 1989), herein incorporated by reference).

[0153] Immunostimulatory CpG oligonucleotides can be divided into three types A, B and C, which differ in their immunostimulatory activities. Type A stimulatory CpG oligonucleotides are characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3' poly-G string. Following activation of TLR9, these CpG oligonucleotides induce high IFN-a production from plasmacytoid dendritic cells (pDC). Type A CpG oligonucleotides weakly stimulate TLR9-dependent NF-KB signaling.

[0154] Type B stimulatory CpG oligonucleotides contain a full phosphorothioate backbone with one or more CpG dinucleotides. Following TLR9 activation, these CpG- oligonucleotides strongly activate B cells. In contrast to Type A CpG-ODNs, Type B CpG- ODNS weakly stimulate IFN-a secretion.

[0155] Type C stimulatory CpG oligonucleotides comprise features of Types A and B. Type C CpG oligonucleotides contain a complete phosphorothioate backbone and a CpG containing palindromic motif. Similar to Type A CpG ODNs, Type C CpG ODNs induce strong IFN-a production from pDC. Similar to Type B CpG ODNs, Type C CpG ODNs induce strong B cell stimulation.

[0156] Examples of stimulatory CpG oligonucleotides comprise, but are not limited to, ODN 1585, ODN 1668, ODN 1826, ODN 2006, ODN 2006-G5, ODN 2216, ODN 2336, ODN 2395, ODN M362 (all InvivoGen).

[0157] In some embodiments, the CpG dinucleotide is selected from the group consisting of CpG, C*pG, CpG*, and C*pG*, wherein C is 2'-deoxycytidine, C* is an analog thereof, G is 2'-deoxyguanosine, and G* is an analog thereof, and p is an internucleoside linkage selected from the group consisting of phosphodiester, phosphorothioate, and phosphorodithioate. In some embodiments C* is selected from the group consisting of 2'- deoxythymidine, arabinocytidine, 2'-deoxythymidine, 2'-deoxy-2'-substituted arabinocytidine, 2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine, 2’-deoxy-N4- alkyl-cytidine, 2'-deoxy-4-thiouridine. In some embodiments, G* is 2' deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine, 2'-deoxy-2'substituted- arabinoguanosine, 2'-O-substituted-arabinoguanosine, 2'-deoxyinosine.

[0158] In some embodiments, the TLR9 agonist can include lefitolimod, tilsotolimod, or a cytidine-phosphate-guanosine (CpG) oligonucleotide.

[0159] In some embodiments, the lipid nanoparticles can include CpG ODN and R848 complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids.

[0160] The lipid nanoparticles can be loaded with different ratios of the TLR7 agonist, TLR8 agonist, or TLR7 / 8 agonist (e.g., R848) and / or TLR9 agonist (e.g., CpG). In some embodiments, lipid nanoparticles described herein can include a TLR7 / 8 agonist (e.g., R848) and a TLR9 agonist (CpG ODNs) loaded into the lipid nanoparticles at a TLR7 / 8 agonist / TLR9 agonist (w / w) ratio of about 10:1 to about 1 :10, preferably about 5: 1 to about 1:1, for example, about 5:1, about 3:1, and about 1:1.

[0161] In some embodiments, the lipid nanoparticles can include TLR7 / 8 agonist concentration of at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM or more.

[0162] In other embodiments, the lipid nanoparticles can include a TLR9 agonist concentration of at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM or more.

[0163] In some embodiments, the lipid nanoparticles can further include a nucleic acid encoding a cancer antigen or neoantigen that is complexed or conjugated with and / or encapsulated by the pH sensitive protonatable or ionizable lipids for expression of the cancer antigen or neoantigen in a cell, such as an immune cell. The nucleic acid can be an isolated ribonucleic acid (RNA), such as messenger RNA (mRNA), comprising an open reading frame (ORF) encoding the cancer or neoantigen. The cancer antigen or neoantigen can be a peptide encoded by a mutated gene selected from ABL1, BRAF, CDKN1A, EPHA3, FGFR4, IKZF1, MCL1, NKX2-1, PMS2, RNF43, TET2, ACVR1B, BRCA1, CDKN1B, EPHB1 , FH, INPP4B, MDM2, NOTCH1, POLDI, ROS1, TGFBR2, AKT1, BRCA2, CDKN2A, EPHB4, FLCN, IRF2, MDM4, NOTCH2, POLE, RPTOR, TIP ARP, AKT2, BRD4, CDKN2B, ERBB2, FLT1, IRF4, MED12, NOTCH3, PPARG, SDHA, TNFAIP3, AKT3, BRIP1, CDKN2C, ERBB3, FLT3, IRS2, MEF2B, NPM1, PPP2R1A, SDHB, TNFRSF14, ALK,BTG1, CEBPA, ERBB4, F0XL2, JAK1, MEN1, NRAS, PPP2R2A, SDHC, TP53, AL0X12B, BTG2, CHEK1, ERCC4, FUBP1, JAK2, MERTK, NT5C2, PRDM1, SDHD, TSC1, AMER1, BTK, CHEK2, ERG, GABRA6, JAK3, MET, NTRK1, PRKAR1A, SETD2, TSC2, APC, Cllorf30, CIC, ERRFI1, GATA3, JUN, MITF, NTRK2, PRKC1, SF3B1, TYR03, AR, CALR, CREBBP, ESRI, GATA4, KDM5A, MKNK1, NTRK3, PTCHI, SGK1, U2AF1 , ARAF, CARD1 1 , CRKL, EZH2, GATA6, KDM5C, MLH1, P2RY8, PTEN, SMAD2, VEGFA, ARFRP1, CASP8, CSF1R, FAM46C, GID4, (C17orf39), KDM6A, MPL, PALB2, PTPN11, SMAD4, VHL, ARID1A, CBFB, CSF3R, FANCA, GNA11, KDR, MRE11A, PARK2, PTPRO, SMARCA4, WHSCI, ASXL1, CBL, CTCF, FANCC, GNA13, KEAP1, MSH2, PARP1, QKI, SMARCB1, WHSC1L1, ATM, CCND1, CTNNA1, FANCG, GNAQ, KEL, MSH3, PARP2, RAC1, SMO, WT1, ATR, CCND2, CTNNB1, FANCL, GNAS, KIT, MSH6, PARP3, RAD21, SNCAIP, XPO1, ATRX, CCND3, CUL3, FAS, GRM3, KLHL6, MST1R, PAX5, RAD51, SOCS1, XRCC2, AURKA, CCNE1, CUL4A, FBXW7, GSK3B, KMT2A, (MLL), MTAP, PBRM1, RAD51B, SOX2, ZNF217, AURKB, CD22, CXCR4, FGF10, H3F3A, KMT2D, (MLL2), MTOR, PDCD1, RAD51C, SOX9, ZNF703, AXIN1, CD274, CYP17A1, FGF12, HDAC1, KRAS, MUTYH, PDCD1LG2, RAD51 D, SPEN, AXL, CD70, DAXX, FGF14, HGF, LTK, MYC, PDGFRA, RAD52, SPOP, BAP1, CD79A, DDR1, FGF19, HNF1A, LYN, MYCL, PDGFRB, RAD54L, SRC, BARD1, CD79B, DDR2, FGF23, HRAS, MAF, MYCN, PDK1, RAFI, STAG2, BCL2, CDC73, DIS3, FGF3, HSD3BI, MAP2KI, MYD88, PIK3C2B, RARA, STAT3, BCL2L1, CDH1, DNMT3A, FGF4, ID3, MAP2K2, NBN, P1K3C2G, RBI, STK11, BCL2L2, CDK12, DOT1L, FGF6, IDH1, MAP2K4, NF1, PIK3CA, RBM10, SUFU, BCL6, CDK4, EED, FGFR1, IDH2, MAP3K1, NF2, P1K3CB, REL, SYK, BCOR, CDK6, EGFR, FGFR2, IGF1R, MAP3K13, NFE2L2, PIK3R1, RET, TBX3, BCORL1, CDK8, EP300, FGFR3, IKBKE, MAPK1, NFKBIA, PIM1, RICTOR, TEK, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, MYB, NUTM1, RSPO2, SDC4, SLC34A2, TERC, TERT, or TMPRSS2.

[0164] For example, a the mRNA can encode a peptide comprising at least one of KRAS G12A (KLVVVGAAGVGKSALTI (SEQ ID NO: 1)), KRAS G12C (KLVVVGACGVGKSALTI (SEQ ID NO: 2)), KRAS G12D (KLVVVGADGVGKSALTI (SEQ ID NO: 3)), KRAS G12R (KLVVVGARGVGKSALTI (SEQ ID NO: 4)), KRAS G12S (KLVVVGASGVGKSALTI (SEQ ID NO: 5)), KRAS G12V (KLVVVGAVGVGKSALTI(SEQ ID NO: 6)), KRAS G13D (KLVVVGAGDVGKSALTI (SEQ ID NO: 7)), or KRAS G13C (KLVVVGAGCVGKSALTI (SEQ ID NO: 8)).

[0165] mRNAs can be synthesized according to any of a variety of known methods. Various methods are described in published U.S. Application Publication No. US 2018 / 0258423, and can be used to practice the present invention, all of which are incorporated herein by reference. For example, mRNAs may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions will vary according to the specific application.

[0166] In some embodiments, the mRNA sequence is codon optimized for efficient expression human cells. In some embodiments, the mRNA sequence is naturally -occurring or a wild-type sequence. In some embodiments, the mRNA sequence encodes a cancer antigen or neoantigen protein or a peptide that contains one or mutations in amino acid sequence.

[0167] In some embodiments, the mRNA may contain backbone modifications, sugar modifications and / or base modifications. For example, modified nucleotides may include, but not be limited to, modified purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6- isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl- cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2- methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5- carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5 -bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl- uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5- methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5 -methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5 -oxy acetic acid (v), 1 -methyl-pseudouracil, queosine, P-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5 -methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g., from the U.S.Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosures of which are incorporated by reference in their entirety.

[0168] In some embodiments, mRNAs may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g., cytidine 5'-O-(l-thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

[0169] In some embodiments, mRNAs may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'- deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5 '-triphosphate, 2'-fluoro-2'- deoxyuridine 5 '-triphosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'- deoxycytidine 5 '-triphosphate, 2'-amino-2'-deoxyuridine 5 '-triphosphate), 2'-O- alkyloligoribonucleotide, 2'-deoxy-2'-C-alkyloligoribonucleotide (2'-O-methylcytidine 5'- triphosphate, 2 '-methyluridine 5 '-triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5'-triphosphate, 2'-arauridine 5'-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5 '-triphosphate, 2'-azido-2'-deoxyuridine 5 '-triphosphate).

[0170] Typically, a 5' cap and / or a 3' tail may be added after the synthesis. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0171] A 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5'5'5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G. Additional cap structures are described in published U.S. Application No. US 2016 / 0032356 and published U.S.Application No. US 2018 / 0125989, which are incorporated herein by reference.

[0172] Typically, a tail structure includes a poly(A) and / or poly(C) tail. A poly-A or poly-C tail on the 3' terminus of mRNA typically includes at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, at least 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb adenosine or cytosine nucleotides, respectively. In some embodiments, a poly A or poly C tail may be about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10 to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine or cytosine nucleotides, about 10 to 400 adenosine or cytosine nucleotides, about 10 to 500 adenosine or cytosine nucleotides, about 10 to 550 adenosine or cytosine nucleotides, about 10 to 600 adenosine or cytosine nucleotides, about 50 to 600 adenosine or cytosine nucleotides, about 100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosine or cytosine nucleotides, about 200 to 600 adenosine or cytosine nucleotides, about 250 to 600 adenosine or cytosine nucleotides, about 300 to 600 adenosine or cytosine nucleotides, about 350 to 600 adenosine or cytosine nucleotides, about 400 to 600 adenosine or cytosine nucleotides, about 450 to 600 adenosine or cytosine nucleotides, about 500 to 600 adenosine or cytosine nucleotides, about 10 to 150 adenosine or cytosine nucleotides, about 10 to 100 adenosine or cytosine nucleotides, about 20 to 70 adenosine or cytosine nucleotides, or about 20 to 60 adenosine or cytosine nucleotides) respectively. In some embodiments, a tail structure includes a combination of poly (A) and poly (C) tails with various lengths described herein. In some embodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, a tail structure includes at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0173] As described herein, the addition of the 5' cap and / or the 3' tail facilitates the detection of abortive transcripts generated during in vitro synthesis because without cappingand / or tailing, the size of those prematurely aborted mRNA transcripts can be too small to be detected. Thus, in some embodiments, the 5' cap and / or the 3' tail are added to the synthesized mRNA before the mRNA is tested for purity (e.g., the level of abortive transcripts present in the mRNA). In some embodiments, the 5' cap and / or the 3' tail are added to the synthesized mRNA before the mRNA is purified as described herein. In other embodiments, the 5' cap and / or the 3' tail are added to the synthesized mRNA after the mRNA is purified as described herein.

[0174] mRNA synthesized as described herein may be used without further purification. In some embodiments, mRNA synthesized may be further purified. Various methods may be used to purify mRNA synthesized as described herein. For example, purification of mRNA can be performed using centrifugation, filtration and for chromatographic methods. In some embodiments, the synthesized mRNA is purified by ethanol precipitation or filtration or chromatography, or gel purification or any other suitable means. In some embodiments, the mRNA is purified by HPLC. In some embodiments, the mRNA is extracted in a standard phenol: chloroform: isoamyl alcohol solution, well known to one of skill in the art. In some embodiments, the mRNA is purified using Tangential Flow Filtration. Suitable purification methods include those described in published U.S.Application No. US 2016 / 0040154, published U.S. Application No. US 2015 / 0376220, published U.S. Application No. US 2018 / 0251755, published U.S. Application No. US 2018 / 0251754, U.S. Provisional Application No. 62 / 757,612 filed on Nov. 8, 2018, and U.S. Provisional Application No. 62 / 891,781 filed on Aug. 26, 2019, all of which are incorporated by reference herein.

[0175] The mRNA described herein is substantially free of contaminants comprising short abortive RNA species, long abortive RNA species, double- stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzymes, residual solvent and / or residual salt.

[0176] The mRNA described herein can have a purity of about between 60% and about 100%. Accordingly, in some embodiments, the purified mRNA has a purity of about 60%. In some embodiments, the purified mRNA has a purity of about 65%. In some embodiments, the purified mRNA has a purity of about 70%. In some embodiments, the purified mRNA has a purity of about 75%. In some embodiments, the purified mRNA has a purity of about 80%. In some embodiments, the purified mRNA has a purity of about 85%. In someembodiments, the purified mRNA has a purity of about 90%. In some embodiments, the purified mRNA has a purity of about 91 %. In some embodiments, the purified mRNA has a purity of about 92%. In some embodiments, the purified mRNA has a purity of about 93%. In some embodiments, the purified mRNA has a purity of about 94%. In some embodiments, the purified mRNA has a purity of about 95%. In some embodiments, the purified mRNA has a purity of about 96%. In some embodiments, the purified mRNA has a purity of about 97%. In some embodiments, the purified mRNA has a purity of about 98%. In some embodiments, the purified mRNA has a purity of about 99%. In some embodiments, the purified mRNA has a purity of about 100%.

[0177] In some embodiments, the surfaces of the lipid nanoparticles can be modified with a stabilizing amount of the polymer, polyethylene glycol, or polysaccharide. A polymer, such as polyethylene glycol, can be covalently incorporated on the lipid nanoparticles by reacting unpolymerized free thiol of the nanoparticle to reduce non-specific tissue uptake in vivo. For example, PEG-maleimide reacts rapidly with free thiol groups. The molecular weight of the PEG can vary depending upon the desired amount of hydrophilicity to be imparted on the lipid nanoparticles. PEG-modification of the lipid nanoparticles can also protect nanoparticles composed of the TLR agonists and optionally mRNA from enzymatic degradation upon uptake by the cell (e.g., endonucleases).

[0178] In some embodiments, the polymer or stabilizing polymer can include polyethylene glycol (PEG). The PEG can have an average molecular weight of about 1,000 Daltons to about 100,000 Daltons, preferably about 1,000 Daltons to about 50,000 Daltons, more preferably about 1,000 Daltons to about 20,000 Daltons, or about 2,000 Daltons to about 10,000 Daltons.

[0179] In some embodiments, the polysaccharide or stabilizing polysaccharide can include dextran. The dextran can have an average molecular weight of about 1 ,000 to about 50,000 Daltons, more preferably about 1,000 Daltons to about 20,000 Daltons, or about 2,000 Daltons to about 10,000 Daltons.

[0180] In some embodiments, the dextran includes at one least side chain functionalized with a maleimide linker that is conjugated to a thiol group of formula (I) and / or a terminal end functionalized with a maleimide linker that is conjugated to a thiol group of formula (I).

[0181] In some embodiments, the plurality of pH sensitive protonatable or ionizable lipids include a plurality of ECO and / or ECLn lipids and a plurality of ECLn and / or ECO modified with PEG and / or dextran.

[0182] In some embodiments, the plurality of pH sensitive protonatable or ionizable lipids include about 1 mol % to about 30 mol %, about 1 mol % to about 25 mol %, or about 1 mol % to about 20 mol %, about 1 mol % to about 15 mol %, or about 2.5 mol % to about 15 mol % of ECLn and / or ECO modified with PEG and / or dextran.

[0183] In other embodiments, the stabilizing polysaccharide is hyaluronic acid that is complexed with the plurality of pH sensitive protonatable or ionizable lipids and mRNA. The hyaluronic acid can provide a surface modification of lipid nanoparticle and have an average molecular weight of about 1,000 Daltons to about 100,000 Daltons, preferably about 1,000 Daltons to about 50,000 Daltons, more preferably about 1,000 Daltons to about 20,000 Daltons, or about 2,000 Daltons to about 10,000 Daltons. The stabilized lipid nanoparticle can include, for example, about 1 mol % to about 30 mol %, about 1 mol % to about 25 mol %, or about 1 mol % to about 20 mol %, about 1 mol % to about 15 mol %, about 2.5 mol % to about 15 mol %, about 2.5 mol % to about 10 mol %, or about 2.5 mol % to about 5 mol % hyaluronic acid.

[0184] In some embodiments, the lipid nanoparticle can further include one or more structural lipids. Structural lipids can be selected from cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alphatocopherol, or a mixture thereof. In some embodiments, the structural lipid is cholesterol.

[0185] In some embodiments, the structural lipid can include cholesterol. For example, the stabilized lipid nanoparticle can include about 1 mol % to about 30 mol %, about 1 mol % to about 25 mol %, or about 1 mol % to about 20 mol %, about 1 mol % to about 15 mol %, about 2.5 mol % to about 15 mol %, about 2.5 mol % to about 10 mol %, or about 2.5 mol % to about 5 mol % cholesterol.

[0186] The pH sensitive protonatable or ionizable lipids having the general formula I can be synthesized using solid phase techniques known in the art. In general, the approach involves the systematic protection / elongation / deprotection to produce a dithiol compound. The hydrophobic group is produced by reacting oleic acid with the amino group present on the cysteine residue.

[0187] Any of the pH sensitive protonatable or ionizable lipids described herein can exist or be converted to the salt thereof. In one aspect, the salt is a pharmaceutically acceptable salt. The salts can be prepared by treating the free acid with an appropriate amount of a chemically or pharmaceutically acceptable base. Representative chemically or pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2- dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, and the like. In one aspect, the reaction is conducted in water, alone or in combination with an inert, water- miscible organic solvent, at a temperature of from about 0°C to about 100°C, such as at room temperature. The molar ratio of the compound to base used is chosen to provide the ratio desired for any particular salts. For preparing, for example, the ammonium salts of the free acid starting material, the starting material can be treated with approximately one equivalent of base to yield a salt.

[0188] If the pH sensitive protonatable or ionizable lipids possess carboxylic acid groups, these groups can be converted to pharmaceutically acceptable esters or amides using techniques known in the art. Alternatively, if an ester is present on the dendrimer, the ester can be converted to a pharmaceutically acceptable ester using transesterification techniques.

[0189] The pH sensitive protonatable or ionizable lipids described herein have numerous applications with respect to the delivery of nucleic acids to a subject. In some embodiments, the stabilized lipid nanoparticles described herein can be used in gene therapy to deliver nucleic acid or genetic materials to cells and tissues.

[0190] The TLR agonists and optionally the mRNA can be complexed to the pH sensitive protonatable or ionizable lipids described herein by admixing the TLR agonists, optional mRNA, and the pH sensitive protonatable or ionizable lipids or corresponding disulfide oligomer or polymer. The pH of the reaction can be modified to convert the amino groups present on the pH sensitive protonatable or ionizable lipids described herein to cationic groups. For example, the pH can be adjusted to protonate the amino group. With the presence of cationic groups on the compound, the negatively charged TLR agonist and optional mRNA can electrostatically bond (i.e., complex) with the pH sensitive protonatable or ionizable lipids. In one aspect, the pH is from 1 to 7.4.

[0191] In another aspect, the N / P ratio of the pH sensitive protonatable or ionizable lipids complexed with the nucleic acid TLR agonists (e.g., CpG TLR9 agonists) and optional other nucleic acids, such as mRNA, can be from 0.5 to 100, where N is the number of nitrogen atoms (e.g., amines) present on the lipids that can form a positive charge and P is the number of phosphate groups present on the nucleic acid TLR agonists and optional mRNA. Thus, by modifying the pH sensitive protonatable or ionizable lipids with the appropriate number of amino groups in the head group, it is possible to tailor the bonding e.g., type and strength of bond) between the nucleic acid TLR agonists and optional mRNA and the pH sensitive protonatable or ionizable lipids. The N / P ratio can be adjusted depending on the cell type to which the nucleic acid TLR agonists and optional mRNA is to be delivered. In some embodiments, the N / P ratio can be at least about 6, at least about 10, or at least about 15. In other embodiments, the N / P ratio can be from about 6 to about 20, about 10 to about 20, about 12 to about 20, or about 6 about 14. In still other embodiments, the N / P ratio can be about 2 to about 20, about 2 to about 18, about 2 to about 16, about 2 to about 14, about 2 to about 12, or about 4 to about 12.

[0192] In one aspect, the lipid nanoparticles can have an average particle diameter of about 100 nm to less than about 500 nm, about 100 nm to about 400 nm, about 100 nm to about 300 nm, or about 100 nm to about 200 nm. In another aspect, the lipid nanoparticles can have a diameter of about 1000 nanometers or less, for example, about 50 nm to about 200 nm, about 60 nm to about 180 nm, about 70 nm to about 160 nm, about 80 nm to about 140 nm, or about 90 nm to about 120 nm.

[0193] In other aspects, the lipid nanoparticles described herein can be designed to escape endosomal and / or lysosomal compartments at the endosomal-lysosomal pH. For example, the pH sensitive protonatable or ionizable lipids forming nanoparticles with the TLR agonists and optional mRNA can be designed such that its structure and amphiphilicity changes at endosomal-lysosomal pH (5.0-6.0) and disrupts endosomal-lysosomal membranes, which allows entry of the nanoparticle into the cytoplasm. In one aspect, the ability of specific endosomal-lysosomal membrane disruption of the pH sensitive protonatable or ionizable lipids described herein can be tuned by modifying their pH sensitive amphiphilicity by altering the number and structure of protonatable amines and lipophilic groups. For example, decreasing the number of protonatable amino groups can reduce the amphiphilicity of a nanoparticle produced by the pH sensitive protonatable or ionizable lipids at neutral pH.In one aspect, the pH sensitive protonatable or ionizable lipids described herein have 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 2 protonatable amino or substituted amino groups. The pH-sensitive amphiphilicity of the pH sensitive protonatable or ionizable lipids and nanoparticles produced by the pH sensitive protonatable or ionizable lipids can be used to fine-tune the overall pKa of the nanoparticle. Low amphiphilicity of the nanoparticles at physiological pH can minimize non-specific cell membrane disruption and nonspecific tissue uptake of the nucleic acid. In certain aspects, it is desirable that the lipid nanoparticles have low amphiphilicity at the physiological pH and high amphiphilicity at the endosomal-lysosomal pH, which will only cause selective endosomal-lysosomal membrane disruption with the nanoparticles.

[0194] The amount of a therapeutic and / or prophylactic combinatorial cancer vaccine comprising a plurality of lipid nanoparticles described herein may depend on the size, composition, desired target and / or application, or other properties of the lipid nanoparticles as well as on the properties of the therapeutic and / or prophylactic. The relative amounts of a therapeutic and / or prophylactic and other elements (e.g., pH sensitive protonatable or ionizable lipids) in a nanoparticle composition may also vary. In some embodiments, the wt / wt ratio of the pH sensitive protonatable or ionizable lipids to the TLR agonist and optional mRNA may be from about 5:1 to about 60:1, such as 5: 1, 6: 1, 7:1, 8: 1, 9: 1, 10: 1, 1 1: 1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18: 1, 19:1, 20:1, 25: 1, 30:1, 35:1, 40: 1, 45: 1, 50: 1, and 60:1. For example, the wt / wt ratio of the pH sensitive protonatable or ionizable lipids to the TLR agonist and optional mRNA may be from about 10:1 to about 40:1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of a the TLR agonist and optional mRNA in lipid nanoparticles may, for example, be measured using absorption spectroscopy e.g., ultraviolet-visible spectroscopy).

[0195] The combinatorial cancer vaccine may be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical composition may include one or more lipid nanoparticles and one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington’s The Science and Practice of Pharmacy, 21. sup. st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and accessory ingredients may be used in any pharmaceutical composition, except insofar as anyconventional excipient or accessory ingredient may be incompatible with one or more components of a nanoparticle composition. An excipient or accessory ingredient may be incompatible with a component of a nanoparticle composition if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.

[0196] In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a nanoparticle composition. For example, the one or more excipients or accessory ingredients may make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0197] Relative amounts of the one or more nanoparticle compositions, the one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more nanoparticle compositions.

[0198] In certain embodiments, a pharmaceutical composition that includes the combinatorial cancer vaccine includes a cryoprotectant, such as sucrose, that is provided in the pharmaceutical composition at an amount effective to enhance the stability of the composition when cryopreserved. However, any cryoprotectant will suffice (e.g., trehalose). The concentration of the cryo-protectant can be from 4%-32% v / v.

[0199] In certain embodiments, the vaccine and / or pharmaceutical composition can be refrigerated or frozen for storage and / or shipment, e.g., being stored at a temperature of 4°C or lower, such as a temperature between about -150°C and about 0°C or between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C).

[0200] In certain embodiments, a method of increasing stability of the combinatorial cancer vaccine can include storing the nanoparticle compositions and / or pharmaceutical compositions at a temperature of 4° C or lower, such as a temperature between about -150°C and about 0°C or between about -80°C and about -20°C, e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C. For example, the combinatorial cancer vaccine described herein and / or pharmaceutical compositions disclosed herein are stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months, e.g., at a temperature of 4°C or lower (e.g., between about 4°C and -20°C).

[0201] In certain embodiments, the combinatorial cancer vaccine can include the lipid nanoparticles described herein and a pharmaceutically acceptable carrier selected from one or more of Tris, an acetate (e.g., sodium acetate), an citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the carrier may be at a concentration of 1-100 mM (e.g., including but not limited to any numerical value or range within the range of 1-100 mM such as 1, 2, 3, 4, ...97, 98, 99, 100, 10-90 mM, 20-80 mM, 30-70 mM and so on).

[0202] The combinatorial cancer vaccine and / or pharmaceutical compositions including one or more nanoparticle compositions may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of a therapeutic and / or prophylactic to one or more particular cells, tissues, organs, or systems or groups thereof. Although the descriptions provided herein of nanoparticle compositions and pharmaceutical compositions including nanoparticle compositions are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are notlimited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and / or rats.

[0203] A pharmaceutical composition described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if desirable or necessary, dividing, shaping, and / or packaging the product into a desired single- or multi-dose unit.

[0204] A pharmaceutical composition may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., nanoparticle composition). The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.

[0205] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0206] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0207] The combinatorial cancer vaccine described herein can be administered to a subject using techniques known in the art. For example, pharmaceutical compositions can be prepared with the complexes. It will be appreciated that the actual preferred amounts of the complex in a specified case will vary according to the specific compound being utilized, theparticular compositions formulated, the mode of application, and the particular sites and subject being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate conventional pharmacological protocol. Physicians and formulators, skilled in the art of determining doses of pharmaceutical compounds, will have no problems determining dose according to standard recommendations (Physicians’ Desk Reference, Barnhart Publishing (1999).

[0208] In some embodiments, the combinatorial cancer vaccine can be used in a method of treating or preventing cancer in a subject in need thereof. The method can include administering to the subject a therapeutically effective or prophylactically effective amount of combinatorial cancer vaccine described herein.

[0209] While not intending to be bound by theory, it appears that simultaneously targeting cancer antigen or neoantigen in combination with administration of a TLR 7,8 agonist, and TLR9 agonist elicits a functional synergy resulting in activation of innate immune cells into T cell-stimulatory cells driving antitumor immune responses to recruit systemic immunity. For example, in a pilot experiment, C57BL / 6 mice bearing orthotopic KPC1242 (LSL-Krasr,12D:LSL-Trp53R172H:Pdxl-Cre) PDAC were treated by i.v. injecting the combinatorial cancer vaccine (5pg CpG + 8.5pg R848 + 20pg G12D peptide per mouse), cocktail vaccine (20 pg CpG + 25|ig R848 + 20 pg G12D per mouse, positive control), or PBS after tumor initiation. Approximately 70% mice treated with the combinatorial cancer vaccine had complete tumor rejection, while the cocktail vaccine showed less therapeutic efficacy in this strain of KPC1242 PDAC model with only 20% disease- free survival.

[0210] In some embodiments, the cancer to be treated is selected from cancers of: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, and lipoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor- associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal system, for example, esophagus (squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), gastric, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); gastrointestinal stromal tumors and neuroendocrine tumors arising at any site; genitourinary tract, for example, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and / or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver, for example, hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma); bone, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system, for example, neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); reproductive system, for example, gynecological, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), placenta, vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma) and other sites associated with female genital organs; penis, prostate, testis, and other sites associated with male genital organs; hematologic system, forexample, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin’s lymphoma [malignant lymphoma]; oral cavity, for example, lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx; skin, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids; adrenal glands: neuroblastoma; and other tissues comprising connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa, breast, head or neck, anal region, thyroid, parathyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites. In some embodiments, the cancer is a colon cancer, colorectal cancer or rectal cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is an adenocarcinoma, an adenocarcinoma, an adenoma, a leukemia, a lymphoma, a carcinoma, a melanoma, an angiosarcoma, or a seminoma.

[0211] In other embodiments, the disclosed methods and combinatorial cancer vaccine can herein can be used to treat, inhibit, reduce, decrease, ameliorate, and / or prevent any disease where uncontrolled cellular proliferation occurs, such as lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostatic cancer, non- small cell lung cancer (NSCLC), or pancreatic cancer.

[0212] In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is not a solid tumor. In further embodiments, the cancer is a leukemia cancer. In someembodiments, the cancer is from a carcinoma, a sarcoma, a myeloma, a leukemia, or a lymphoma. In some embodiments, the cancer is a colon cancer, colorectal cancer or rectal cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a pancreatic cancer.

[0213] In some embodiments, the cancer is a primary cancer or a metastatic cancer. In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer reaches a remission, but can relapse. In some embodiments, the cancer is unresectable.

[0214] In some embodiments, the cancer expresses a gene or mutation of a gene as described herein, and the lipid nanoparticles of the combinatorial vaccine include a cancer antigen or neoantigen or a nucleic encoding a cancer antigen or neoantigen corresponding to the gene or mutated gene. For example, the cancer can include a lung adenocarcinoma, a mucinous adenoma, a ductal carcinoma of the pancreas, a colorectal carcinoma; a rectal cancer, a follicular thyroid cancer, an autoimmune lymphoproliferative syndrome, a Noonan syndrome, a juvenile myelomonocytic leukemia; a bladder cancer, a follicular thyroid cancer, or an oral squamous cell carcinoma that express a mutated KRAS, and the combinatorial cancer vaccine can include KRAS antigen or neoantigen corresponding to the mutated KRAS.

[0215] In some embodiments, the mutation can be detected by sequencing a biopsy of the cancer, a Southern Blotting, a Northern Blotting, or by contacting with an antibody specifically binding to the mutation, such as KRas (G12D Mutant) Monoclonal Antibody (HL10) available from ThermoFisher, or anti-KRas (mutated G12D) antibody (ab221163) available from abeam.

[0216] In some embodiments, a method of treating the cancer includes administering the combinatorial cancer vaccine described herein in combination with an immune checkpoint therapy. Immune checkpoint therapy for cancer encompasses strategies that target immunity regulatory pathways in order to enhance immunity activity against tumor cells. In some embodiments, the immune checkpoint therapy can include the administration to a subject of one or more immune checkpoint modulating agents, such as immune checkpoint inhibitors. The immune checkpoint modulating agents can target the same immune checkpoint or can target two or more immune checkpoints.

[0217] An immune checkpoint modulating agent for use in a method described herein can include an agent that either inhibits negative regulators of the immune system responseagainst cancer cells, such as programmed cell death protein 1 (PD-1), or agents that act as agonists for positive regulators, such as 0X40 (CD134). In some embodiments, the immune checkpoint modulating agents can include an immune checkpoint-targeting antibody such as anti-PD-1 or agonistic OX40-specific monoclonal antibodies.

[0218] The programmed death 1 (PD-1) immune checkpoints are negative regulators of T-cell immune function and inhibition of PD-1 , results in increased activation of the immune system. PD- 1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0219] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0220] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD- 1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Pat. Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference.Other PD- 1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0221] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, andpidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PDL1 inhibitor comprises AMP-224. Nivolumab, also known as MDX- 1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121 168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO2011 / 066342. Additional PD- 1 inhibitors include MEDIO680, also known as AMP-514, and REGN2810.

[0222] In some embodiments, PD-1 inhibitory monoclonal antibodies for use in a combination therapy described herein include, but are not limited to, Pembrolizumab, Nivolumab, and Cemiplimab, and MEDI0608. Examples of PD-L1 inhibitory monoclonal antibodies include, but are not limited to, Atezolizumab, Avelumab, Vonlerolizumab, and Durvalumab.

[0223] In some embodiments, the additional immune checkpoint modulating agent is another agent capable of inhibiting or blocking engagement / interaction with VISTA. In addition to the nucleic acid inhibitor of VISTA, agents targeting VISTA can include human monoclonal antibody JNJ-61610588 and CA-170, an oral inhibitor of both PD-L1 / PD-L2 and VISTA.

[0224] Additional examples of immune checkpoint modulating agents that target negative regulators of the immune system response against cancer cells can include agents capable of inhibiting or blocking engagement / interaction with Cytotoxic T-lymphocyte- associated antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3, CD223), T cell immunoglobulin-3 (TIM-3), T cell immunoglobulin and ITIM domain (TIGIT), and B7 homolog 3 (B7 / H3).

[0225] For example, an immune checkpoint modulating agent targeting CTLA-4 can include the anti-CTLA-4 antibodies Tremelimunab, BMS-986249, and Ipilimumab, which is approved for the treatment of advanced or unresectable melanoma. Agents targeting LAG-3 can include the IMP321 fusion protein and monoclonal antibodies targeting LAG-3, such as Relatlimab or LAG525. An agent targeting TIM-3 can include the anti-TIM-3 monoclonalantibody MBG453. An agent targeting TIGIT can include the anti-TIGIT monoclonal antibody OMP-31M32. Agents targeting B7-H3, also known as CD276, can include Enoblituzumab (MGA271) which is an engineered Fc humanized IgGl monoclonal antibody against B7-H3, the humanized DART protein MGD009, and 8H9 which is an antibody against B7-H3 labeled with radioactive iodine (1-131) which, after internalization, promotes cancer cell death.

[0226] In some embodiments, the immune checkpoint modulating agents can include a positive regulator of the immune system response against cancer cells. In some embodiments, immune checkpoint modulating agents that act as positive regulators of the immune system response against cancer cells can include 0X40 agonistic agents. In one embodiment, an 0X40 agonistic agent can include a monoclonal antibody capable of promoting the engagement / interaction of 0X40 with OX40L ligand to promote the NF-KB signaling pathway and T cell clonal expansion and activation. 0X40 agnostic agents for use in a method described herein can include, but are not limited to, MED16368 fusion protein, MEDI0562, MEDI6469, BMS986178, Pf-04518600 (PF-8600), GSK3174998 and MOXR0916.

[0227] Additional immune checkpoint modulating agents for use in a method described herein can include agonistic agents targeting positive regulators of the immune system response against cancer cells such as, but not limited to, Inducible co-stimulator (ICOS), Glucocorticoid-induced TNF receptor family-related protein (GITR), 4-1BB, CD27 / CD70 pathway, and CD40. GITR agonists can include TRX-518, an aglycosylated human mAb, BMS-986156, AMG 228, MEDI1873, MK-4166, INCAGN01876, and GWN323. ICOS agonists can include JTX-2011, GSK3359609, and MEDI-570. 4-1BB (CD137) agonists can include Utomilumab (PF-05082566) and Urelumab. Agonists of the CD27 / CD70 pathway can include ARGX-110, BMS-936561 (MDX-1203), and Varlilumab. CD40 agonists can include CP-870893, APX005M, ADC-1013, lucatumumab, Chi Lob 7 / 4, dacetuzumab, SEA- CD40, and R07009789 monoclonal antibodies.

[0228] Additional exemplary immune checkpoint modulating agents can include, but are not limited to, agents capable of inhibiting or blocking engagement / interaction with adenosine A2a receptor (A2aR), CD73, B and T cell lymphocyte attenuator (BTLA, CD272), or non-T cell-associated inhibitory molecules such as transforming growth factor P(TGF-P),Killer immunoglobulin-like receptors (KIRs, CD158), Phosphoinositide 3-kinase gamma (PI3Ky), and CD47 (integrin-associated protein).

[0229] Further immune checkpoint modulating agents can include molecules targeting tumor microenvironment components like Indoleamine 2,3-dioxygenase (IDO), Toll-like receptors (TLRs), IL2R, as well as arginase inhibitors such as CB-1158 or oncolytic peptides, such as LTX-31 . For example, agents targeting (IDO) can include BMS-986205, and Indoximod, and the oral agent epacadostat. Agents targeting TLRs for use as an immune checkpoint modulating agent in a method described herein can include MEDI9197, PG545 (pixatimod, pINN), and Poly inosinic -poly cytidylic acid polylysine carboxy methylcellulose (poly-ICLC). For example, an IL-2R inhibitory agent can include NKTR-214 (bempeg) and an IL-10 inhibitory agent can include AM0010 (pegilodecakin).

[0230] The combinatorial cancer vaccine described herein, and compositions containing the same, and / or additional therapeutic agents may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical, and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art. For example, a suitable amount and dosing regimen is one that causes at least transient protein (e.g., enzyme) production.

[0231] The combinatorial cancer vaccine and / or additional therapeutic agents described herein can be administered via intravenous delivery, subcutaneous delivery, oral delivery, subdermal delivery, ocular delivery, intratracheal injection pulmonary delivery (e.g., nebulization), intramuscular delivery, intrathecal delivery, or intraarticular delivery.

[0232] Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, or intranasal.

[0233] In some embodiments, the combinatorial cancer vaccine and / or additional therapeutic agents is administered by intravenous delivery. In some embodiments, the combinatorial cancer vaccine and / or additional therapeutic agents is administered by subcutaneous delivery. In some embodiments, the combinatorial cancer vaccine and / or additional therapeutic agents is administered by oral delivery. In some embodiments, the combinatorial cancer vaccine and / or additional therapeutic agents is administered by subdermal delivery.

[0234] Alternatively or additionally, the combinatorial cancer vaccine and / or additional therapeutic agents described herein may be administered in a local rather than systemic manner, for example, via injection of the combinatorial cancer vaccine and / or additional therapeutic agents directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted. For example, aerosols containing the combinatorial cancer vaccine and / or additional therapeutic agents can be inhaled (for nasal, tracheal, or bronchial delivery); the combinatorial cancer vaccine and / or additional therapeutic agents can be injected into the site of injury, disease manifestation, or pain, for example; the combinatorial cancer vaccine and / or additional therapeutic agents can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection. Formulations containing the combinatorial cancer vaccine and / or additional therapeutic agents can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.

[0235] Provided methods contemplate single as well as multiple administrations of a therapeutically effective amount of the combinatorial cancer vaccine and / or additional therapeutic agents described herein. Therapeutic agents can be administered at regular intervals, depending on the nature, severity and extent of the subject's condition. In some embodiments, a therapeutically effective amount of the combinatorial cancer vaccine and / or additional therapeutic agents may be administered intrathecally periodically at regular intervals (e.g., once every year, once every six-months, once every five-months, once every three-months, bimonthly (once every two-months), monthly (once every month), biweekly(once every two-weeks), twice a month, once every 30-days, once every 28-days, once every 14-days, once every 10-days, once every 7-days, weekly, twice a week, daily, or continuously).

[0236] In some embodiments, the combinatorial cancer vaccine and / or additional therapeutic agents are formulated such that they are suitable for extended-release of the TLR agonist contained therein. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. For example, in one embodiment, the compositions of the present invention are administered to a subject twice a day, daily, or every other day. In a preferred embodiment, the compositions of the present invention are administered to a subject twice a week, once a week, once every 7-days, once every 10-days, once every 14-days, once every 28-days, once every 30-days, once every two-weeks, once every three-weeks, or more-preferably once every four-weeks, once-a-month, twice-a-month, once every six-weeks, once every eight-weeks, once every other month, once every three- months, once every four-months, once every six-months, once every eight-months, once every nine-months, or annually. Also contemplated are the combinatorial cancer vaccine and / or additional therapeutic agents that are formulated for depot administration(e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release TLR agonists over extended periods of time. Preferably, the extended-release means employed are combined with modifications made to enhance stability.

[0237] As used herein, the term “therapeutically effective amount” is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject e.g., treating, modulating, curing, preventing and / or ameliorating a disease or disorder). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. Generally, the amount of a therapeutic agent e.g., mRNA) administered to a subject in need thereof will depend upon the characteristics of the subject. Such characteristics include the condition, disease severity, general health, age, sex and body weight of the subject. One of ordinary skill in the art will be readily able to determine appropriate dosages depending on these and other related factors. In addition, both objective and subjective assays may optionally be employed to identify optimal dosage ranges.

[0238] A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0239] In some embodiments, the therapeutically effective dose ranges from about 0.005 mg / kg body weight to 500 mg / kg body weight, e.g., from about 0.005 mg / kg body weight to 400 mg / kg body weight, from about 0.005 mg / kg body weight to 300 mg / kg body weight, from about 0.005 mg / kg body weight to 200 mg / kg body weight, from about 0.005 mg / kg body weight to 100 mg / kg body weight, from about 0.005 mg / kg body weight to 90 mg / kg body weight, from about 0.005 mg / kg body weight to 80 mg / kg body weight, from about 0.005 mg / kg body weight to 70 mg / kg body weight, from about 0.005 mg / kg body weight to 60 mg / kg body weight, from about 0.005 mg / kg body weight to 50 mg / kg body weight, from about 0.005 mg / kg body weight to 40 mg / kg body weight, from about 0.005 mg / kg body weight to 30 mg / kg body weight, from about 0.005 mg / kg body weight to 25 mg / kg body weight, from about 0.005 mg / kg body weight to 20 mg / kg body weight, from about 0.005 mg / kg body weight to 15 mg / kg body weight, from about 0.005 mg / kg body weight to 10 mg / kg body weight.

[0240] In some embodiments, the therapeutically effective dose is greater than about 0.1 mg / kg body weight, greater than about 0.5 mg / kg body weight, greater than about 1.0 mg / kg body weight, greater than about 3 mg / kg body weight, greater than about 5 mg / kg body weight, greater than about 10 mg / kg body weight, greater than about 15 mg / kg body weight, greater than about 20 mg / kg body weight, greater than about 30 mg / kg body weight, greater than about 40 mg / kg body weight, greater than about 50 mg / kg body weight, greater than about 60 mg / kg body weight, greater than about 70 mg / kg body weight, greater than about 80 mg / kg body weight, greater than about 90 mg / kg body weight, greater than about100 mg / kg body weight, greater than about 150 mg / kg body weight, greater than about 200 mg / kg body weight, greater than about 250 mg / kg body weight, greater than about 300 mg / kg body weight, greater than about 350 mg / kg body weight, greater than about 400 mg / kg body weight, greater than about 450 mg / kg body weight, greater than about 500 mg / kg body weight. In a particular embodiment, the therapeutically effective dose is 1.0 mg / kg. In some embodiments, the therapeutically effective dose of 1.0 mg / kg is administered intramuscularly or intravenously.

[0241] The following examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto Example 1Development of a cancer vaccine

[0242] The vaccine includes 3 major components: a TLR 7 and TLR8 agonist, resiquimod (R848); a TLR 9 agonist, CpG-oligonucleotides C-type; and short Kras targeting peptide, KRAS G12D. Components were mixed using pipette mechanical action. As illustrated in Fig. 1, the vaccine works by the following mechanism: KRAS G12D peptide targets the KRAS mutation, G12D. KRAS mutations occur in 90% of PDAC tumors and G12D is a common mutation that occurs in 35-40% of all PDACs, with some estimates as high as 50%. The G12D peptide targets the glycine-12 (G12) mutation and R848 and CpG- ODN are internalized by endosomes / lysosomes. Once they reach the late endosome / lysosome they interact with the intercellular TLR-7 / 8 / 9. CpG-ODN then stimulate an innate and adaptive immune response by binding to the TLR and stimulate B cells to secret IL-6 and pDCs as well as to produce IFN-alpha. R848 similarly, induces a pathogen associated molecular pattern and results in the induction of cytokines, and specifically IFN- alpha and IL- 12 production. When tested in vivo using PDAC mice bearing orthotopic KPC tumors we were able to determine the efficacy of our cancer vaccine through prolonged survival, reductions in tumor size (compared to i.p. PBS dosed controls) and expression of anti-tumor mRNA.Nanoparticle vaccine

[0243] A unique capability of ECO lipid nanoparticles (ELNP) is the pH-sensitive amphiphilicity of the lipid ECO, which facilitates pH-sensitive amphiphilic endosomal escape. In addition, the nanoparticles are stabilized by the disulfide cross-links that areformed via autooxidation of thiols on ECO during the ELNP formulation. Consequently, ELNP of siRNA or miRNA is stable and safe for systemic administration for cancer therapy. The disulfide bonds in ELNP can be reduced by the high concentration of intracellular glutathione (-15 pM) to allow dissociation of ELNP and release of the CpG+R848 into the cytosol of target cells to stimulate anti-cancer immunity.

[0244] Fig. 2 shows a K-RasG12Dpeptide (KLVVVGADGVGKSALTT) (SEQ ID NO: 3) can be conjugated to a PEG spacer (MW = 3,400 Da) by reacting the 3-amine of the lysine residue at N-terminal with the active ester of a PEG derivative with a maleimide group (I) to give G12D-PEG-MAL. The 5-amine has a low steric hindrance and can specifically conjugate to the PEG spacer without affecting the function of the peptide vaccine. PEG is a biocompatible synthetic polymer and has commonly been used to modify nanoparticle surface to minimize non-specific uptake in normal tissues and to enhance the uptake in the tumors and immune cells. Next, G12D-PEG-MAL modified CpG / R848-ELNP (G12D- CpG / R848-ELNP) is prepared using the method for RGD modified ECO / miR-200c nanoparticles. Briefly, G12D-PEG-MAL in RNase-free water first reacts with one of the thiol groups of a small portion (< 3 mol-%) of ECO molecules in ethanol for 30 min under gentle agitation, which is subsequently mixed with a predetermined amount of R848, then CpG oligonucleotides in RNase- free water at the N / P ratio of 8 or 10 by 2-min vortexing. This is followed by gentle agitation for an additional 30 min with sufficient aeration to allow autoxidation of the remaining thiols of ECO to form disulfide cross-links to stabilize the ELNP. The final volume of ethanol is controlled to be less than 5% of the total volume of final ELNP formulation. Free peptide derivative is removed from G12D-CpG / R848-ELNP by ultrafiltration (Nanosep, MWCO = 100 KDa, 5000 g).

[0245] ELNPs carrying RGD model peptide and CpG were prepared using methods described above. Gel electrophoresis shows complete encapsulation of CpG in RGD-CpG- ELNP, RGD-CpG / R848- ELNP, and reference ECO / CpG / R848 with no free CpG (Fig. 3). The size of RGD-CpG-ELNP, RGD-CpG / R848-ELNP, and ECO / CpG / R848 is 83+23, 76+21, and 76+19 nm, respectively. This result demonstrates the feasibility to generate ELNP vaccine.Example 2

[0246] In a pilot experiment, C57BL / 6 mice bearing orthotopic KPC1242 (LSL- KrasG12D:LSL-Trp53R172H:Pdxl-Cre) PDAC were treated by i.v. injecting the nanovaccine(5 g CpG + 8.5pg R848 + 20pg G12D peptide per mouse), cocktail or batch vaccine (20 pg CpG + 25pg R848 + 20 pg G12D per mouse, positive control), or PBS on day 10 and day 17 after tumor initiation. Approximately 70% mice had complete tumor rejection, while the cocktail vaccine showed less therapeutic efficacy in this strain of KPC1242 PDAC model with only 20% disease-free survival, Fig. 4. Compared with the KPC-K8484 model, the cocktail vaccine resulted in lower disease-free survival in this KPC1242 model. We speculate that this is due to the heterogeneity of the tumor microenvironment, as these two syngeneic cell line models were obtained from different sources. Altogether, these results support our hypothesis that the ELNP nanovaccine is therapeutically more effective than the cocktail vaccine formulation.

[0247] Rechallenge experiments showing immune memory in mice completely responded to nanovaccines. We demonstrated that the long-term immune memory of the C57BL / 6 mice bearing orthotopic KPC-K8484 pancreatic cancer. The tumor bearing mice were treated by intraperitoneal injections of nanovaccines. MT218-MRM1 revealed tumor eradication in over 50% of the tumor-bearing mice treated with the nanovaccine, Fig. 5 A. The mice showed complete tumor-free response after the treatment with the ELNP nanovaccine targeting the KRASG12Dwere injected with KPC-K8484 cancer cells. Naive C57BL / 6 mice received no treatment were used as a control. The mice in both groups were age matched to prevent age based effects on tumor growth. MRMI detected no tumor recurrence in the treated mice, whereas large tumors formed in the control group, Fig. 5B. Fig. 6 shows the survival curve of the mice after rechallenging of the mice with cancer cells. These results indicate that the proposed ELNP nanovaccines have the potential to induce effective, long-term, target-specific anti-tumor immunity, eradicating target tumors and preventing tumor recurrence.Example 3MRMI of fibronectin can be used to evaluate efficacy of a pro-inflammatory nanovaccine adjuvant

[0248] A vaccine adjuvant (i.e., combinatorial cancer vaccine or nanovaccine) was created using a mixture of TLR agonists and a mKRAS targeting peptide. Vaccine was produced both as a batch for i.p. injections, s.q. injections and as a nanoparticle encapsulated in ECO for systemic delivery. The batch solution combined CpG-ODN (5’-tccatgacgttcctgacgtt-3’ (SEQ ID NO: 39) (20 mer)), R848 and G12D peptide. Vaccine nanoparticles were formulated at an N / P ratio of 8 with synthetic CpG-ODN, a murine TLR9 agonist. R848 for solubilized in ethanol and combined with self- assembling ECO and CpG- ODN at a concentration ratio of 1: 1. Briefly, the ECO stock solution (50 mM in ethanol) and CpG-ODN stock solution (1 mg / mL) at predetermined amounts based on the N / P ratio were mixed and vortexed for 20 min at room temperature. Either RGD-PEG-MAL or ZD2-PEG- MAL (0.625 mM) was added to the ECO / mRNA nanoparticle complex solution at 2.5 mol% for PEGylation. The mixture was vortexed for at least 30 min allowing complete PEGylation. Complexation was confirmed by agarose gel electrophoresis, whereby particles were run in a 1% agarose gel at 100 V for 20 mins. Size and zeta potential were determined using dynamic light scattering (DLS) with an Anton Paar Litesizer 500. Vaccine adjuvant was measured immediately after formulation.

[0249] Figs. 7(A-D) illustrate (A) Agarose gel encapsulation with 3 nanoparticle formulations: NP1 : CpG / ECO-PEG-RGD, NP2: CpG-R848 / ECO and NP3: CpG-R848 / ECO- PEG-RGD. (B-D) size distribution of particles measured by DLS. (E) Free CpG in solution vs nanoparticle formulations.

[0250] Fig. 8 illustrates size and zeta-potentials of ECO / CpG-ODN nanoparticle formulations with different targeting moieties.

[0251] 6-week-old male and female immunocompetent C57BL / J6 mice were used for in vivo experimentation and housed in the Animal Facility at CWRU, as per protocols approved by IACUC. These mice were split into groups for subcutaneous injection and orthotopic implantation. Mice had tumors initiated with KPC cells in a 1 : 1 Matrigel mixture. Prior to the start of treatment, mice were imaged at day 10 following recovery from surgery and once tumors had reached sizes of 100-150 mm3. IP injection of vaccine was conducted at days 10 (after imaging) and 17. Mice were imaged again at days 27 and 45, to determine the short- and longer-term effects of vaccine therapy. Each mouse was imaged in 2D using (1) fast-spin echo sequences in the coronal and axial planes, (2) dynamic contrast enhancement sequences in the axial plane and (3) T1 mapping. Additionally, 3D scans were performed using a FLASH sequence. Mice were scanned in 10 min increments to determine contrast agent effects over time. Analysis was conducted on scans to determine signal intensity, contrast-to-noise ratios, signaLto-noise ratios, parameters (e.g., rate of dissociation and washin) and features for extraction over each scan timepoint and longitudinally through the study.Animal were euthanized after day 45 and tissues were extracted. Acquired tissue samples were then used for qRT-PCR, Western Blotting, flow cytometry, and histochemical staining.

[0252] Results from in vivo therapy showed that vaccine therapy reduced overall tumor size and had two distinct effects on tumor composition, which could be detected by MRMI. First, respondent mice had a decreased tumor burden as indicated by impeded tumor growth. Notably, in MRMT scans there was increased signal in the tumors, suggesting an increase in scaffolding proteins in the tumor including fibronectin. Histopathology using immunohistochemistry of an EDB-FN specific antibody, G4, corroborated this result. Second, mice that had larger tumor sizes >300 mm3at the termination of the study had weaker signals and less strong staining for G4 in fixed tissue samples. In both cases, MRMI at an earlier timepoint, day 27 (17 days after treatment start) MRMI was able delineate between a more and less responsive tumor through MT218 accumulation and consequently a change in signal intensity.Example 4MRMI with a fibronectin targeting contrast agent can be used in tandem with combinational mAb immunotherapy for single timepoint and longitudinal imaging

[0253] Lastly, a combination of immunotherapies including immune checkpoint inhibitors and / or vaccine adjuvants can be used, and all elicit different signal patterns.

[0254] As described above, C57J / B6 mice were implanted orthotopically and dosed with vaccine adjuvant on day 10 and 17. Additionally, mice were treated with VISTA mAb (200ug) starting on day 20 every 2 days for a total of 12 doses. Mice were similarly imaged in 2D, and 3D as described above with MT218 contrast.

[0255] MRMI of VISTA mAb and vaccine adjuvant treated mice also revealed two patterns that can be distinguished by responders and non-responders to therapy. Responders, where tumors abrogated had robust signal enhancement at day 27 with no signal and no evidence of adenocarcinoma at day 45. Furthermore, hematoxylin and eosin staining showed intact pancreatic tissue free from cancerous cells, indicating a complete pathological response, and thus corroborating the results from MRMI. Non-responders, who had tumors at the conclusion of the study had lower enhancement (2-fold CNR at 10 min) compared to VISTA responders (5-fold CNR at 10 min). The lower pattern of enhancement and signalintensity was also seen at day 45. IHC staining with G4 showed a more diffuse pattern of staining across the tissues.

[0256] Fig. 9 illustrates axial slices of mice over time. Mouse 1 shows a non-responder who developed a tumor similar to that of the i.p. saline controls. Mouse 2 had a major pathological response and the MRI demonstrates the associated size reduction. Mouse 3 shows cancer vaccine mouse with strong signal enhancement at days 10 and 27 but no signs of tumor at day 45. Mice 2 and 3 that are responders have higher signal (characterized by contrast-to-noise ratio) at day 27. Comparatively, mouse 3 with vaccine therapy has with less signal enhancement at 10 min within the tumor than responder mice 2 and 3.

[0257] Fig. 10 illustrates axial slices of mice over time. Mouse 1 shows cancer vaccine + VISTA mAb mouse with strong signal enhancement at days 10 and 27 but no signs of tumor at day 45. Mice 2 and 3 show tumors that have a partial response to therapy with less signal enhancement at 10 min within the tumor than responder mouse 1.

[0258] Fig. 11 illustrates tumor growth from 3D MRM1 scans over time in immunotherapy treated groups.

[0259] Fig. 12 illustrates survival proportions of groups treated with vaccine combination alone IP and PBS treated groups

[0260] Figs. 13(A-C) illustrate physical characterization of nanovaccine particlesA. encapsulation of 2 batches of nanovaccine in lanes 2 and 3 versus free CpG in lane 1.B. Nanovaccine size distribution as quantified by DLS by intensity weighted measurement.C. Charge distribution of nanovaccine via zeta-potential.

[0261] Figs. 14(A-C) illustrate A. MRMI of mice over time, left is saline controls before and after contrast administration, middle is vaccine and right is nanovaccine. Nanovaccine shows clear hinderance of tumor growth and reduction in signal at day 45 compared to control and soluble vaccine. B. Quantification of CNR at pre-contrast and postcontrast 15-minute timepoint. C. Tumor growth based on 2D and 3D imaging of tumors with MRMI.

[0262] Figs. 15(A-C) illustrate evaluation of nanovaccine efficacy in C57BL / 6 mice bearing orthotopic pancreatic tumors (red dashed lines). T1 -weighted contrast-enhanced MRI was performed at days 14 and 45 post-implantation. A. Nanovaccine administration (days 5 and 12) resulted in visible tumor suppression by day 14, with complete tumor regression observed at day 45. Control mice exhibited progressive tumor growth, withextensive abdominal cavity involvement by day 45. B. Final tumor and metastatic burden showed a trend toward significance between nanovaccine-treated and control groups. C. In survival studies, nanovaccine treatment extended median survival to 88 days compared to 66 days in controls, with 50% overall survival in the treatment group.

[0263] Figs. 16(A-C) illustrate evaluation of vaccine memory response in C57BL / 6 mice using subcutaneous pancreatic tumor rechallenge (white arrows, red dashed lines). Following initial orthotopic tumor treatment (day 75), mice received subcutaneous tumor implants. T2-weighted MRI monitoring occurred biweekly post-implantation. A.Nanovaccine-treated mice showed reduced tumor burden 30 days post-rechallenge, while controls exhibited progressive growth with peritoneal invasion by day 60 in surviving control mice. B. Terminal tumor and pancreas mass demonstrated highly significant differences between treatment groups. C. Survival analysis established 100% disease-free survival in nanovaccine-treated mice versus median survival of 50 days in controls.Summary

[0264] Fig. 17 illustrates plots and graph showing therapeutic outcomes of mice administered the vaccine.

[0265] Tumor growth is substantially higher in s.q. controls compared to mice rechallenged with s.q. PDAC tumors.

[0266] Rechallenged mice did not have intrapancreatic tumors nor s.q. tumor growth during the monitoring period

[0267] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.

Claims

Having described the invention, we claim:

1. A combinatorial cancer vaccine for cancer therapy comprising a lipid nanoparticle that includes: a) a plurality of pH sensitive protonatable or ionizable lipids having the structure of formula (T):wherein R1is an alkylamino group, a hydroxylalkyl group, or a group containing at least one aromatic group;R2and R3are independently an aliphatic group or a hydrophobic group;R4and R5are independently H, an alkyl group, an alkenyl group, an acyl group, or an aromatic group, or each R4or R5independently includes a polymer which is optionally linked to a targeting group or linked to at least one of a cancer antigen or neoantigen; a, b, c, and d are independently an integer from 1 to 10; and pharmaceutically acceptable salts thereof; b) at least one of a TLR7 agonist, TLR8 agonist, or a TLR9 agonist complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids, preferably a TLR7 / 8 and TLR9 agonist complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids; c) optionally a nucleic acid encoding a cancer antigen or neoantigen complexed or conjugated with and / or encapsulated by the pH sensitive protonatable or ionizable lipids; and d) optionally a stabilizing amount of at least one stabilizing polymer, polyethylene glycol or polysaccharide, or structural lipid that is conjugated to and / or complexed with the pH sensitive protonatable or ionizable lipids; andwherein at least one R4or R5includes a polymer linked to the cancer antigen or neoantigen and / or the lipid nanoparticle includes the nucleic acid encoding the cancer antigen or neoantigen.

2. The vaccine of claim 1, wherein the TLR9 agonist includes lefitolimod, tilsotolimod, or a cytidine-phosphate-guanosine (CpG) oligonucleotide.

3. The vaccine of claim 1 or 2, wherein the TLR7 agonist or TLR8 agonist comprises 4-amino-2-(ethoxymethyl)-a,a-dimethyl-lH-imidazo[4,5-c]quinoline-l-ethanol (R848), imiquimod, gardiquimod, their lipid derivatives, or mixtures thereof, preferably R848.

4. The vaccine of any of claims 1 to 3, including CpG ODN and R848 complexed with and / or encapsulated by the pH sensitive protonatable or ionizable lipids.

5. The vaccine of any of claims 1 to 4, wherein the neoantigen is a peptide encoded by a mutation of gene selected from ABL1, BRAF, CDKN1A, EPHA3, FGFR4, IKZF1, MCL1, NKX2-1, PMS2, RNF43, TET2, ACVR1B, BRCA1, CDKN1B, EPHB1, FH, INPP4B, MDM2, N0TCH1, POLDI, ROS1, TGFBR2, AKT1, BRCA2, CDKN2A, EPHB4, FLCN, IRF2, MDM4, N0TCH2, POLE, RPTOR, TIP ARP, AKT2, BRD4, CDKN2B, ERBB2, FLT1, IRF4, MED12, N0TCH3, PPARG, SDHA, TNFAIP3, AKT3, BRIP1, CDKN2C, ERBB3, FLT3, IRS2, MEF2B, NPM1, PPP2R1A, SDHB, TNFRSF14, ALK, BTG1, CEBPA, ERBB4, F0XL2, JAK1, MEN1, NRAS, PPP2R2A, SDHC, TP53, AL0X12B, BTG2, CHEK1, ERCC4, FUBP1, JAK2, MERTK, NT5C2, PRDM1, SDHD, TSC1, AMER1, BTK, CHEK2, ERG, GABRA6, JAK3, MET, NTRK1, PRKAR1A, SETD2, TSC2, APC, Cllorf30, CIC, ERRFI1, GATA3, JUN, MITF, NTRK2, PRKC1, SF3B1, TYR03, AR, CALR, CREBBP, ESRI, GATA4, KDM5A, MKNK1, NTRK3, PTCHI, SGK1, U2AF1, ARAF, CARD11, CRKL, EZH2, GATA6, KDM5C, MLH1, P2RY8, PTEN, SMAD2, VEGFA, ARFRP1, CASP8, CSF1R, FAM46C, GID4, (C17orf39), KDM6A, MPL, PALB2, PTPN11, SMAD4, VHL, ARID1A, CBFB, CSF3R, FANCA, GNA11, KDR, MRE11A, PARK2, PTPRO, SMARCA4, WHSCI, ASXL1, CBL, CTCF, FANCC, GNA13,KEAP1, MSH2, PARP1, QKI, SMARCB1, WHSC1L1, ATM, CCND1, CTNNA1, FANCG, GNAQ, KEL, MSH3, PARP2, RAC1, SMO, WT1, ATR, CCND2, CTNNB1, FANCL, GNAS, KIT, MSH6, PARP3, RAD21, SNCAIP, XPO1, ATRX, CCND3, CUL3, FAS, GRM3, KLHL6, MST1R, PAX5, RAD51, SOCS1, XRCC2, AURKA, CCNE1, CUL4A, FBXW7, GSK3B, KMT2A, (MLL), MTAP, PBRM1, RAD51B, SOX2, ZNF217, AURKB, CD22, CXCR4, FGF10, H3F3A, KMT2D, (MLL2), MTOR, PDCD1, RAD51C, SOX9, ZNF703, AXIN1, CD274, CYP17A1, FGF12, HDAC1, KRAS, MUTYH, PDCD1LG2, RAD51 D, SPEN, AXL, CD70, DAXX, FGF14, HGF, LTK, MYC, PDGFRA, RAD52, SPOP, BAP1, CD79A, DDR1, FGF19, HNF1A, LYN, MYCL, PDGFRB, RAD54L, SRC, BARD1, CD79B, DDR2, FGF23, HRAS, MAF, MYCN, PDK1, RAFI, STAG2, BCL2, CDC73, DIS3, FGF3, HSD3BI, MAP2KI, MYD88, PIK3C2B, RARA, STAT3, BCL2L1, CDH1, DNMT3A, FGF4, ID3, MAP2K2, NBN, P1K3C2G, RBI, STK11, BCL2L2, CDK12, DOT1L, FGF6, IDH1, MAP2K4, NF1, PIK3CA, RBM10, SUFU, BCL6, CDK4, EED, FGFR1, 1DH2, MAP3K1, NF2, P1K3CB, REL, SYK, BCOR, CDK6, EGFR, FGFR2, IGF1R, MAP3K13, NFE2L2, PIK3R1, RET, TBX3, BCORL1, CDK8, EP300, FGFR3, IKBKE, MAPK1, NFKBIA, PIM1, RICTOR, TEK, BCR, CD74, ETV4, ETV5, ETV6, EWSR1, EZR, MYB, NUTM1, RSPO2, SDC4, SLC34A2, TERC, TERT, and TMPRSS2.

6. The vaccine of any of claims 1 to 5, wherein the neoantigen comprises at least one of KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12S, KRAS G12V, KRAS G13D, or KRAS G13C.

7. The vaccine of any of claims 1 to 6, wherein the targeting group includes at least one of immune cell or cancer cell targeting peptide.

8. The vaccine of any of claims 1 to 7, wherein R1comprises at least one of:- (CH2)eNR6R7.- (CH2)fN(CH2)gNR9R10r8; orare each independently hydrogen, an alkyl group, a hydrophobic group, a nitrogen containing substituent, or an oxygen containing substituent; and e, f, g, i, j, k, 1, and m are an integer from 1 to 10.

9. The vaccine of any of claims 1 to 8, wherein R1comprises at least one of CH2CH2NH2, CH2CH2OH, CH2CH2OCH2CH2OH, CH2CH2OCH2CH2NH2, CH2CH2NHCH2CH2NHCH2CH2NH2, or CH2CH2NHCH2CH2CH2CH2NHCH2CH2CH2NH2.

10. The vaccine of any of claims 1 to 9, wherein R2and R3are each independently a saturated alkyl with long or branched chains, and or a fatty acid hydrophobic group derived from oleic acid or linoleic acid.

11. The vaccine of any of claims 1 to 10, at least one of R4or R5includes the polymer linked to the cancer antigen or neoantigen.

12. The vaccine of any of claims 1 to 10, wherein the pH sensitive protonatable or ionizable lipids are selected from:polyethylene glycol (PEG) modified lipids thereof, dextran modified lipids thereof, or combinations thereof.

13. The vaccine of claim 12, wherein the plurality of pH sensitive protonatable or ionizable lipids include a plurality of ECO and / or ECLn lipids and a plurality of ECLn and / or ECO modified with PEG linked to the cancer antigen or neoantigen.

14. The vaccine of any of claims 1 to 13, comprising a plurality the lipid nanoparticles.

15. A method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective or prophylactic amount of a vaccine of any of claims 1 to 14.

16. The method of claim 15, further comprising administering at least one immune checkpoint inhibitor in combination with the vaccine.

17. The method of clam 16, wherein the immune checkpoint inhibitor is an inhibitor for any of immune checkpoint molecules selected from the group consisting of PD- 1, CTLA-4, TIM-3, BTLA, LAG-3, A2aR, KIR, VISTA, TIGIT, PD-L1 PD-L2, CD80, CD86, GAL-9, HVEM, CD160, MHC class II, B7-H3, B7-H4, B7-H5. B7-H6, and B7-H7, or a combination of two or more inhibitors therefor.

18. The method of claim 16 or 17, wherein the immune checkpoint inhibitor is selected from an antibody against the immune checkpoint molecule, an antigen-binding fragment of the antibody, or a combination thereof.

19. The method of any of claims 16 to 18, wherein the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, avelumab, atezolizumab, and durvalumab.

20. The method of any of claims 15 to 19, wherein the cancer is selected from colorectal cancer, breast cancer, lung cancer, melanoma, hepatoma, head and neck cancers, squamous cell carcinomas of the lung, ovarian cancer, uterine cancer, prostate cancer, gastric carcinoma, cervical cancer, esophageal carcinoma, bladder cancer, kidney cancer, brain cancer, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular malignant melanoma, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin’s lymphoma, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, adrenal gland cancer, sarcoma of soft tissue, urethra cancer, penis cancer, chronic or acute leukemias solid tumors of childhood, lymphocytic lymphoma, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, medulloblastoma pilomatrixomas, endometrial cancer, multiple myeloma, or T-cell lymphoma.

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