Pancreatic cancer immunotherapy using cationic lipid and polymer nanocarriers for co-delivery of KRAS neoantigens and sting and / or other immunomodulators to boost the cancer immunity cycle and treat liver metastasis

Abstract

Drug delivery nanoparticle compositions and methods for the treatment of a cancer are provided that can be used individually or in combination. These compositions and methods include: (1) exogenous vaccination using neoantigens or tumor-associated antigens to potentiate an anti-tumor immune response; (2) nanoparticle-based drug delivery targeting the liver, aimed at reprogramming the hepatic immune environment to reduce or eliminate tumor antigen tolerance, thereby enhancing the immune system's ability to combat metastatic cancer; and (3) expansion of the liver metastasis treatment platform to target tumor-associated lymphoid structures, supporting the immune response at the primary tumor site. Any of these methods may be optionally combined with chemo-immunotherapy for enhanced efficacy.

Description

P-635332-PC PANCREATIC CANCER IMMUNOTHERAPY USING CATIONIC LIPID AND POLYMER NANOCARRIERS FOR CO-DELIVERY OF KRAS NEOANTIGENS AND STING AND / OR OTHER IMMUNOMODULATORS TO BOOST THE CANCER IMMUNITY CYCLE AND TREAT LIVER METASTASIS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application serial number 63 / 543,705, filed October 11, 2023, which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENTAL SUPPORT

[0002] This invention was made with government support under CA247666 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing conforming the rules of WIPO Standard ST.26 which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on October 7, 2024, is named P-635332-PC_SQL_ST26_07OCT24.xml and is 109,507 bytes in size. BACKGROUND

[0004] Pancreatic ductal adenocarcinoma (PDAC) represents a significant challenge in the field of oncology, ranking as the twelfth most common cancer worldwide with 495,000 new cases in 2020. In the United States, it’s the fourth leading cause of cancer-related deaths where patients diagnosed with PDAC face an alarming median survival of less than six months, and the five-year survival rate remains distressingly in the single-digit range. One of the most challenging aspects of PDAC is its tendency to be detected at an advanced stage, often limiting the feasibility of surgical intervention, which is crucial for successful treatment of other cancer types. Consequently, patients are frequently left with limited treatment options. Despite extensive research efforts, the response to traditional chemotherapy or immunotherapy, including the use of checkpoint blocking antibodies, has been disappointing. Over the years, several investigators, including our own laboratory, have introduced several nanotechnology-enabled strategies for the treatment of PDAC, aimed at improving drug delivery by nanocarriers that provide improved pharmacokinetics and the use of single or synergistic drug combinations that trigger cytotoxic cell death and / or immunogenic cell death (ICD) for chemo-immunotherapy strategies.

[0005] The generation of ICD by select chemotherapeutic agents constitute a means of overcoming the low neoantigen burden in PDAC, making a major contribution to poor immunogenic immune landscapes in contrast to the immunogenic “hot” landscape scene non-small cell lung cancer, colon cancer, melanoma, etc. This constitutes the basis for introducing nanocarriers, such as the silicasome to deliver ICD-inducing chemo agents such as irinotecan or Pt-based agents to boost the immune response.

[0006] ICD refers to tumor cell killing by these cancer drugs, such that the dying cancer cells display “eat me” signals for the APC, such as dendritic cells, appearing at the primary cancer site. This allows the full complement of endogenous tumor antigens in dying cancer cells to be taken up by the APC for transport to regional lymph nodes and the spleen, where cytotoxic T-cells (CTL) are educated and recruited back to the primary cancer site. This participation between the primary cancer site, and regional lymphoid tissue is also known as the cancer immunity cycle and is equivalent to an endogenous vaccination response. SUMMARY

[0007] In one aspect, a drug delivery vehicle is provided for reprogramming the tolerogenic liver immune landscape thereby reducing or eliminating tumor antigen tolerance and / or enhancing immune defense against metastatic spread, said vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle configured for preferential liver uptake; one or more STING agonists; and a nucleic acid encoding one or more tumor neoantigens or encoding one or more tumor associated antigens, or one or more a tumor neoantigens or one or more tumor-associated antigens.

[0008] In one embodiment, the nanoparticle comprises a cationic lipid nanoparticle. In one embodiment, said cationic lipid is selected from the group consisting of dilinoleylmethyl-4 dimethyl aminobutyrate (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), didodecyl-dimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3- trimethylammonium propane chloride (DOTAP), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), C12-200 (Lipid 5), 3-(dimethylamino) propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa- 12,15-dienoate (DMAP-BLP), and (2Z)-non-2-en-1-yl 10-[(Z)-(1-methylpiperidin-4 yl)carbonyloxy]nonadecanoate (L101), dicetylphosphate-tetraethylenepentaamine-basedpolycation lipid, YSK05, YSK12-C4, YSK13-C4, and YSK15-C4, or any combination thereof.

[0009] In one embodiment, said lipid comprises a lipid composition that favors hepatic biodistribution. In one embodiment, said lipid comprises MC3, DSPC, Cholesterol, and PEG-DMG2000. In one embodiment, said lipid comprises in mol percent: MC350%, DSPC 10%, Cholesterol, 38.5%, and PEG-DMG20001.5%.

[0010] In one embodiment, the one or more STING agonists are selected from the group consisting of 2′,3′-cGAMP, 3′,3′-cGAMP, c-di-GMP, and c-di-AMP, Rp,Rp-2′,3′-c-di- AMPSS (ADU-S100), amidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'-Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'- cGAMP), 3',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), CMA, MK- 1454 and CRD5500, or any combination thereof. In one embodiment, said STING agonist is 2’,3’-cGAMP.

[0011] In one embodiment, said tumor neoantigen is selected from the group consisting of a mutant KRAS peptide, a mutant P53, or an immunogenic fragment thereof. In one embodiment, said tumor neoantigen comprises one or more KRAS mutants. In one embodiment, said tumor neoantigens comprises one or more KRAS mutants selected from among SEQ ID Nos:2-13, 15-19, 22-24, 27, 29, 30-36, 50, 52-56, 74, or 81-86. In one embodiment, said one or more KRAS mutants comprise G12D.

[0012] In one embodiment, said drug delivery vehicle comprises a nucleic acid encoding said one or more tumor neoantigens such as SEQ ID Nos:37-43, 57, 59-63 or 77. In one embodiment, said nucleic acid encodes an MHC-I targeting signal. In one embodiment, said nucleic acid encodes an MHC-II targeting signal.

[0013] In one embodiment, said drug delivery vehicle comprises a tumor associated antigen comprising MUC1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof, ERBB2 or an immunogenic fragment therein, carcinoembryonic antigen (CEA) or an immunogenic fragment thereof, surviving or an immunogenic fragment thereof, VEGF receptor (VEGFR) or an immunogenic fragment thereof, and human telomerase reverse transcriptase and (HTRT) or an immunogenic fragment thereof. In one embodiment, said drug delivery vehicle comprises a tumor associated antigen that comprises or consists of an amino acid sequence of SEQ ID Nos:44-49, 65-71 or 73.

[0014] In one embodiment, said drug delivery vehicle comprises a nucleic acid encoding said one or more tumor associated antigens. In one embodiment, said nucleic acid encodes an MHC-I targeting signal. In one embodiment, said nucleic acid encodes an MHC-II targeting signal.

[0015] In one embodiment, said drug delivery vehicle further comprises a TLR agonist. In one embodiment, the TLR agonist is a TLR7 / 8 agonist. In one embodiment, said TLR7 / 8 agonist is a lipid compatible or lipidated TLR7 / 8 agonist. In one embodiment, said TLR7 / 8 agonist selected from the group consisting of resiquimod (R848), imiquimod, 852A (PF- 4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD-8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX-2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD-8848) and lipidated selgantolimod (GS-9688).

[0016] In one embodiment, said nanoparticle further comprises an inducer of immunogenic cell death (ICD inducer). In one embodiment, said ICD inducer is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

[0017] In one embodiment, the drug delivery vehicle comprises a STING agonist and a TLR7 / 8 agonist. In one embodiment, the drug delivery vehicle comprises 2’,3’-cGAMP and resiquimod.

[0018] In one aspect, a drug delivery vehicle is provided for enhancing an anti-tumor immune response by targeting tumor-associated lymphoid structures, said drug delivery vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle that comprises a lipid composition and / or targeting molecules that facilitate uptake by lymphoid organs, including lymph nodes, spleen and / or tertiary lymphoid structures; one or more STING agonists; and one or more a tumor associated antigens or tumor neoantigens or a nucleic acid encoding one or more a tumor associated antigens or tumor neoantigens.

[0019] In one embodiment, said nanoparticle comprises a cationic lipid nanoparticle. In one embodiment, said cationic lipid is selected from the group consisting of dilinoleylmethyl-4 dimethyl aminobutyrate (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylaminopropane(DODAP), didodecyl-dimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3- trimethylammonium propane chloride (DOTAP), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), C12-200 (Lipid 5), 3-(dimethylamino) propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa- 12,15-dienoate (DMAP-BLP), and (2Z)-non-2-en-1-yl 10-[(Z)-(1-methylpiperidin-4 yl)carbonyloxy]nonadecanoate (L101), dicetylphosphate-tetraethylenepentaamine-based polycation lipid, YSK05, YSK12-C4, YSK13-C4, and YSK15-C4, or any combination thereof.

[0020] In one embodiment, said lipid comprises MC3, DSPC, Cholesterol, and PEG- DMG2000. In one embodiment, said lipid comprises in mol percent: MC350%, DSPC 10%, Cholesterol, 38.5%, PEG-DMG20001.5%. In one embodiment, said lipid comprises DODAP, DOPE, Cholesterol, and PEG-DMG2000. In one embodiment, said lipid comprises in mol percent: DODAP 18.5%, DOPE 60%, Cholesterol, 20%, and PEG- DMG20001.5%.

[0021] In one embodiment, said one or more STING agonists are selected from the group consisting of 2′,3′-cGAMP, 3′,3′-cGAMP, c-di-GMP, and c-di-AMP, Rp,Rp-2′,3′-c-di- AMPSS (ADU-S100), amidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'-Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'- cGAMP), 3',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), CMA, MK- 1454 and CRD5500, or any combination thereof. In one embodiment, said delivery vehicle comprises the STING agonist 2’,3’-cGAMP.

[0022] In one embodiment, said drug delivery vehicle comprises a tumor neoantigen selected from the group consisting of a mutant KRAS peptide, a mutant P53, or an immunogenic fragment thereof. In one embodiment, said neoantigens comprise one or more KRAS mutants. In one embodiment, said KRAS mutant is a peptide selected from among SEQ ID Nos: 2-13, 15-19, 22-24, 27, 29, 30-36, 50, 52-56, 74, or 81-86. In one embodiment, said one or more KRAS mutants comprise G12D.

[0023] In one embodiment, said nanoparticle comprises a nucleic acid encoding said one or more neoantigens such as SEQ ID NOs:37-43, 57, 59-63 or 77. In one embodiment, saidnucleic acid encodes an MHC-I targeting signal. In one embodiment, said nucleic acid encodes an MHC-II targeting signal.

[0024] In one embodiment, said drug delivery vehicle comprises a tumor associated antigen comprising MUC1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof, ERBB2 or an immunogenic fragment therein, carcinoembryonic antigen (CEA) or an immunogenic fragment thereof, surviving or an immunogenic fragment thereof, VEGF receptor (VEGFR) or an immunogenic fragment thereof, and human telomerase reverse transcriptase and (HTRT) or an immunogenic fragment thereof. In one embodiment, said drug delivery vehicle comprises a tumor associated antigen that comprises or consists of an amino acid sequence of SEQ ID Nos:44-49, 65-71 or 73.

[0025] In one embodiment, said drug delivery vehicle further comprises targeting ligands that facilitate uptake by lymphoid organs, including lymph nodes, spleen and tertiary lymphoid structures. In one embodiment, said delivery vehicle comprises a targeting ligand that binds to a mannose receptor. In one embodiment, said targeting ligand comprises a mannose targeting ligand or a complex oligomannoside. In one embodiment, said drug delivery vehicle comprises a cationic lipid that shows preferential distribution to a lymphoid organ. In one embodiment, said nanoparticle comprises dioleoyl-3-trimethylammonium propane (DOTAP) and / or dioleoyl-dimethylammonium propane (DODAP), and optionally dioleoylphosphatidylethanolamine (DOPE).

[0026] In one embodiment, said drug delivery vehicle further comprises a TLR agonist. In one embodiment, said TLR agonist is a TLR7 / 8 agonist. In one embodiment, said drug delivery vehicle comprises a lipid compatible TLR7 / 8 agonist. In one embodiment, said drug delivery vehicle comprises a TLR7 / 8 agonist selected from the group consisting of resiquimod (R848), imiquimod, 852A (PF-4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD-8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX-2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD- 8848) and lipidated selgantolimod (GS-9688).

[0027] In one embodiment, said nanoparticle further comprises an inducer of immunogenic cell death (ICD inducer). In one embodiment, said ICD inducer is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

[0028] In one embodiment, the drug delivery vehicle of claim 29 comprises a STING agonist and a TLR7 / 8 agonist. In one embodiment, the drug delivery vehicle comprises 2’,3’-cGAMP and resiquimod.

[0029] In one embodiment, a method is provided for reprogramming the tolerogenic liver immune landscape to reduce or eliminate tumor antigen tolerance, leading to lack if immune prevention against metastatic spread, said method comprising administering to a subject in need thereof a drug delivery vehicle according to the above aspect and any of its embodiments.

[0030] In one aspect. a drug delivery vehicle is provided for enhancing an anti-tumor immune response by targeting tumor-associated lymphoid structures, said drug delivery vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle that comprises a lipid composition and / or targeting molecules that facilitate uptake by lymphoid organs, including lymph nodes, spleen and / or tertiary lymphoid structures; one or more TLR7 / 8 agonists; and one or more a tumor associated antigens or tumor neoantigens, or a nucleic acid encoding one or more a tumor associated antigens or tumor neoantigens.

[0031] In one embodiment, said nanoparticle comprises a cationic lipid nanoparticle. In one embodiment, said cationic lipid is selected from the group consisting of cationic lipids selected from the group consisting of dilinoleylmethyl-4 dimethyl aminobutyrate (DLin- MC3-DMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), didodecyl- dimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), C12-200 (Lipid 5), 3- (dimethylamino) propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa-12,15- dienoate (DMAP-BLP), and (2Z)-non-2-en-1-yl 10-[(Z)-(1-methylpiperidin-4 yl)carbonyloxy]nonadecanoate (L101), dicetylphosphate-tetraethylenepentaamine-based polycation lipid, YSK05, YSK12-C4, YSK13-C4, and YSK15-C4, or any combination thereof.

[0032] In one embodiment, said lipid comprises MC3, DSPC, Cholesterol, and PEG- DMG2000. In one embodiment, said lipid comprises in mol percent: MC350%, DSPC10%, Cholesterol, 38.5%, PEG-DMG20001.5%. In one embodiment, said lipid comprises DODAP, DOPE, Cholesterol, and PEG-DMG2000. In one embodiment, said lipid comprises in mol percent: DODAP 18.5%, DOPE 60%, Cholesterol, 20%, and PEG- DMG20001.5%.

[0033] In one embodiment, said TLR7 / 8 agonist is a lipid compatible TLR7 / 8 agonist. In one embodiment, said TLR7 / 8 agonist is selected from the group consisting of resiquimod (R848), imiquimod, 852A (PF-4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD-8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX- 2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD-8848) and lipidated selgantolimod (GS-9688).

[0034] In one embodiment, said drug delivery vehicle comprises a tumor-associated neoantigen selected from the group consisting of a mutant KRAS peptide, a mutant P53, or an immunogenic fragment from one or more of these neoantigens. In one embodiment, said neoantigens comprise one or more KRAS mutants. In one embodiment, said one or more KRAS mutants are selected from SEQ ID Nos: 2-13, 15-19, 22-24, 27, 29, 30-36, 50, 52-56, 74, or 81-86. In one embodiment, said one or more KRAS mutants comprise G12D.

[0035] In one embodiment, said nanoparticle comprises a nucleic acid encoding said one or more neoantigens such as SEQ ID Nos:37-43, 57, 59-63 or 77. In one embodiment, said nucleic acid encodes an MHC-I targeting signal. In one embodiment, said nucleic acid encodes an MHC-II targeting signal.

[0036] In one embodiment, said drug delivery vehicle comprises a tumor associated antigen comprising MUC1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof, ERBB2 or an immunogenic fragment therein, carcinoembryonic antigen (CEA) or an immunogenic fragment thereof, surviving or an immunogenic fragment thereof, VEGF receptor (VEGFR) or an immunogenic fragment thereof, and human telomerase reverse transcriptase and (HTRT) or an immunogenic fragment thereof. In one embodiment, said drug delivery vehicle comprises a tumor associated antigen that comprises or consists of an amino acid sequence of SEQ ID Nos:44-49, 65-71 or 73.

[0037] In one embodiment, said drug delivery vehicle comprises targeting ligands that facilitate uptake by lymphoid organs, including lymph nodes, spleen and tertiary lymphoid structures. In one embodiment, said delivery vehicle comprises a targeting ligand that binds to a mannose receptor. In one embodiment, said targeting ligand comprises a mannose targeting ligand or a complex oligomannoside.

[0038] In one embodiment, said drug delivery vehicle comprises a cationic lipid that shows preferential uptake by a lymphoid organ. In one embodiment, said nanoparticle comprises dioleoyl-3-trimethylammonium propane (DOTAP) and / or dioleoyl-dimethylammonium propane (DODAP), and optionally dioleoylphosphatidylethanolamine (DOPE).

[0039] In one embodiment, the drug delivery vehicle further comprises one or more STING agonists. In one embodiment, said one or more STING agonists are selected from the group consisting of 2′,3′-cGAMP, 3′,3′-cGAMP, c-di-GMP, and c-di-AMP, Rp,Rp-2′,3′-c-di- AMPSS (ADU-S100), amidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'-Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'- cGAMP), 3',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), CMA, MK- 1454 and CRD5500, or any combination thereof. In one embodiment, said delivery vehicle comprises the STING agonist 2’,3’-cGAMP.

[0040] In one embodiment, said nanoparticle further comprises an inducer of immunogenic cell death (ICD inducer). In one embodiment, said ICD inducer is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

[0041] In one embodiment, the drug delivery vehicle further comprises a TLR agonist.

[0042] In one embodiment, the drug delivery vehicle comprises a STING agonist and a TLR7 / 8 agonist. In one embodiment, the drug delivery vehicle comprises 2’,3’-cGAMP and resiquimod.

[0043] In one embodiment, a method is provided for enhancing an anti-tumor immune response in a subject in need thereof, said method comprising administering to said subject a drug delivery vehicle according to the foregoing aspect and its embodiments.

[0044] In one aspect. a drug delivery vehicle is provided for enhancing an anti-tumor immune response by targeting tumor-associated lymphoid structures, said drug delivery vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle that comprises a lipid composition and / or targeting molecules that facilitate uptake by lymphoid organs, including lymph nodes, spleen and / or tertiary lymphoid structures;one or more a tumor associated antigens or tumor-associated neoantigens or a nucleic acid encoding one or more a tumor associated antigens or tumor- associated neoantigens.

[0045] In one embodiment, said nanoparticle comprises a cationic lipid nanoparticle. In one embodiment, said cationic lipid nanoparticle comprises a cationic lipid selected from the group consisting of cationic lipids selected from the group consisting of dilinoleylmethyl-4 dimethyl aminobutyrate (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), didodecyl-dimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3- trimethylammonium propane chloride (DOTAP), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), C12-200 (Lipid 5), 3-(dimethylamino) propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa- 12,15-dienoate (DMAP-BLP), and (2Z)-non-2-en-1-yl 10-[(Z)-(1-methylpiperidin-4 yl)carbonyloxy]nonadecanoate (L101), dicetylphosphate-tetraethylenepentaamine-based polycation lipid, YSK05, YSK12-C4, YSK13-C4, and YSK15-C4, or any combination thereof.

[0046] In one embodiment, said lipid comprises MC3, DSPC, Cholesterol, and PEG- DMG2000. In one embodiment, said lipid comprises in mol percent: MC350%, DSPC 10%, Cholesterol, 38.5%, PEG-DMG20001.5%. In one embodiment, said lipid comprises DODAP, DOPE, Cholesterol, and PEG-DMG2000. In one embodiment, said lipid comprises in mol percent: DODAP 18.5%, DOPE 60%, Cholesterol, 20%, and PEG- DMG20001.5%.

[0047] In one embodiment, said drug delivery vehicle further comprises a TLR agonist. In one embodiment, the TLR agonist is a TLR7 / 8 agonist. In one embodiment, the TLR7 / 8 agonist is a lipid compatible TLR7 / 8 agonist. In one embodiment, said TLR7 / 8 agonist is selected from the group consisting of resiquimod (R848), imiquimod, 852A (PF-4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD-8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX-2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD-8848) and lipidated selgantolimod (GS-9688).

[0048] In one embodiment, said drug delivery vehicle further comprises one or more STING agonists. In one embodiment, said one or more STING agonists are selected from the group consisting of 2′,3′-cGAMP, 3′,3′-cGAMP, c-di-GMP, and c-di-AMP, Rp,Rp-2′,3′-c-di-AMPSS (ADU-S100), amidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'-Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'-cGAMP), 3',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), CMA, MK- 1454 and CRD5500, or any combination thereof. In one embodiment, the STING agonist is 2’,3’-cGAMP.

[0049] In one embodiment, said nanoparticle further comprises an inducer of immunogenic cell death (ICD inducer). In one embodiment, said ICD inducer is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

[0050] In one embodiment, said drug delivery vehicle comprises a tumor-associated neoantigen selected from the group consisting of a mutant KRAS peptide, a mutant P53, or an immunogenic fragment from one or more of these neoantigens. In one embodiment, said neoantigens comprise one or more KRAS mutants. In one embodiment, the mutant KRAS peptide is a peptide selected from among SEQ ID Nos: 2-13, 15-19, 22-24, 27, 29, 30-36, 50, 52-56, 74, or 81-86. In one embodiment, said one or more KRAS mutants comprise G12D.

[0051] In one embodiment, said nanoparticle comprises a nucleic acid encoding said one or more neoantigens such as SEQ ID Nos:37-43, 57, 59-63 or 77. In one embodiment, said nucleic acid encodes an MHC-I targeting signal. In one embodiment, said nucleic acid encodes an MHC-II targeting signal.

[0052] In one embodiment, said drug delivery vehicle comprises a tumor associated antigen comprising MUC1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof, ERBB2 or an immunogenic fragment therein, carcinoembryonic antigen (CEA) or an immunogenic fragment thereof, surviving or an immunogenic fragment thereof, VEGF receptor (VEGFR) or an immunogenic fragment thereof, and human telomerase reverse transcriptase and (HTRT) or an immunogenic fragment thereof. In one embodiment, said drug delivery vehicle comprises a tumor associated antigen that comprises or consists of an amino acid sequence of SEQ ID Nos: 44-49, 65-71 or 73.

[0053] In one embodiment, said drug delivery vehicle comprises targeting ligands that facilitate uptake by lymphoid organs, including lymph nodes, spleen and tertiary lymphoid structures. In one embodiment, said delivery vehicle comprises a targeting ligand that bindsto a mannose receptor. In one embodiment, said targeting ligand comprises a mannose targeting ligand or a complex oligomannoside.

[0054] In one embodiment, said drug delivery vehicle comprises a cationic lipid that shows preferential uptake by a lymphoid organ. In one embodiment, said nanoparticle comprises dioleoyl-3-trimethylammonium propane (DOTAP) and / or dioleoyl-dimethylammonium propane (DODAP), and optionally dioleoylphosphatidylethanolamine (DOPE).

[0055] In one embodiment, a method of for enhancing an anti-tumor immune response in a subject in need thereof, said method comprising administering to said subject a drug delivery vehicle according to the foregoing aspect and its embodiments.

[0056] In one aspect, a drug delivery vehicle is provided for reprogramming the tolerogenic liver immune landscape thereby reducing or eliminating tumor antigen tolerance and / or enhancing immune defense against metastatic spread, said vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle configured for preferential liver uptake; one or more TLR7 / 8 agonists; and a nucleic acid encoding one or more tumor neoantigens or encoding one or more tumor associated antigens, or one or more a tumor neoantigens or one or more tumor-associated antigens.

[0057] In some embodiments, the selection of nanoparticle components, TLR7 / 8 agonists, tumor neoantigen or tumor associated antigen or a nucleic acid encoding the tumor neoantigen or tumor associated antigen, and optionally included STING agonist, ICD inducer, TLR agonist or components are described herein above.

[0058] In one aspect, a pharmaceutical formulation is provided comprising: a drug delivery vehicle according to any one of the foregoing aspects or embodiments; and a pharmaceutically acceptable carrier.

[0059] In one embodiment, the pharmaceutical formulation is formulated for administration via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation, intracranial administration via a cannula, intratumoral administration and subcutaneous or intramuscular depot deposition.

[0060] In one aspect, a method of treating a cancer is provided, said method comprising administering to a subject in need thereof a drug delivery vehicle according to any one of the preceding aspects or embodiments, or a pharmaceutical formulation thereof.

[0061] In one embodiment, a method of treating cancer in a subject is provided, said method comprising administering to a subject that is receiving or that has received a chemotherapeutic agent, a drug delivery vehicle according to any one of the preceding aspects and their embodiments. In one embodiment, said chemotherapeutic agent is an immunogenic cell death (ICD) inducing chemotherapeutic agent. In one embodiment, said chemotherapeutic agent is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

[0062] In one embodiment, said method is an adjunct therapy to a primary chemotherapy regime.

[0063] In one embodiment, said subject is a human or a non-human mammal that has a KRAS dependent cancer. In one embodiment, said KRAS dependent cancer comprises a cancer selected from the group consisting of cancer of the pancreas, lung, breast, colon, rectum, prostate, stomach, esophagus, cervix, uterus, thyroid, lung and bladder. In one embodiment, said KRAS dependent cancer comprises pancreatic ductal adenocarcinoma (PDAC). DEFINITIONS

[0064] The terms "subject," "individual," and "patient" may be used interchangeably and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, and the like). In various embodiments, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker.

[0065] As used herein, the phrase "a subject in need thereof" refers to a subject, as described infra, that suffers from, or is at risk for a cancer as described herein. Thus, for example, in certain embodiments the subject is a subject with a cancer (e.g., pancreatic ductal adenocarcinoma (PDAC), breast cancer (e.g., drug-resistant breast cancer), colon cancer, brain cancer, lung cancer, rectal cancer, prostate cancer, stomach cancer, esophaguscancer, cervical cancer, uterine cancer, thyroid cancer, lung cancer, bladder cancer, and the like). In certain embodiments the methods described herein are prophylactic and the subject is one in whom a cancer is to be inhibited or prevented. In certain embodiments the subject for prophylaxis is one with a family history of cancer and / or a risk factor for a cancer (e.g., a genetic risk factor, an environmental exposure, and the like).

[0066] The term "treat" when used with reference to treating, e.g., a pathology or disease refers to the mitigation and / or elimination of one or more symptoms of that pathology or disease, and / or a delay in the progression and / or a reduction in the rate of onset or severity of one or more symptoms of that pathology or disease, and / or the prevention of that pathology or disease. The term treat can refer to prophylactic treatment which includes a delay in the onset or the prevention of the onset of a pathology or disease.

[0067] The terms "coadministration" or " administration in conjunction with" or "cotreatment" when used in reference to the coadministration of a first compound (or component) (e.g., a nanoparticle as disclosed herein) and a second compound (or component) (e.g., a chemotherapeutic agent) indicates that the first compound (or component) and the second compound (or component) are administered so that there is at least some chronological overlap in the biological activity of first compound and the second compound in the organism to which they are administered. Coadministration can include simultaneous administration or sequential administration. In sequential administration there may even be some substantial delay (e.g., minutes or even hours) between administration of the first compound and the second compound as long as their biological activities overlap. In certain embodiments, the coadminstration is over a time frame that permits the first compound and second compound to produce an enhanced therapeutic or prophylactic effect on the organism. In certain embodiments the enhanced effect is a synergistic effect.

[0068] The term "immunogenic cell death" or "ICD" refers to a unique form of cell death caused by some cytostatic agents such as anthracyclines (Obeid et al. (2007) Nature Med., 13(1): 54-61), oxaliplatin, irinotecan and bortezomib, or radiotherapy and photodynamic therapy (PDT). Unlike regular apoptosis, which is mostly non-immunogenic or even tolerogenic, immunogenic apoptosis of cancer cells can induce an effective antitumor immune response through activation of dendritic cells (DCs) and consequent activation of specific T cell response (Spisek and Dhodapkar (2007) Cell Cycle, 6(16): 1962-1965). Endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) production and induction of autophagy are key intracellular response pathways that govern ICD (Krysko et al. (2012) Nat. Rev. Canc.12(12): 860-875). In addition to facilitating tumor cell death thatfacilitates antigen presentation by dendritic cells, ICD is characterized by secretion or release of damage-associated molecular patterns (DAMPs), which exert additional immune adjuvant effects. Calreticulin (CRT), one of the DAMP molecules, which is normally in the lumen of the ER, is translocated to the surface of dying cell where it functions as an “eat me” signal for phagocytes. Other important surface exposed DAMPs are heat-shock proteins (HSPs), namely HSP70 and HSP90, which under stress condition are also translocated to the plasma membrane. On the cell surface they have an immunostimulatory effect, based on their interaction with number of antigen-presenting cell (APC) surface receptors like CD91 and CD40 and also facilitate cross-presentation of antigens derived from tumor cells on MHC class I molecule, which then triggers CD8+ T cell activation and expansion. Other important DAMPs, characteristic for ICD are secreted high mobility group box 1 (HMGB1) and ATP (see, e.g., Apetoh et al. (2007) Nature Med.13(9): 1050-1059; Ghiringhelli et al. (2009) Nature Med.15(10): 1170-1178). HMGB1 is considered to be a late apoptotic marker and its release to the extracellular space appears to be required for the optimal release and presentation of tumor antigens to dendritic cells. It binds to several pattern recognition receptors (PRRs) such as Toll-like receptor (TLR) 2 and 4, which are expressed on APCs. The most recently found DAMP released during immunogenic cell death is ATP, which functions as a “find-me” signal for monocytes when secreted and induces their attraction to the site of apoptosis (see, e.g., Garg et al. (2012) EMBO J.31(5): 1062-1079). ATP binds to purinergic receptors on APCs.

[0069] The terms "nanocarrier" and "nanoparticle drug carrier" are used interchangeably and refer to a nanostructure capable of carrying one or more drugs. In certain embodiments the nanocarrier comprises a lipid (lipidic) nanoparticle (e.g., vesicle or liposome), a silicasome (a porous nanoparticle, e.g., mesoporous silica nanoparticle, encased in a lipid bilayer), a polymeric nanoparticle (e.g., comprising one or more biocompatible polymers, and optionally comprising one or more lipids), and the like.

[0070] As used herein, the term “lipid” refers to conventional lipids, phospholipids, cholesterol, chemically functionalized lipids for attachment of PEG and ligands, etc.

[0071] As used herein, the terms “lipid bilayer” or "LB" refers to any double layer of oriented amphipathic lipid molecules in which the hydrocarbon tails face inward to form a continuous non-polar phase.

[0072] As used herein, the terms “liposome” or "lipid vesicle" or "vesicle" are used interchangeably to refer to an aqueous compartment enclosed by a lipid bilayer, as beingconventionally defined (see, e.g., Stryer (1981) Biochemistry, 2d Edition, W. H. Freeman & Co., p.213).

[0073] As used herein, the term “selective targeting” or “specific binding” refers to use of targeting ligands on the surface of a drug delivery nanocarrier (e.g., a LB-coated nanoparticle). In certain embodiments the targeting ligands are on the surface of a lipid bilayer of LB-coated nanoparticle. Typically, the ligands interact specifically / selectively with receptors or other biomolecular components expressed on the target, e.g., a cell surface of interest. The targeting ligands can include such molecules and / or materials as peptides, antibodies, aptamers, targeting peptides, polysaccharides, and the like.

[0074] The term "about" or "approximately" as used herein refers to being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e. the limitations of the measurement system, i.e. the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5% and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0075] The term "drug" as used herein refers to a chemical entity of varying molecular size, small and large, naturally occurring or synthetic, that exhibits a therapeutic effect in animals and humans. A drug may include, but is not limited to, an organic molecule (e.g., a small organic molecule), a therapeutic protein, peptide, antigen, or other biomolecule, an oligonucleotide, an siRNA, a construct encoding CRISPR cas9 components and, optionally one or more guide RNAs, and the like.

[0076] A "pharmaceutically acceptable carrier" as used herein is defined as any of the standard pharmaceutically acceptable carriers. The pharmaceutical compositions of the subject disclosure can be formulated according to known methods for preparing pharmaceutically useful compositions. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the disclosure. Examples include, but are not limited to: phosphate buffered saline,physiological saline, water, and emulsions, such as oil / water emulsions. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W

[1995] Easton Pa., Mack Publishing Company, 19th ed.) describes formulations which can be used in connection with the drug delivery nanocarriers described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figs.1A-1E depict mRNA design and chemistry of cGAMP and D-Lin-DMA. Fig. 1A: Common KRAS mutant sequences, including for the 17-peptide RAS5-21 sequence. Fig.1B: Comparison of wild type RAS5-21 and G12D mutant KRAS5-21, used for mRNA design by including 2 amino acids upstream and downstream as flanking sequences, before transverse translation into the respective to nucleic acid sequences by the online GeneScript tool. Fig.1C: The open reading frame included 5 repetitions of each nucleotide sequence, with the addition of start codon and stop codons, 5’ Cap, 5’ UTR, 3’ UTR, and 3’ PolyA tail. Fig.1D: Structure of 2’,3’-cGAMP. Fig.1E: Structure of D-Lin-MC3-DMA lipid.

[0078] Figs.2A-2B depict mRNA design. Fig.2A: Open reading frame for constructing mKRAS and mRASWT strands. The GC contents were 52.08% and 53.65%, respectively. Fig.2B: Agarose gel electrophoresis showing the construction of the corresponding mRNA strands by Trilink.

[0079] Figs.3A-3G depict the preparation and characterization of LNP. Fig.3A: LNP with cGAMP and KRAS G12D mRNA cargo was prepared by blending the organic phase, containing the lipids MC3, DSPC, cholesterol, DMG-PEG2000, with the aqueous phase containing cGAMP and mKRAS in a microfluidics blender. The cGAMP / mKRAS / LNPs were dialyzed against PBS at 4oC for 1 h before filtering (220 nm). Fig.3B: the particle size, PDI and surface charge of cGAMP / mKRAS / LNP were 91.6 ± 0.4 nm, 0.173 ±0.012, and 91.3%. Fig.3C: Representative cryoEM image of cGAMP / mKRAS / LNP. Scale bar is 100 nm. Fig.3D: Change in particle size during storage at 4oC for 15 days. Fig.3E: Change of EE during storage at 4oC for 15 days. Fig.3F: Physical characterization of LNPs. The size and the surface potential of the purified LNP samples were determined by dynamic light scattering (DLS). The concentration and encapsulation efficiency (EE, %) of polyA were determined using a RiboGreen assay kit (Thermo Fisher). The concentration of cGAMP was determined by cGAMP ELISA kit (Thermo Fisher). Briefly, the size ofcGAMP / LNP, PolyA / LNP and cGAMP / PolyA / LNP were 81.6 nm, 92.2 nm and 89.7 nm, respectively, with relatively low PDI (< 0.2) and neutral surface potential. The EE of PolyA in PolyA / LNP and cGAMP / PolyA / LNP are all higher than 90%, and the concentration of cGAMP in cGAMP / LNP and cGAMP / PolyA / LNP were16.76 μg / mL and 11.93 μg / mL. Fig. 3G illustrates particle physicochemical characterization. DLS measurement of the hydrodynamic sizes of the carriers for cGAMP / LNP, mKRAS / LNP, cGAMP / mCtrl / LNP and cGAMP / mKRAS / LNP, yielded measurements of 83.1, 90.2, 95.5 and 96.7 nm, respectively. The encapsulation concentrations of the mRNA’s were quantified by a RiboGreen Assay, while the cGAMP concentration was quantified by an ELISA kit. There is no interference of these analogs with each other.

[0080] Fig.4 depicts parameters for microfluidic synthesis of LNPs delivering dual cargo. Preparation of the stock solution containing cGAMP and mRNA was carried by dissolving these components in 0.1 M NaAc medium at 5 mg / mL. The stock solution containing the lipid phase was prepared by dissolving the MC3 lipid, DSPC, cholesterol and DMG- PEG2000 in ethanol at 20 mg / ml, yielding molar ratio of 50:10:38.5:1.5. The resulting N:P ratio was 4, while the volume ratio of the aqueous phase vs. organic phase was 3. The tables show calculation of analyte concentrations and volumes of the stock solutions used in microfluidic synthesis.

[0081] Figs.5A-5B depicts a hemolysis assay for investigating the hexagonal lipid phase. Fig.5A: Images of the plate wells containing 1% RBC, incubated with different concentrations of mKRAS / LNP or cGAMP / mKRAS / LNP. The pH of the incubation media was adjusted to 5.5 and 7.4. The positive control for cell lysis was 0.1% Triton X-100. Fig. 5B: Use of the supernatant absorbance at 570 nm to express % hemolysis against a blank control (0% lysis) or Triton X (100% lysis).

[0082] Figs.6A-6E depicts activation of the cellular STING pathway by encapsulated cGAMP. Fig.6A: Free 2’,3’-cGAMP is incapable of permeating cell membranes. However, intracellular uptake can be achieved by encapsulating the agonist in LNP. This allows cytosolic 2’,3’-cGAMP to serve as a messenger for activating STING, which is incorporated into the APC reticuloendothelial membrane. STING activation triggers the TBK1 / interferon regulatory factor 3 (IRF3) and NF-κB pathways, leading to transcriptional of type I IFNs and other pro-inflammatory cytokines(17). Figs.6B-E: KPC cells were incubated with saline, free cGAMP, cGAMP / LNP, mKRAS / LNP, cGAMP / mRASWT / LNP and cGAMP / mKRAS / LNP for 24 h, delivering cGAMP and mRNA at concentrations of 1 μg / mL and 4 μg / mL, respectively. Following cellular extraction and preparation of mRNA,RT-PCR was performed to establish the fold increase in the activation in the expression of IFN-β1 mRNA (Fig.6B), CCL5 mRNA (Fig.6C), CXCL9 mRNA (Fig.6D), and CXCL10 mRNA (Fig.6E). *** p<0.001.

[0083] Figs.7A-7D depict assessment of cellular activation of STING. Fig.7A: Schematic to explain the contributions of MHC-I and CD80 / CD86 expression on the APC in activating CD8+ T-cells, which recognize the KRAS mutant peptide presented by MHC-I. CD80 / CD86 delivers a co-stimulatory signal (“signal 2”) upon binding to CD28 to assist the T-cell antigen receptor. Fig.7B: Flow cytometry assessment of DC maturation, gating on CD11c+ cells to assess surface expression of CD80 and CD86. Fig.7C: Flow cytometry analysis to show the impact of different cellular treatments on CD80 and CD86 expression. The concentration of cGAMP and mRNA in each group was 1 μg / mL and 4 μg / mL, respectively. Fig.7D: Statistical analysis of the % CD80 and CD86 expression. *** p < 0.001.

[0084] Figs.8A-8G depict biodistribution and in vivo mRNA translation of cGAMP / mGFP LNP. Fig.8A: Fluorescent labeled Cy5-cGAMP as well as GFP-encoding mRNA (mGFP) were used for the synthesis of Cy5-cGAMP / mGFP / LNP to determine LNP distribution, liver uptake and mRNA expression after IV injection. Fig.8B: Size distribution and representative cryoEM images of the synthesized particles. Scale bar is 100 nm. Fig.8C: The experimental planning involved IV injection into female B6129SF1 / J mice for in vivo IVIS imaging, followed by animal sacrifice and harvesting the major organs (heart, liver, spleen, lung and kidneys) for ex vivo IVIS imaging. The liver tissues were sent for immunofluorescence staining to study GFP expression. The nucleus, GFP and LSECs were stained with DAPI, anti-GFP antibody and anti-CD146 antibody respectively. The co- localization index of GFP with LSECs was 12.3%. Scale bar is 100 μm. *** p<0.001. Fig. 8D: The Cy5 signal was discernible in the liver only of animals receiving encapsulated but not free Cy5-cGAMP. Fig.8E: Quantitative analysis of Cy5 radiance is shown. No significant uptake of encapsulated particles was seen in other organs. Fig.8F: Harvested liver tissues were sectioned and subjected to immunofluorescence microscopy, which confirmed GFP expression throughout the liver, including uptake by fluorescent-labeled (red) sinusoidal endothelial cells (LSECs). Fig.8G: Demonstration of the merged fluorescence views at 4X magnification, including calculation of the GFP increase compared to animals not receiving encapsulated GFP. All considered, these results show effective delivery of both the STING agonist as well as the GFP mRNA by a cationic LPN that was designed for liver biodistribution.

[0085] Figs.9A-9J depict the immune reprogramming of non-parenchymal liver cells (NPCs) and activation of cytotoxic and memory T-cells by LNPs. Fig.9A: Experimental design to study the effect of cGAMP / mKRAS / LNP in NPCs. Briefly, two particle doses (0.3 mg / kg cGAMP and 1.25 mg / kg mKRAS each) were IV injected in female B6129SF1 / J mice on days 1 and 7. Fig.9B: NPCs were extracted from in vivo perfused liver tissues (collagenase buffer), before animal sacrifice and harvesting NPCs used for RT-PCR and flow cytometry. Fig.9C: RT-PCR to assess the fold increase in IFN-β1 mRNA expression, following treatment with saline or cGAMP / mKRAS / LNP (n=3). Fig.9D: Representative plot to display fluorescence intensity distribution as well as statistical histograms for CD80, CD86, MHC-I and MHC-II expression in NPCs. Fig.9E: Schematic to describe the in vivo assessment of immune activation parameters in mice receiving IV injection of 4 doses of saline or cGAMP / mKRAS / LNP. Animals were sacrificed to separate splenocytes for ELISPOT assays and flow cytometry. Fig.9F: ELISPOT assays were conducted by treating the splenocytes with the G12D or wildtype peptide for 24 h and assessing IFN-γ producing colonies. Fig.9G: Quantitative analysis of ELISPOT colonies (n=3). Fig.9H: Flow cytometry to assess CD44 and CD62L expression in CD3+ / CD8+ splenocytes. CD44+CD62L+ T-cells represent central memory T-cells, while CD44+CD62L-cells are considered as effector memory T-cells. (Figs.9I and 9J) Statistical expression of the % central memory and effector memory T-cells (n=3). *** p<0.001.

[0086] Figs.10A-10B depict flow cytometry assessment of central and memory T-cells. Fig.10A: Schematic to explain the generation of effector as well as memory T-cells. Fig. 10B: Gating method to assess relative content of central memory (TCM) as well as effector memory (TCM) T-cells. CD44 is involved in T-cell migration while CD62L is involved in homing to specific tissue environments.

[0087] Figs.11A-11L depict in vivo therapeutic effect against PDAC metastasis. Fig.11A: Diagram to explain hemispleen injection of KPC cells to obtain liver metastases. IVIS images in vivo day 14 after tumor inoculation, showing the signal from the liver. Photograph showing the liver metastases. Fig.11B: Schematic describing the investigation of the therapeutic effect in mice receiving IV injection of 4 doses of saline or cGAMP / LNP, mKRAS / LNP, cGAMP / mRASWT / LNP or cGAMP / mKRAS / LNP (0.3 mg / kg cGAMP and 1.25 mg / kg mKRAS or mRASWT). Tumor growth was monitored by IVIS imaging and body weight was recorded every 3 d. All mice were sacrificed on day 18 for the collection of blood and liver tissues. Fig.11C: In vivo IVIS images of the animals on Day 18. Fig. 11D: Summary of average radiance from liver area of each mouse (n=8). Fig.11E: Liverweights on day 18. Fig.11F: Representative H&E images and fluorescence images of the liver in each group. Scale bar is 5 mm. Fig.11G: Statistical analysis of metastatic burden from each H&E image (n=8). Fig.11H: Representative fluorescence images for Ki67 and CD8 staining in liver slides. (Scale bar is 100 μm) Fig.11I: Statistical analysis of the number of CD8+ T-cells representative of each group (n=8). Fig.11J: IHC staining for perforin and IFN-γ. Scale bar is 50 μm. Fig.11K: Statistical analysis of perforin positive cell number from each IHC staining image (n=8). Fig.11L: Statistical analysis of IFN-γ intensity from each IHC staining image (n=8). * p<0.05, ** p<0.01, *** p<0.001, # p<0.05, ## p<0.01, ### p<0.001.

[0088] Figs.12A-12D depict the impact of cGAMP / mKRAS / LNP treatment in mice with established pancreatic cancer liver metastasis. These data are related to the experiment described in Figs.6A-E, looking at the use of IV administration in mice with established liver metastases by hemispleen injection. Fig.12A: Full panel of in vivo IVIS images collected on days 5, 11, 14 and 18. Fig.12B: Body weight plot of animals in the different groups. Fig.12C: Ex vivo images of liver tissues from all mice. Fig.12D: Average radiance efficiency of liver tissues (n=8).

[0089] Figs.13A-13E depict survival in the post-treatment model. Fig.13A: Schematic of the survival study in mice receiving 5×104KPC-luc cells by hemispleen injection. Four doses of cGAMP / LNP, mKRAS / LNP, cGAMP / mRASWT / LNP or cGAMP / mKRAS / LNP (0.3 mg / kg cGAMP and 1.25 mg / kg mKRAS or mRASWT) were IV injected on days 6, 9, 12, and 15. Tumor growth was monitored by IVIS imaging every week. Fig.13B: Survival plots of the different groups (n=8). Fig.13C: Schematic of similar survival study, where the animals received 4 doses of cGAMP / mRASWT / LNP or cGAMP / mKRAS / LNP on days 1, 3, 5, and 7, before hemispleen injection with 1×104KPC-luc cells. Tumor growth was monitored by IVIS imaging every week. Fig.13D: Survival plot of different groups (n=8). * p<0.05, ** p<0.01, *** p<0.001. Fig.13E: LNP characterization for the survival experiment.

[0090] Fig.14 depicts the in vivo IVIS images of the survival experiment described in Figs. 13A-E, for animals receiving a high cell number of KPC-luc cells. This figure shows the full set of IVIS images collected on days 5, 12, 19 and 26 for of mice inoculated with 5×104KPC-luc cells.

[0091] Fig.15 depicts the in vivo IVIS images of the survival experiment described in Figs. 13A-E for animals receiving a high cell number of KPC-luc cells. This figure shows the fullset of IVIS images collected on days 5, 12, 19 and 26 for of mice inoculated with 1×104KPC-luc cells.

[0092] Figs.16A-16D depict the impact of prophylactic treatment and adoptive cell transfer. Fig.16A: Schematic of the survival study of mice pretreated with different formulations, followed by inoculation with 5×104KPC-luc cells by hemispleen injection. Four doses of saline or cGAMP / mKRAS / LNP (0.3 mg / kg cGAMP and 1.25 mg / kg mKRAS) were IV injected on days -14, -11, -7, and -4. Tumor growth was monitored by IVIS imaging. Fig.16B: Survival plots for the two groups (n=8). Fig.16C: Schematic of the survival study in animals receiving adoptive transfer of splenocytes from vaccinated animals. Vaccinated animals received 4 doses of saline or cGAMP / mKRAS / LNP (similar amount of cargo as for A) on days -21, -18, -14, and -11, before euthanasia on day -7 to isolate lymphocytes from the spleen. The cells were used for adoptive transfer into healthy recipient animals (n = 8), each receiving 3×106lymphocytes intravenously.5×104KPC-luc cells were injected into the hemispleen of recipient mice on day 0. Tumor growth was monitored by IVIS imaging. Fig.16D: Survival plots for the animal groups in the adoptive transfer experiment (n=8). * p<0.05, ** p<0.01, *** p<0.001.

[0093] Fig.17 depicts in vivo IVIS images of the survival experiment described in Figs. 8A-G for animals treated prophylactically before establishment of KPC metastases. The mice were inoculated with 5×104KPC-luc before obtaining IVIS images on days 7, 14, 21, 28 and 35.

[0094] Fig.18 depicts in vivo IVIS images of the survival experiment described in Figs. 8A-G, describing the adoptive cell transfer study. The mice were inoculated with 5×104KPC-luc before obtaining IVIS images on days 7, 14, 21, 28 and 35.

[0095] Figs.19A-19H depict a panel of in vivo biosafety assays. Fig.19A: Schematic to explain performance of the in vivo biosafety study. Eight weeks old Female B6129SF1 / J were IV injected with saline, free cGAMP or cGAMP / mKRAS / LNP on days 1, 4, 7, and 10. Blood was collected on days 2, 11, and 24, and body weight was also monitored every other day. Fig.19B: Body weight changes in mice from different groups. The serum was used to quantify the concentration of IL-6 (Fig.19C), TNF-α (Fig.19D), ALT (Fig.19E), AST (Fig.19F), BUN (Fig.19G) and creatinine (Fig.19H).

[0096] Figs.20A-20C illustrate the synthesis and characterization of polymeric nanoparticle vaccines with hydrophobic mutated Kras peptides. Fig.20A: Schematic showing the modified double emulsion solvent evaporation method. Fig.20B: Kras polymeric nanoparticle structures observed under SEM shows largely monodispersenanoparticles with a smooth surface. Scale bar= 200nm. Fig.20C: Table describing characterization of PLGA peptide nanoparticles.

[0097] Figs.21A-21F illustrate the in vivo prophylactic efficacy of the Kras vaccine nanoparticles in a subcutaneous KPC pancreatic cancer model. Fig.21A: Schematic showing vaccination timeline prior to subcutaneous implantation of KPC cells. Figs.21B-C: Mean tumor volume of the challenged mice across the treatment groups over the duration of the study were recorded. (Figs.21D-E) Paraffin embedded tumor sections after the termination of experiment were analyzed for tumor infiltrating T lymphocytes (CD8+) by IHC. and quantified for CD8+ signal by ImageScope software. Fig.21F: H&E and IHC images showing intratumoral TLS like aggregates. Table describing the frequency of their appearance in subcutaneous KPC tumors. Data represents mean ± SEM. Statistical analysis was performed by one way ANOVA. *p < 0.05, ns: not significant. Scale bar= 200um.

[0098] Figs.22A-22F illustrate the in vivo prophylactic efficacy of the Kras vaccine nanoparticles in an orthotopic KPC-luciferase pancreatic cancer model. Fig.22A: Schematic showing vaccination timeline prior to orthotopic implantation of KPC luc cells. Mice were terminated 16 days post tumor challenge. Fig.22B: Visual of the luminescence emission from the luciferin signal in each mouse captured by IVIS imaging. Fig.22C: Quantification of luminescence intensity prior to sacrifice. Fig.22D: Ex vivo imaging and quantification of the luciferin signal from (Fig.22E) tumors and internal organs in the peritoneal cavity. Fig.22F: The weights of the harvested tumors were recorded. Data represents mean ± SEM, n = 8. Statistical analysis was performed by one way ANOVA. *p < 0.05, ns: not significant.

[0099] Figs.23A-23E illustrate the immune landscape and features formed in Kras vaccine treated mice in orthotopic KPC-luc cancer model. Splenocytes harvested from vaccinated mice were stimulated ex vivo and observed for cytotoxic T cell activation in ELISpot assays specific for (Fig.23A) INFγ and (Fig.23B) TNFα. Tumor infiltrating (Fig.23C) CD8+ and (Fig.23D) CD4+ T lymphocytes were analyzed by IHC and quantified with ImageScope. Fig.23E: IHC staining for CD8, CD4 and CD21 showing the structure of intra-tumoral TLS in (M23+R848) PLGA vaccinated mice. The Table describes intra-tumoral tertiary lymphoid structures in orthotopic tumors after vaccination with M23+R848PLGA. Data represents mean ± SEM, n = 8. Statistical analysis was performed by one way ANOVA.

[0100] Figs.24A-24C illustrate different MHC I and II routing strategies as well as an integration of the strategies. Fig.24A: Multi-epitope mRNA vaccine constructed for MHC I presentation to CD8 positive T-cells, using ppCT signal peptide or equivalent. Fig.24B:Multi-epitope mRNA vaccine constructed for MHC II presentation to CD4 positive T cells, using a transferrin receptor (TFR) aa-1-118 or a or an invariant chain(li) chain aa1-80 sequence. Fig.24C: Multi-epitope mRNA vaccine for both MHC I as well as MHC II epitope routing that makes use of self-cleaving linker peptides (T2A) sequence (GSGEGRGSLLTCGDVEENPG (SEQ ID NO:1). T2A peptides are 18–22 amino-acid (aa) viral oligopeptides mediating “cleavage” of polypeptides during translation in eukaryotic cells (see, e.g., Liu et al. (2017) Sci. Rep.7: 2193).

[0101] Fig.25 illustrates the use of mannose targeting ligands in promoting lipid nanoparticle uptake in targeting of the type II mannose receptor, DC SIGN, on the surface of dendritic cells, present in lymphoid structures. Mannosylated particles are capable of increasing antigen uptake by dendritic cells a 100-fold over antigen uptake from a fluid phase. A DSPE-PEG analog can be used for inserting a mannose monosaccharide on the LPN surface. The ability of the binding interaction can be further increased by more complex oligomannosides. The particles can also be endowed with a lipid-tailed TLR7 agonist, 3M-052, that can augment the response by delivering danger signals that increase DC-SIGN expression and promote DC maturation (see, e.g., Li et al. (2019) Frontiers in Chemistry, Volume 7, Article 650; Luo et al. (2022) ACS Nano, 16: 13168-13182).

[0102] Figs.26A-26C illustrate the development of non-decorated and mannose decorated cationic LNPs. Fig.26A: Non-decorated and mannose decorated cationic LNPs. Cationic LNP, with the lipid composition shown on the bottom right-side panel, were synthesized in a microfluidics mixer. The lipids used wereDLin-MC3-DMA, DSPC, cholesterol and either DMG-PEG-2K or DSPE-PEG2000-mannose, dissolved in ethanol in the molar ratio 50:10:38.5:1.5. Briefly, the lipid components were dissolved in an ethanol stock solution, while the mRNA (GFP-mRNA) was diluted in RNAse-free Sodium Acetate Buffer (pH 4.0). Following particle synthesis in a Nanoassemblr. The size and the surface potential of the purified LNP samples were determined by dynamic light scattering (DLS). Fig.26B: This demonstrated particle sizes of 100.6 and 150.2 nm, respectively for non-decorated and decorated LPN, with zeta potentials of – 3.7 and – 5.37, respectively. The encapsulation efficiencies (EE %) of GFP-mRNA LNPs were > 90 %. The particles were also imaged by cryogenic electron microscopy (Cryo-EM), with a representative example shown in Fig. 26C.

[0103] Fig.27 shows an illustrative design of cationic lipid nanoparticles that are mannose decorated and include incorporation of a TLR7 agonist with a lipid tail (3M-052). The bottom panel depicts two cationic lipids that improve LPN biodistribution to the spleen.

[0104] Fig.28 shows peptide and corresponding peptide and nucleotide sequences for human and murine KRAS sequences, including outlining the identical sequence for Kras G12D. This 17 aa sequence was endowed with natural flanking AA sequences on both ends before reverse translation. The comparative peptide sequence for wildtype p21 Ras is shown as well.

[0105] Figs.29A-29B depict the development of spleen-targeting LNPs. Fig.29A depicts the microfluidics mixing for the construction of spleen targeting LNPs. In brief, spleen- targeting LNPs were synthesized by the NanoGeneratorTM Flex-M system (Precigenome LLC) to mix the lipid-containing organic phase with an aqueous phase containing mRNA constructs. The organic phase was comprised of a lipid mix (DODAP / DOPE / cholesterol / DMG-PEG2000) dissolved in ethanol in a molar ratio of 18.5: 60: 20: 1.5. The aqueous phase was comprised of RNase-free sodium acetate buffer (0.1M, pH 4.0) containing the mRNA constructs (polyA, mLuc, mRASwt, or mKRAS). The flow rate ratio of the aqueous vs. organic phase was 3:1. LNPs in the effluent phase were dialyzed against PBS (pH 7.4) to remove ethanol before use. The LNPs were characterized for hydrodynamic size, polydispersity index (PDI), and zeta potential by a ZETAPALS instrument (Brookhaven Instruments Corporation). The encapsulation efficiency (EE%) of mRNA constructs was performed through the RiboGreen assay and determined by UV spectroscopy. Fig.29B depicts the characterization of LNP / polyA for hydrodynamic size, PDI, zeta potential, and EE%.

[0106] Figs.30A-30C demonstrate the biodistribution and spleen-targeting function of LNPs. For the animal imaging studies, we developed fluorescently labeled LNPs to deliver mLuc (DiR-LNP / mLuc) by adding DiIC18(7) (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindo- tricarbocyanine Iodide (DiR dye) into the lipid mix as described above. The weight ratio of DiR to lipid mix was 0.5%. The schematic in Fig.30A depicts the experimental timeline for conducting the biodistribution and luciferase expression of DiR-LNP / mLuc. In brief, healthy B6129SF1 / J mice received a single IV administration of DiR-LNP / mLuc to deliver 0.3 mg / kg luciferase mRNA (n=3). Mice were intraperitoneally injected with D-Luciferin 24 h after LNP injection. Mice were euthanized within 10 min after administration. Ex vivo major organs (heart, lungs, kidneys, spleen and liver) were collected for the bioluminescent imaging of luciferase expression and the fluorescent imaging of DiR dye. Fig.30B depicts the DiR fluorescent imaging and normalized DiR fluorescence intensity of the organs, 24 h after IV injection through the IVIS imaging system. Fig.30C depicts the luciferase expression and normalized luciferase intensity of the same organs described above.

[0107] Figs.31A-31C show the development of 3M-LNPs. Fig.31A depicts the loading of the lipophilic TLR7 / 8 agonist, 3M-052, into the LNP structure by substituting a small fraction of DOPE with this lipid tailed agonist. Fig.31B depicts that 3M-LNPs were formulated as indicated to produce a series of LNPs with 0% to 4.5% 3M-052 (fraction of total lipids). The rest of the synthesis procedure for 3M-LNP / polyA was the same as the LNP / polyA described above. The 3M-LNPs were characterized for size, PDI, zeta potential, EE%, and concentration ratio of 3M / PolyA (Fig.31C).

[0108] Figs.32A-32C depict the biodistribution and spleen-targeting function of 3M-LNPs. For the animal imaging studies, we developed DiR fluorescently labeled LNPs with 4.5% 3M-053 to deliver mLuc (4.5% 3M DiR-LNP / mLuc). The weight ratio of DiR to lipid mix was 0.5%. The schematic in Fig.32A depicts the experimental timeline for conducting the biodistribution and luciferase expression of DiR-LNP / mLuc and 4.5% 3M DiR-LNP / mLuc. In brief, healthy B6129SF1 / J mice received a single IV administration of DiR-LNP / mLuc or 4.5% 3M DiR-LNP / mLuc to deliver 0.3 mg / kg luciferase mRNA (n=3). The rest of the procedure, including intraperitoneal injection with D-Luciferin and euthanasia, was the same as described in Fig.2A. Fig.32B depicts the DiR fluorescent imaging and normalized DiR fluorescence intensity of the organs 24 h after IV injection of DiR-LNP / mLuc or 4.5% 3M DiR-LNP / mLuc. Fig.32C depicts the luciferase expression, normalized luciferase intensity, and bioluminescent radiance of the same organs from DiR-LNP / mLuc or 4.5% 3M DiR-LNP / mLuc groups.

[0109] Figs.33A-33B show the development of LNP / mRNA and 3M-LNP / mRNA carriers. Fig.33A depicts the lipid composition of LNP / mRNA and 3M-LNP / mRNA. The microfluidics mixing procedures were the same as described in Fig.29A. The spleen- targeting LNP / mKRAS, 3M-LNP / mRASwt, and 3M-LNP / mKRAS were synthesized by the NanoGeneratorTM Flex-M system as described in Fig.1A. The obtained LNPs were characterized for hydrodynamic size, PDI, zeta potential, and EE% by a ZETAPALS instrument and UV spectroscopy (Fig.33B).

[0110] Figs.34A-34D depict the therapeutic effects of the spleen-targeting LNP system in an orthotopic KPC pancreatic cancer model. Fig.34A depicts the experimental outline of the study conducted by orthotopic implantation of KPC-luc tumor cells in the pancreatic tail of B6129SF1 / J mice. In brief, 50 μL of PBS / Matrigel (6:4 v / v), containing 0.8 × 106KPC- luc cells, was used for injection into the tail of the pancreas of female B6129SF1 / J mice 6 days before the treatment. Once establishing the KPC-luc tumor model was confirmed by IVIS bioluminescence imaging 1 day before drug injection, tumor-bearing mice wererandomly divided into 4 groups (n=6), namely: saline, 3M-LNP / mRASwt, LNP / mKRAS, and 3M-LNP / mKRAS. These animals received IV injections of the different treatment modalities every 3 days on 4 occasions. The different dose equivalents for mRNA and 3M- 052 amounted to 1.25 mg / kg and 1.75 mg / kg respectively. Animals were monitored daily until reaching moribund status or spontaneous death. Fig.34B depicts the IVIS imaging intensity for each orthotopic tumor in each of the animal groups, recorded on days 5, 11, 14, 18, 25, and 33. Fig.34C depicts the average quantitative tumor bioluminescence in each group on days 14 and 18, as calculated by IVIS software. Fig.34D depicts the Kaplan– Meier plots to display the change in survival for the different animal groups. A summary of the median survival time (MST) and percentage increase in life span (%ILS) for each group are presented in Fig.34D. Data represent mean ± SEM. #p<0.05; ##p<0.01; ###p<0.001; *p<0.05; **p<0.01.

[0111] Figs.35A-35E demonstrate the synergistic therapeutic effect by combining 3M- LNP / mKRAS with an Irinotecan delivering silicasome (Si-IR) in an orthotopic KPC pancreatic cancer model. Fig.35A depicts the experimental outline of the study conducted in an orthotopic KPC pancreatic cancer model. In brief, 50 μL of PBS / Matrigel (6:4 v / v), containing 0.8 × 106KPC-luc cells, was orthotopically injected into the tail of the pancreas of female B6129SF1 / J mice 5 days before administration of treatment. Once establishment of the KPC-luc tumor growth was confirmed by IVIS bioluminescence imaging on day 5, tumor-bearing mice were randomly divided into 5 groups (n=6), namely: saline, Si-IR, 3M- LNP / mRASwt + Si-IR, 3M-LNP / mKRAS, and 3M-LNP / mKRAS + Si-IR. Mice received IV injection of 3M-LNP / mRNA every 3 or 4 days on 4 occasions starting on day 5. Mice were treated with the Si-IR every 4 days on 4 occasions starting on day 9. The different dose equivalents for mRNA, 3M-052, and irinotecan amounted to 1.25 mg / kg, 1.75 mg / kg, and 40 mg / kg, respectively. Animals were monitored daily until reaching moribund status or spontaneous death. Fig.35B depicts the IVIS imaging intensity for each orthotopic tumor in each of the animal groups, recorded on days 5, 11, 15, 19, and 28. Fig.35C depicts the average quantitative tumor bioluminescence in each group on days 15, 19, and 28, as calculated by IVIS software. Fig.35D depicts the Kaplan–Meier plots displaying the survival rate of the different animal groups. A summary of the MST and %ILS for each group was also presented in Fig.35D. The tumors from the animals reaching moribund status or spontaneous death were harvested for immunohistochemistry (IHC) analysis. Fig. 35E depicts the representative IHC images and quantification of CD8+ cells in each group. Bar is 200 μm. Data represent mean ± SEM. #p<0.05; ##p<0.01; ###p<0.001; *p<0.05; **p<0.01; ***p<0.001.

[0112] Figs.36A-36D show that combining 3M-LNP / mKRAS with 3M-Si-IR may further boost the treatment outcome in an orthotopic model. Fig.36A depicts the experimental outline of the study conducted in an orthotopic KPC pancreatic cancer model, which looked at the impact of triggering the endogenous ICD pathway before administration of the exogenous LNP vaccine. In brief, 50 μL of PBS / Matrigel (6:4 v / v), containing 0.8 × 106KPC-luc cells, was injected into the tail of the pancreas of female B6129SF1 / J mice 5 days before the treatment. After establishing KPC-luc tumor growth, as confirmed by IVIS bioluminescence imaging on day 5, tumor-bearing mice were randomly divided into 4 groups (n=6), namely: saline, 3M-Si-IR, 3M-LNP / mKRAS, and 3M-LNP / mKRAS + 3M- Si-IR. Mice received IV injections of 3M-Si-IR as priming vaccination on day 5 and 9. Mice were treated with 3M-LNP / mRNA as booster vaccination every 3 or 4 days on 4 occasions starting on day 13. The different dose equivalents for irinotecan and 3M-052 amounted to 40 mg / kg and 2 mg / kg respectively for 3M-Si-IR treatment, while the different dose equivalents for mRNA and 3M-052 amounted to 1.25 mg / kg and 1.75 mg / kg respectively for 3M-LNP / mKRAS treatment. Animals were monitored daily until reaching moribund status or spontaneous death. Fig.36B depicts the IVIS imaging intensity for each orthotopic tumor in each animal group, recorded on days 5, 10, 15, 19, 26, 33, and 40. Fig. 36C depicts the average quantitative tumor bioluminescence in each group on days 10, 15, 19, and 26, as calculated by IVIS software. Fig.36D depicts the Kaplan–Meier plots to display the different animal groups' survival rates, MST, and %ILS. Data represent mean ± SEM. #p<0.05; ###p<0.001; *p<0.05; **p<0.01.

[0113] Fig.37 depicts the preparation of Spleen-targeting LNP, delivering a cargo comprised of KRAS G12D encoding mRNA, the TLR7 / 8 agonist, 3M-052, and the STING agonist 2’,3’-cGAMP. The particles were synthesized by microfluidic synthesis of DODAP -containing LNP as described in our recent disclosure. Preparation of the stock solution containing cGAMP and mRNA was carried by dissolving these components in 0.1 M NaAc medium at 5 mg / mL. The stock solution containing the lipid phase was prepared by dissolving the DOTAP, DOPE, cholesterol, DMG-PEG2000, and 3M-052 in ethanol at 20 mg / ml, yielding molar ratio of 18.5:57.0:20.0:1.5:3.0. The resulting N:P ratio was 4, while the volume ratio of the aqueous phase vs. organic phase was 3.

[0114] Figs.38A-38H show an in vivo demonstration that the LNPs described in Fig.37 can target the spleen and the liver, while mRNA encoding for luciferase is only expressed in the spleen. Fig.38A: The experimental planning involved IV injection into female B6129SF1 / J mice for in vivo IVIS imaging, followed by animal sacrifice and harvesting themajor organs (heart, liver, spleen, lung and kidneys) for ex vivo IVIS imaging. Fig.38B: Photograph suspensions of mLuc / DiR-3M-LNP and mLuc / cGAMP / DiR-3M-LNP. Fig. 38C: Physiochemical characterizations of mLuc / DiR-3M-LNP and mLuc / cGAMP / DiR-3M- LNP. Fig.38D: Representative ex vivo images of main organs from mice, prior injected with mLuc / DiR-3M-LNP and mLuc / cGAMP / DiR-3M-LNP. The images at the top reflect the DiR Ex / Em wavelength. The lower images reflect GFP luminescence in the spleen. Fig. 38E: Average radiance efficiency of DiR from main organs. Fig.38F: Percentage distribution of DiR in main organs. Fig.38G: Average radiance of luminescence from main organs. Fig.38H: Percentage of luminescence in main organs. In brief, these results demonstrate that the DiR labeled particles localizing both the liver and spleen, but that the expression of luciferase mRNA only occurs in the spleen.

[0115] Figs.39A-39C depict and experiment to adjust the number of KPC cells for examining growth and survival of orthotopic implant in the pancreas mice. Fig.39A: Schematic describing the investigation of the influence of number of KPC cells inoculated to the survival of mice. Mice were divided into three groups and each group received tumor inoculation with 1000k, 100k and 10 k KPC-Luc cells. Fig.39B: Tumor growth was monitored by IVIS imaging. All mice were sacrificed on day 30. Fig.39C: Average radiance of luminescence of all mice. based on these results, it was decided to use a dose of 100,000 KPC cells to establish an orthotopic model with the survival of the animal is sufficient for doing comparative analysis of the impact of LNP’s.

[0116] Figs.40A-40C depict use of an ELISpot Assay for IFN-γ to demonstrate synergy between cGAMP and 3M-052 in generating cytotoxic T cells triggered by mRNA expression of the KRAS epitope. Fig.40A: Schematic to describe the in vivo assessment of immune activation parameters in mice receiving IV injection of 2 doses of saline, mKRAS / LNP, cGAMP / mKRAS / LNP, mKRAS / 3M-LNP or cGAMP / mKRAS / 3M-LNP. Animals (n = 3 in each group) were sacrificed to separate splenocytes for conducting ELISPOT assays on the splenocytes seven days later. Fig.40B: ELISPOT assays were conducted by treating the splenocytes with the G12D or wildtype peptide for 24 h and assessing IFN-γ producing colonies. Fig.40C: Quantitative analysis of ELISPOT colonies (n=3).

[0117] Figs.41A-41E depict an assessment of the therapeutic effect in an orthotopic PDAC, established by injecting 100,000 KPC cells into the pancreas. Fig.41A: Schematic describing the investigation of the therapeutic effect in mice receiving IV injection of 4 doses of saline or cGAMP / LNP, mKRAS / LNP, cGAMP / mKRAS / LNP, mKRAS / 3M-LNPor cGAMP / mKRAS / 3M-LNP (0.3 mg / kg cGAMP, 0.3 mg / kg 3M and 1.25 mg / kg mKRAS). Tumor growth was monitored by IVIS imaging and body weight was recorded every 7 d. Cell number inoculated was 100k. Fig.41B: Characterization of different LNPs. Fig.41C: In vivo IVIS images of the animals on Day 6 and Day 13 (experiment still ongoing. Fig.41D: Summary of average radiance from liver area of each mouse (n=6). Fig. 41E: Statistical analysis of the radiance efficiency for luciferase expression by the growing KPC cells four days 6 and 13. These data demonstrate a statistically significant reduction in orthotopic tumor cell growth by a combination of cGAMP plus 3M-052, compared to the individual agonists. DETAILED DESCRIPTION

[0118] An approach was developed for the treatment of cancer (e.g., pancreatic cancer) using, in one embodiment, a combination of vaccination with KRAS mutants that can be delivered with nanocarriers that can also be endowed with one or more STING and / or TLR7 / 8 agonists, as well as, in other embodiments, including one or more immunomodulators and / or surface targeting ligands. This approach facilitates treatment of treat PDAC liver metastases as well as augmenting the cancer immunity cycle, triggered by chemo-immunotherapy.

[0119] A dominant KRAS peptide epitope (e.g., any one of SEQ ID NOs: 2-13, 15-19, 22- 23, 27, 29-36, 50, 52-56, 74, or 81-86, or nucleic acid encoding a dominant KRAS epitope, e.g., any one of SEQ ID NOs:37-43, 57, 59-63 or 77) delivered by nanoparticles, leads to successful immune-mediated cytotoxicity and shrinkage of subcutaneous and orthotopic tumors in a KRAS pancreatic cancer (KPC) model. A novel finding was the appearance of tertiary lymphoid follicles at the primary cancer site, playing a role in the recruitment of immune cells. In one embodiment, this vaccination was performed using a PLGA nanoparticle construct that co-delivered a TLR7 agonist. This finding sets the opportunity for using cationic lipid nanoparticles to deliver single tandem repeats or a compilation of multiple KRAS epitopes, that can be targeted to lymphoid tissue that participate in the cancer immunity cycle.

[0120] PDAC liver metastasis is often fatal due to the protective immune environment in the liver, resulting from the presence of tolerogenic APC that favor suppression of the immune response to metastatic spread in cancer cells. We demonstrated in another embodiment that the tolerogenic effect of liver APCs could be reversed by encapsulating and delivering a STING agonist via non-decorated cationic lipid nanoparticles, which preferentially target the liver. This reprogrammed the tolerogenic immune landscape in theliver, with significant impact observed in a liver metastasis model, where KPC cells were injected into the spleen.

[0121] Furthermore, incorporating a tandem repeat of the dominant KRAS mRNA epitope (e.g., SEQ ID NO:77) further enhanced the immune response in the metastatic liver model. This combination led to a synergistic prolongation of animal survival, achieved through intravenous administration of the cationic lipid nanoparticles following metastasis establishment.

[0122] The cationic LNPs delivering the STING agonist and KRAS mRNA can also be utilized in an orthotopic KPC model to enhance the endogenous immune response triggered by chemo-immunotherapy using silicasome carriers. These particles can be further optimized with a TLR7 agonist and a lipid composition designed to preferentially target lymphoid tissues in the spleen and tertiary lymphoid structures at the tumor site, amplifying the immune response.

[0123] While endogenous tumor-associated antigens (TAAs), released from dying cancer cells exposed to chemotherapy agents, contribute to initiating an immune "hot-start," the supply of these TAAs (e.g., MUC1 or mesothelin) or neoantigens (e.g., KRAS, p53) may, in some cases, be insufficient to fully educate high-affinity CTLs at regional lymphoid instruction sites. To overcome this limitation, exogenous vaccination with expressed TAAs and / or neoantigens was employed to enhance the generation of high-affinity CTL clones capable of participating in the immunogenic cell death (ICD)-induced immune cycle.

[0124] In a first approach to the treatment of a cancer (e.g., KPC), we made use of neoantigenic KRAS peptides, frequently expressed in PDAC, for the performance of exogenous vaccination to investigate the impact on PDAC growth in subcutaneous and orthotopic model. In any of the embodiments disclosed herein, a KRAS peptide may be N- terminally acetylated, C-terminally amidated, or both acetylated and amidated.

[0125] In a second approach, we investigated anti-PDAC immunity with a focus on metastatic spread to the liver, aiming to reprogram the liver's immune landscape, which creates a protective immunological niche that hinders immune recognition. It is important to highlight that PDAC is often diagnosed at an advanced stage, with over 40% of patients presenting with liver metastases at diagnosis. While patients with resectable disease can undergo surgery, achieving a 5-year survival rate of 20–25%, those with liver metastases face a median survival of less than 6 months, regardless of treatment. This poor prognosis is closely linked to the liver's tolerogenic immune environment, making it a prime target formetastasis from various solid tumors, including but not limited to pancreatic, lung, breast, colorectal, prostate, gastric, esophageal, cervical, thyroid, and bladder cancers.

[0126] The liver contains unique antigen-presenting cells (APCs) that are programmed to maintain an immunosuppressive environment, designed to prevent excessive immune responses to foreign antigens originating from the gut and portal circulation. This immune regulation is achieved through the specialized function of liver APCs, which differ in how they uptake and present antigens to naïve T-cells, often leading to the development of regulatory T-cells (Tregs). While Tregs are essential for suppressing immune responses to dietary antigens, they can also hinder protective immunity, facilitating metastatic spread. Additionally, the liver is a common site for cancer recurrence following "curative" treatment.

[0127] Several APCs in the liver, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), dendritic cells, and even hepatocytes, play key roles in fostering a tolerogenic immune environment. LSECs possess a large sinusoidal endothelial surface area that promotes Treg generation, contributing to the liver's immune-suppressive landscape. Notably, these tolerogenic LSECs use scavenger receptors, such as the mannose receptor, to recruit and internalize foreign antigens, promoting Treg development. Utilizing this mechanism, mannose-decorated cationic lipid nanoparticles (LPNs) have been developed to target liver APCs and induce immune tolerance to food anaphylaxis. This approach includes delivering encapsulated peanut peptides or nucleic acid epitopes to generate Tregs capable of suppressing peanut anaphylaxis.

[0128] Considering this background, we pursued liver-targeted drug delivery nanoparticles (e.g., cationic LNPs) for reprogramming the tolerogenic status of liver APCs by nanocarriers that deliver encapsulated small molecule Stimulator of Interferon Genes (STING) agonists (e.g., cyclic dinucleotides; see, for example, Zhu Y, An X, Zhang X, Qiao Y, Zheng T, Li X. STING: a master regulator in the cancer-immunity cycle. Mol Cancer. 2019;18(1):152; Van Herck S, Feng B, Tang L. Delivery of STING agonists for adjuvanting subunit vaccines. Adv Drug Deliv Rev.2021;179:114020; Wehbe M, Wang-Bishop L, Becker KW, et al. Nanoparticle delivery improves the pharmacokinetic properties of cyclic dinucleotide STING agonists to open a therapeutic window for intravenous administration. J Control Release.2021;330:1118-1129; Garland KM, Sheehy TL, Wilson JT. Chemical and Biomolecular Strategies for STING Pathway Activation in Cancer Immunotherapy. Chem Rev.2022;122(6):5977-6039). STING agonists are strong inducers of an intracellular sensor pathway that turn on type I interferon production, which provides a strong stimulus to APCsfor mounting protective rather than tolerogenic T-cell responses in the liver. The encapsulation of the agonists by a nanocarrier is an important design principle, allowing these impermeable small agonists to cross the APC membrane for intracellular release where the STING sensor is located.

[0129] In PDAC spread to the liver, the immune landscape serves as a protective niche that interfere in eliminating the metastases, often resulting in mortality from this event. The mode of action of the STING pathway is dependent on several different steps: The first is the detection of cytosolic DNA when APCs are infected by viruses or other intracellular pathogens, releasing their DNA into the cytoplasm. Cyclic GMP-AMP synthase (cGAS) acts as a sensor protein that recognizes cytosolic DNA and produces cyclic GMP-AMP (cGAMP). cGAMP acts as a secondary messenger that binds to and activates the STING protein, located on the endoplasmic reticulum (ER) membrane. Activated STING undergoes a conformational change, which leads to the recruitment and activation of downstream signaling molecules, including TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). TBK1 and IRF3 activation results in the production of type I interferons (such as IFN-β) and other proinflammatory cytokines. Cyclic dinucleotides, such as 2’,3’- cGAMP could theoretically be used to reprogram the tolerogenic liver APC by acting as a STING agonist. However, the negative charge of the CDN prevents cellular interest, which can be promoted by encapsulation into a cationic LNP for intracellular delivery to the APCs.

[0130] In a third approach, the successful design of a nanoparticle drug carrier (e.g., a cationic LNP that delivers a STING agonist) for preferential targeting of liver metastases can be modified to also address the primary PDAC site via redirected targeting to secondary lymphoid tissue that cooperate with the primary cancer site by way of the cancer immunity cycle. In certain embodiments this can be in combination with chemo-immunotherapy. This strategy allows a combination of the features of endogenous (chemotherapy-induced) and exogenous vaccination (nucleic acid neoantigens) strategies that intersect at the cancer immunity cycle. While the secondary lymphoid tissue participating in the cancer immunity cycle includes lymph nodes and possibly the tertiary lymphoid structures that are induced at the cancer site by mutant KRAS peptides, for experimental purposes we focused on the role of the spleen as a major participant in PDAC immunity cycle. Not only do the secondary lymphoid structures and the spleen act as a filtering station for released tumor antigens, which can be presented to T-cells but are also an excellent potential target for nanocarriers that can be used to augment activation of antigen-specific T-cell subsets that are educated torecognize prominent neoantigens. We hypothesized that generation of an oligoclonal immune response to an exogenous delivered KRAS neoantigen epitopes will synergize with the polyclonal T-cell responses to a wider pool of endogenous ICD induced of neoantigens and tumor-associated antigens (TAA).

[0131] Accordingly, in various embodiments, provided herein are three approaches to the treatment of a cancer that can be used in individually or in combination. Examples of these approaches comprise, but need not be limited to: 1) Exogenous vaccination with a tumor associated antigen or neoantigen to enhance an anti-PDAC immune response; 2) Use of drug delivery nanoparticles for preferential liver targeting, APC uptake and reprogramming of the liver immune landscape to reduce or eliminate tumor antigen tolerance and defend against metastatic spread; and 3) Modification of the liver metastasis treatment platform to allow targeting to the PDAC-associated secondary lymphoid structures participating in the cancer immunity cycle to also address the primary PDAC site, including the option to combine that with chemo-immunotherapy.

[0132] In some embodiments, these approaches include incorporation of one or more STING agonists. In some embodiments, these approaches include incorporating one or more TLR7 / 8 agonists. In some embodiments, these approaches include incorporating one or more other immunomodulatory agents such as an inducer of immunogenic cell death (ICD) or a TLR agonists that is not a TLR7 / 8 agonist. In some embodiments, these approaches include incorporation of one or more STING agonists and an inducer of ICD. In some embodiments, these approaches include incorporation of one or more TLR7 / 8 agonists and an inducer of ICD. In some embodiments, these approaches include incorporating both a STING agonist and a TLR7 / 8 agonist. In some embodiments, these approaches include incorporating both a STING agonist and a TLR7 / 8 agonist and an inducer of ICD.

[0133] These approaches and drug delivery nanoparticles suitable for implementing these approaches are described in more detail below. 1) Use of Exogenous Neoantigen Vaccination to Boost Pancreatic Cancer Immunotherapy by polymeric Nanocarriers.

[0134] The use of polymeric nanoparticles to for generating an effective immune response to the mutant KRAS G12D epitope, suffice for tumor shrinking of a KRAS-induced pancreatic cancer (KPC) model, has been demonstrated. This example establishes thefeasibility of using a dominant KRAS peptide mutant sequence for tumor shrinking, in murine subcutaneous and orthotopic KPC models, similar to the ability to generate antigen specific immune responses in humans. Noteworthy, we also observed that the immune response in KPC mice included generation of tertiary lymphoid structures at the cancer site, a feature also observed in human cancers. This supports the use of an exogenous vaccination strategy to boost the impact of the ICD-related endogenous vaccination response, as discussed below. The rest of the data provided in this section are directly derived from data described in Example 2.

[0135] KRAS frequently presents as driver mutations in PDAC, colorectal and lung cancers, with mutation frequencies of 97.7, 44.7, and 30.9% respectively. Four mutations (G12, G13, Q61, G12D), cover more than 99% of the PDAC mutations, with KRAS G12D contributing up to 45%. Among others, G12C, 1.7%; G12A / S / L / I, 1.4%; G13C / D / P / H / R 1.2%; Q61H, 4.8%; Q61R, 1.2%; Q61K, 0.5%; and others, 0.5%. Common KRAS oncogenic point mutations seen in PDAC patients KRAS mutant peptide Peptide sequence KRA WT MTEYKLVVV A V K ALTI LIModified and unmodified KRAS mutant peptides for vaccination KRAS mutant peptide Peptide sequence

[0136] Not only do these PDAC mutations drive the development of pancreatic intraepithelial neoplasia but the ability to select immunogenic KRAS peptides for performance of vaccination has greatly improved. This includes evidence that KRAS G12Dcan be expressed on type I murine MHC and human HLA (e.g., HLA-A*11:01 or HLA- C*8:02) antigen presenting complexes, with the ability to induce PDAC targeting by CTLs. This has resulted in demonstrating therapeutic responses to mutated KRAS peptides in human PDAC trials. Thus, while some phase I and II clinical trials have shown longer survival in vaccine responders compared to non-responders, no therapeutic benefit was obtained in advanced-stage pancreatic cancer patients. Vaccine efficacy can be improved in combination with ICD therapy, thereby allowing CTLs with higher affinity to participate in the cancer immunity cycle. From a vaccination perspective, it is also relevant that the administration of an irradiated allogeneic PDAC vaccine (GVAX) could boost the generation of tertiary lymphoid structures in a human PDAC trial. 2) Use of drug delivery nanoparticles for preferential liver uptake and reprogramming of the liver immune landscape to reduce or eliminate tumor antigen tolerance and to defend against metastatic spread

[0137] The STING (Stimulator of Interferon Genes) agonist pathway plays a crucial role in the immune response, particularly in the function of APCs responsible for capture, processing, and presenting antigens to T-cells. The canonical role of the STING signaling pathway is to sense cytoplasmic double-stranded DNA (dsDNA) including host cytoplasmatic chromatin, mitochondrial DNA, and foreign dsDNA, such as viral dsDNA. The STING pathway is involved in detecting cytosolic DNA and activating an immune response against viral infections, intracellular pathogens, and cancer cells. The mode of action of the STING pathway is dependent on several different steps: The first is the detection of cytosolic DNA when APCs are infected by viruses or other intracellular pathogens, releasing their DNA into the cytoplasm. Cyclic GMP-AMP synthase (cGAS) acts as a sensor protein that recognizes cytosolic DNA and produces cyclic GMP-AMP (cGAMP). cGAMP acts as a second messenger that binds to and activates the STING protein, located on the endoplasmic reticulum (ER) membrane. Activated STING undergoes a conformational change, which leads to the recruitment and activation of downstream signaling molecules, including TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3). TBK1 and IRF3 activation results in the production of type I interferons (such as IFN-β) and other proinflammatory cytokines. These cytokines play a pivotal role in initiating antiviral and anti-tumor immune responses, attracting immune cells, and enhancing the adaptive immune response. All considered, the impact of STING activation in APCs, particularly dendritic cells, enhances antigen presentation by upregulating majorhistocompatibility complex class I (MHC-I) molecules and co-stimulatory molecules (such as CD80 and CD86) that promote T cell activation.

[0138] Leveraging the STING agonist pathway for therapeutic use has shown promise in the development of vaccination strategies and cancer immunotherapy. First, this approach has proven particularly beneficial for developing vaccines against challenging pathogens, where the STING pathway has been used for promoting robust antigen presentation, leading to improved T cell activation and memory responses. Many cancer cells also express genetic abnormalities that lead to the production of cytosolic DNA. By using synthetic STING agonists (molecules that mimic the role of cGAMP), researchers have demonstrated that it is possible to activate the STING pathway in tumor-infiltrating dendritic cells, capable of initiating strong antitumor immune responses that can control and eliminate cancer cells. This is particularly relevant for the improvement of antigen presentation of unique cancer antigens that are capable of triggering immune responses, but present in low abundance or not effectively presented by DC. For pancreas cancer, several neoantigens (e.g., KRAS and TP53 mutagenic regions) or tumor-associated antigens (TAA, e.g., MUC1, or mesothelin) have been identified as potentially useful vaccinating antigens that can be introduced to the immune system as peptide vaccines (synthetic fragments of the antigens), viral vectors (delivering antigens using modified viruses), or nucleic acid-based vaccines (delivering genetic encoded antigens).

[0139] Among the natural cyclic dinucleotide (CDN) molecules available as cGAMP mimics there are 2′,3′-cGAMP, 3′,3′-cGAMP, c-di-GMP, and c-di-AMP. While 2’,3′- cGAMP is produced by cGAS in mammalian cells, the latter three are produced by bacterial cGAS. The biology of these CDNs involves cGAMP binding to STING in an "open-state" shape, triggering a conformational change to a "closed-lid" state, leading to oligomerization and activation. However, using cGAMP or other natural CDNs in vivo has significant challenges due to their poor pharmacokinetic profile. Their net negative charge and high polarity result in limited cellular internalization. Additionally, their susceptibility to enzymatic degradation, caused by rapid hydrolysis of the phosphodiester linkage, leads to a short half-life. To address these issues, one approach has been directed toward creating hydrolysis-resistant derivatives, which mainly involves phosphothioate analogues. For instance, replacement of the non-bridging oxygen atoms in the phosphate linkage with sulfur atoms has significantly increased the half-life, extending it up to 24 hours while maintaining high potency. In addition, substituting the 2'-hydroxyl group with chlorine or fluorine has proven effective in enhancing stability while preserving activity. Otherstability-enhancing modifications encompass substitutions in the ribose or the bases. Among the phosphothioate analogues, Rp,Rp-2′,3′-c-di-AMPSS (ADU-S100) has emerged as one of the most promising ligands that has undergone evaluation in clinical trials. While these modifications have significantly improved the hydrolytic stability of CDNs, they still do not fully address the challenges associated with their polar nature.

[0140] In addition to the chemical modifications mentioned earlier to enhance stability and cellular internalization of CDN STING agonists, another promising approach involves incorporating these agonists into nanocarriers that can encapsulate and protect therapeutic molecules from degradation during their journey in the body. In addition, nanocarriers can be engineered to improve the cellular internalization of CDN STING agonists and to release the agonists in a controlled manner into the cytosol. Another advantage is the potential to provide targeting to specific tissues or organs, such as the liver. This minimizes off-target side effects and reduces the dose required for therapeutic efficacy. The exploitation of nano- enabled delivery of STING agonists to improve cancer immunotherapy has been demonstrated for several material compositions, including polymeric nanoparticles, liposomes, and micelles. Noteworthy, it has also been shown that applied mRNA lipid nanoparticles (LNP) can be used to deliver a permanent-active gain-of-function STING- R284S mutant into PDAC cells, useful for inducing intratumoral infiltration of CD8+ positive T cells, leading to robust cancer cell death. While this study did not involve incorporation of a STING agonist, a STING agonist-derived LNP-mRNA vaccine has been demonstrated to be able to enhance protective immunity against SARS-CoV‑2. This STING agonist involved a library of non-nucleotide STING agonist-derived amino lipids instead of CDN.

[0141] Considering this background, cationic LPN will be useful for reprogramming the tolerogenic status of liver APCs by encapsulating CDNs. Our initial choice focuses on 2’,3’-cGAMP, chosen as a CDN that can be incorporated in the LPN matrix by interacting with a cationic lipid, which can serve to bind to mRNA, as well as contributing to endosomal release in the intracellular APC environment. Reprogramming of the tolerogenic APC response for mounting a cytotoxic T-cell response to metastasized pancreatic cancer cells, could be further boosted by inclusion of mRNA constructs for the expression of mutated KRAS epitopes in the liver. For this purpose, we have chosen G12D as one or more frequent mutant KRAS epitopes, expressed in human PDAC as well as being displayed with an identical sequence in the KRAS pancreatic cancer (KPC) animal model.

[0142] Cationic LNP were developed to deliver 2’,3’-cGAMP plus or minus a polyA nucleotide sequence. LNP synthesis was accomplished using a microfluidics synthesizer (Nanoassemblr, Precision NanoSystems Inc) that mixes the ingredients in an aqueous flow stream that include the polyA nucleotide sequence (plus or minus cGAMP) with a flow stream containing an appropriate lipid mix, dissolved in ethanol. PolyA was used as an inexpensive nucleic acid source for practicing particle synthesis prior to including the more expensive custom designed KRAS mRNA strand. The initial particle design made use of a lipid composition that favors hepatic biodistribution, without particle surface decoration with a specific targeting ligand. The liver biodistribution is achieved based on the formation of a protein corona that favors binding to the apoB receptor. Fig.3F shows calculations for preparing aqueous and organic phases to be blended in the microfluidics mixer. (i) 2’,3’- cGAMP and PolyA were dissolved in PBS at 5 mg / mL respectively, to prepare the aqueous phase. Ionizable cationic lipid DLin-MC3-DMA, together with neutral lipids DSPC, cholesterol and DMG-PEG2000, were dissolved in ethanol at 20 mg / mL respectively, to prepare the organic phase at a molar ratio of 50 / 10 / 38.5 / 1.5 for DLin-MC3- DMA / DSPC / cholesterol / DMG-PEG2000. (ii) The N / P ratio, defined as the molar ratio of the cationic lipid (MC3) versus the negatively charged phosphate groups present in cGAMP and polyA, was set at a 4:1 ratio. The flow rate ratio for mixing of aqueous vs organic phase was set at a 3:1 ratio to produce a two mL LNP volume. To calculate the exact quantities and volumes of cGAMP, polyA and lipids, cGAMP and polyA were dissolved in 100 mM sodium acetate buffer (pH 4.0), while the lipids were dissolved in ethanol. Fig.3A provides a schematic diagram showing LNP construction by the microfluidics mixer. The flow rate was 12 mL / min. The primary LNP samples were dialyzed against PBS (pH 7.4) at 4oC for 1 h, followed by filtration through a 0.2 μm filter. For comparative analysis, three different LNPs were constructed, namely PolyA / LNP, cGAMP / LNP and cGAMP / PolyA / LNP. Fig. 3G illustrates physical characterization of LNPs. The size and the surface potential of the purified LNP samples were determined by dynamic light scattering (DLS), using a Zetasizer Nano ZS from Malvern Instruments Ltd. The concentration and encapsulation efficiency (EE, %) of polyA were determined using a RiboGreen assay kit (Thermo Fisher). The concentration of cGAMP was determined by cGAMP ELISA kit (Thermo Fisher). Briefly, the size of cGAMP / LNP, PolyA / LNP and cGAMP / PolyA / LNP were 81.6 nm, 92.2 nm and 89.7 nm, respectively, with relatively low PDI (< 0.2) and neutral surface potential. The EE of PolyA in PolyA / LNP and cGAMP / PolyA / LNP are all higher than 90%, and the concentration of cGAMP in cGAMP / LNP and cGAMP / PolyA / LNP were16.76 μg / mL and 11.93 μg / mL. These results establish successful synthesis of single drug- loaded LNP(cGAMP / LNP, PolyA / LNP) as well as a dual cGAMP / PolyA loaded LNP (cGAMP / PolyA / LNP) with good encapsulation efficiency.

[0143] Design and synthesis of a Kras G12D mRNA strand for the construction of a cationic LNP that co-delivers cGAMP: The RAS family of human proto-oncogenes encode mutated versions of 21-kD guanine nucleotide-binding wildtype p21 RAS, serving as key mediators that regulate cell growth and differentiation. PDAC express mutant KRAS epitopes in over 90% of tumors, with the most frequent single amino acid missense mutations appearing at G12 (98%), G13 (21%) or Q61 (28%) (See: http: / / cancer.sanger.ac.uk / cancergenome / projects / cosmic). Eight different amino acid substitutions have been identified at G12, with G12D the predominant mutation (51%). Reference Target Peptide sequence Length Gjerstsen MK Lancet 1995 G12D KLVVVGAXGVGKSALTI wherein X 17

[0144] Not only do these KRAS mutations serve as drivers of pancreatic intraepithelial neoplasia but exert immunogenic effects that allow performance of vaccination studies,including the example shown for a G12D peptide. In addition, several human clinical trials have been launched using different KRAS mutant sequences, including a clinical trial making use of a mixture of 7 synthetic RAS peptides (17 aa peptides combined in a formulation designated as TG01), which represent the most common codon 12 and 13 oncogenic KRAS mutations.

[0145] Based on our efficacy demonstration for the M23 peptide as well as the human vaccination studies discussed above, we selected a 17 amino acid (aa) sequence for constructing a liver biodistribution team cationic LPN carrier. There is a high level of homology between the human and murine KRAS peptide sequence, in which the first 21 amino acids (including the G12D sequence) are identical (Fig.28). We added to natural flanking amino acids to the 17 aa G12D sequence, on both the N- and C-terminal sides to provide peptide stability and enhancing immunogenicity by improving antigen presentation. The mutant peptide RAS3-23 was reverse translated into nucleotide sequence, using the GenScript online tool. We also included a non-immunogenic control, choosing the corresponding AA sequences for wildtype p21Ras. The control sequence was reverse translated as shown.

[0146] For the design of a codon-optimized mRNA strand to express Kras G12D and wildtype Ras epitope sequences (SEQ ID NO:77), five tandem repeats of each flanked nucleotide sequence were incorporated into the nucleotide sequences. Start and stop codons were added to the cDNA, before the performance of codon optimization, using the GenSmartTM Codon Optimization tool. This yielded GC contents of 52.08% and 53.65% for the mutant and wildtype cDNA sequences, respectively. Trilink was contracted for in vitro synthesis of a codon-optimized expression mRNA strand, also endowed with 5’ and 3’ untranslated region (UTR) sequences. While the 5' UTR is important for ribosome recognition, the 3' UTR is targeted by microRNAs and RNA-binding proteins post- transcriptionally. All uridine bases were substituted with N1-methyl-pseudouridine for enhancement of RNA stability as well as eliminating innate immune danger signals that could impact the immune response. The co-transcriptional procedure used for attachment of the cap sequences as well as a PolyA (120) tail utilized CleanCap® technology, premised on the natural Cap 1 structure. Following in vitro transcription, DNase and phosphatase treatment were performed, including silica membrane purification to obtain single-stranded mRNA strands of the expected size (582 nt). The final products were packaged and delivered in 1 mM sodium citrate buffer, pH 6.4.

[0147] LNP synthesis was carried out by using the microfluidics synthesis procedure described in Fig.4, which was adapted for the synthesis of cationic LNP that encapsulate cGAMP only, KRAS G12D mRNA only, cGAMP plus KRAS G12D mRNA or cGAMP plus p21p Ras mRNA (Fig.3). Fig.3A shows the lipids used for NP synthesis, including cationic ionizable DLin-MC3-DMA (MC3), neutral lipids DSPC and cholesterol, and PEGylated lipid DMG-PEG2000, in the molar ratio 50:10:38.5:1.5. MC3 is important for encapsulating negatively charged cGAMP and mRNA, as well as their intracellular release. Particle physicochemical characterization was carried out as described in Fig.3G. DLS was used to measure the hydrodynamic size of cGAMP / LNP, mKRAS / LNP, cGAMP / mCtrl / LNP and cGAMP / mKRAS / LNP, yielding measurements of 83.1, 90.2, 95.5 and 96.7 nm, respectively. All PDI values were less than 0.2, with negatively charged zeta potentials. The encapsulation concentrations of the mRNA’s were quantified by a RiboGreen Assay, while the cGAMP concentration was quantified by an ELISA kit. We verified that there is no interference of these analogs with each other.

[0148] Figs.6A-E show a demonstration of in vitro STING activation by free and encapsulated cGAMP in KPC cells. The schematic explains the conducting of a cellular experiment in KPC cells to assess the pro-inflammatory STING affects, using real-time RT- qPCR to quantify interferon beta-1 (Ifn-β1), CCL5, CXCL9, CXCL10. KRAS, pancreatic cells (KPC) cells were plated in 6 well plates, 50,000 cells per well. After 24 h incubation at 37oC, cells were incubated with saline, free cGAMP, cGAMP / LNP, mKRAS / LNP, cGAMP / mCtrl / LNP, and cGAMP / mKRAS / LNP. The LNP synthesis and physicochemical characterization is the same as described above. The concentrations of cGAMP and mRNA (mKRAS, mCtrl) were 1 μg / mL and 4 μg / mL, respectively. After 24 h, cells were collected and total RNA isolated by TRIzol extraction according to the manufacturer’s instruction, followed by the RNA quantification, using a Nanodrop instrument. These extracts were used as templates for RT-PCR analysis, using specific sets of primer pairs to quantify the mRNA levels for GAPDH Ifn-β1, CCL5, CXCL9, and CXCL10 (STING-responsive cytokines and chemokines). Fig.6B shows a comparative increase in the abundance of Ifnb1 mRNA expression in KPC after exposure to the saline control and list of stimuli. The expression level for the saline control was set at 1.0. In contrast, there was no increase in the relative abundance of GAPDH mRNA, used as an internal control. Fig.6C shows relative increase in the abundance of CCL5 mRNA expression by the same stimuli. Fig.6D shows relative increase in CXCL9 mRNA expression. Fig.6E shows relative increase in CXCL10 mRNA expression.

[0149] No statistically significant increases in mRNA abundance for each of the four cytokine / chemokines were observed in KPC cells treated with free cGAMP or the mKRAS / LNP compared to the saline control. This confirms that the negatively charged cGAMP do not gain access to the living KPC cells and that mKRAS expression has no impact on the STING pathway. In contrast, the three LNPs containing encapsulated cGAMP, increased mRNA expression of the cytokine / chemokines, without any significant difference between particles containing cGAMP only versus the CDN plus a mRNA strand.

[0150] Tandem experimentation was conducted to determine the impact of post-metastatic implant administration of free and encapsulated cGAMP, plus or minus, co-delivery of KRAS mRNA. For logistical reasons, the experimentation was carried out as two separate experiments for which the data were ultimately combined to allow comparative analysis among five animal groups. Hemispleen injection was carried out prior to IV administration of the treatment modalities used in each experiment. On day 0, 50, 0000 KPC-luc cells were injected into a hemispleen section in 8 weeks old female B6129SF1 / J mice. Animals were randomly divided into 3 groups, with 8 mice per group, receiving intravenous saline, cGAMP / mCtrl / LNP, or cGAMP / mKRAS / LNP injections on days 6, 9, 12, and 15. The doses for cGAMP and mRNA were 0.3 mg / kg and 1.25 mg / kg, respectively. The body weight was monitored every 3 days after the first dose. Tumor growth was monitored by in vivo IVIS imaging on days 5, 11, 14, and 18. Following the last image collection, the mice were sacrificed after blood collection, and the liver tissues were isolated for weighing, ex vivo imaging, and histological analysis. Serum IL-6 and IL-10 were quantified by ELISA. Essentially the same regimen was followed for conducting another experiment, with the exception that the treatment groups were comprised of animals receiving saline, cGAMP / LNP and mKRAS LNP. In vivo IVIS images were collected for each mouse on days 5, 11, 14, and 18. Two mice in the saline group died before day 18.

[0151] Luminescence signals, reflecting the growth of liver metastases, were collected from day 5 onwards, followed by subsequent slowdown in growth with specific treatments. The most intense luminescence was obtained in animals treated with saline, while the least intense signals were seen in in cGAMP / mKRAS / LNP group. Calculation of the radiance flux and statistical analysis demonstrated that average value of the saline group was significantly higher than cGAMP / mCtrlLNP group (p< 0.05) as well as the cGAMP / mKRAS / LNP group (p < 0.001). The radiance for the cGAMP / mCtrl / LNP group was also significantly higher than cGAMP / mKRAS / LNP group (p < 0.05), proving that the therapeutic effect of cGAMP / mKRAS / LNP is superior to cGAMP / mCtrl / LNP. Whileanimals maintained a stable body weight until day 15, the subsequent loss of body weight in the saline treated group reflects declining health and ultimate fatality due to the fastest growing tumors. Moreover, the lower liver weights on day 18 in the cGAMP / mKRAS / LNP group, in combination with the weakest luminescence signals obtained in explanted livers reflect the therapeutic impact of this combined treatment modality., No obvious changes were seen in serum IL-6 and IL-10 levels, in keeping with the lack of a systemic inflammatory response during LNP treatment.

[0152] Comparing single cargo LNP delivery (cGAMP / LNP and mKRAS / LNP) with saline in the hemispleen model, was performed with the same methodology as described above. Four doses of saline, cGAMP / LNP and mKRAS / LNP were intravenously injected into mice in different groups on days 6, 9, 12, and 15. The doses for cGAMP and mRNA were 0.3 mg / kg and 1.25 mg / kg, respectively. Body weights were monitored every 3 days, while tumor growth was monitored by in vivo IVIS imaging on days 5, 11, 14, and 18.

[0153] Luminescence signals were captured for each mouse from day 5 onwards, reflecting the growth of the metastatic tumors. The strongest signal was seen in the saline treated group, with lowering of the signal intensity during treatment with the cGAMP / LNP for mKRAS / LNP carriers. Moreover, calculation of average radiance intensity was significantly higher for the saline compared to the cGAMP / LNP (p < 0.05) and mKRAS / LNP groups (p < 0.05). No significant difference was observed between cGAMP / LNP group and mKRAS / LNP groups, showing that both encapsulated entities had significant effects on pancreatic cancer liver metastasis. Like the first experiment, there was a drop in body weight in the saline treated group after 15 days, reflecting the impact of rapid cancer growth on general health status. Ex vivo imaging of the liver tissue showed the strongest luminescence in the saline group, which was significantly higher than other two groups (p < 0.05). The average liver weight of the saline group was also significantly higher than the treatment groups (p < 0.05). No obvious changes in serum IL-6 and IL-10 levels were observed, like the first experiment.

[0154] Integrated data for the tandem experimentation was performed to assess the post- treatment effect of KPC cell growth in the liver following hemispleen injection. The data from experiments 1 and 2 were integrated, using metastatic tumor growth in animals receiving saline injection for data reconciliation. Following injection of 50 k KPC-luc cells into the hemi-spleen of female B6129SF1 / J mice, 4 doses of saline, cGAMP / LNP, mKRAS / LNP, cGAMP / mCtrl / LNP, or cGAMP / mKRAS / LNP were intravenously injected on days 6, 9, 12, and 15. The body weight was monitored every 3 days after the first dose.The tumor growth was monitored by in vivo IVIS imaging on days 5, 11, 14, and 18. After the final IVIS imaging data were obtained, mice were sacrificed, blood collected, and the liver tissues harvested for weighing, ex vivo imaging, and histological study. The serum IL- 6 and IL-10 were quantified by corresponding ELISA kits.

[0155] Use of the integrated luminescence signals confirm that the highest signaling intensity is from saline group, while the signal from the dual delivery carrier, cGAMP / mKRAS / LNP, is the lowest. Statistical analysis showed that the average radiance in the cGAMP / mKRAS / LNP group is significantly lower than saline group (p < 0.001), cGAMP / LNP group (p < 0.05), mKRAS / LNP group (p < 0.05), and cGAMP / mCtrl / LNP group (p < 0.05). This confirms a synergistic contribution by cGAMP as well as mKRAS to metastasis inhibition, implying that both reprogramming of the immune landscape as well as generating an epitope specific cellular response is involved in the combined outcome. The decline in body weight in the saline treated group after day15 was discussed above. Ex vivo IVIS imaging also confirmed that mice treated with cGAMP / mKRAS / LNP showed significant reduced metastatic tumor growth, also reflected in the lowest liver weights. There was no change in serum IL-6 and IL-10 levels with LNP treatment.

[0156] For further verification of the therapeutic contribution of cGAMP during encapsulated delivery, harvested liver tissues were homogenized and used for RNA isolation using the TRIzol reagent before performance of qPCR quantification of IFN-β1 and GAPDH mRNA expression. While all LNPs increased the IFN-β1 mRNA expression, the biggest increase was obtained for the cGAMP / mKRAS / LNP group, which was statistically significant from saline group (p<0.001), cGAMP / LNP group (p<0.01), mKRAS / LNP (p<0.05), and cGAMP / mCtrl / LNP (p<0.01). Interestingly, the delivery of mKRAS as well as cGAMP could increase IFN-β1 expression, with evidence of a combinatorial effect.

[0157] Animal survival experiments compare the outcomes for groups 1-5 using post- metastatic treatment. We hypothesized the likelihood of survival extension in animals receiving encapsulated delivery of cGAMP and KRAS mRNA, individually and in combination. Hemispleen injection commenced with 50,000 KPC-luc cells being injected on days zero, followed by treatment on days 6, 9, 12 and 15. The tumor growth was monitored every week by in vivo IVIS imaging, while the animals were being observed for health status. Animals developing hypoactivity and signs of defining health status were euthanized as per the approved institutional protocol. The first animal deaths in the saline group occurred on day 15, with all for animals being sacrificed by day 23. There wassignificant survival for the animals receiving treatment with LNPs, with the last two animals being euthanized after five weeks. Characterization of the LNP prepared for this experiment is provided.

[0158] The Kaplan-Meier plot shows significant prolongation of median survival up to 24 d, 24 d, 25.5 d and 31 d, respectively, for the cGAMP / LNP, mKRAS / LNP, cGAMP / mCtrl / LNP, or cGAMP / mKRAS / LNP groups, compared to 19 d for the saline group. Statistical analysis indicates that the best survival for the cGAMP / mKRAS / LNP treated group, which was statistically significant to all other encapsulated deliveries. This includes p values < 0.001, compared to groups 1-3 and a p < 0.005 against group 4.

[0159] Accordingly, in certain embodiments a drug delivery vehicle for reprogramming the liver immune landscape to reduce or eliminate tumor antigen tolerance and to defend against metastatic spread is provided. In certain embodiments the vehicle comprises: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle configured for preferential liver uptake wherein said nanoparticle comprises: one or more STING agonists; and one or more a tumor-associated neoantigens or a nucleic acid encoding one or more a tumor neoantigens. 3) Extending the liver metastasis treatment platform, e.g., by targeting tumor- associated lymphoid structures to also address the primary PDAC site, optionally in combination with chemo-immunotherapy.

[0160] Successful design of a cationic LNP that delivers a STING agonist plus nucleic acid KRAS epitopes introduces the possibility of extending the liver metastasis treatment platform to also address the primary PDAC site, including in combination with chemo- immunotherapy. This strategy allows combination of the features of endogenous (chemotherapy-induced) and exogenous vaccination (nucleic acid neoantigens) strategies in the cancer immunity cycle. In addition to the role of STING in priming APC responses, with the potential to reprogram the tolerogenic effects of liver APC, STING also facilitates cancer cell death and release of cancer cell antigens. In this context, we are particularly interested in the contribution of regional lymph nodes and the spleen in the PDAC immunity cycle, as well as the potential role of tertiary lymphoid structures that are induced at the primary cancer site by mutant KRAS peptides, as demonstrated in Figs.21 and 23. Not only do these lymphoid structures act as a filtering station for released tumor antigens, which can be presented to T-cells but are also an excellent potential target for nanocarriers that can be used to augment activation of antigen-specific T-cell subsets that are educated to recognize prominent neoantigens. This aspect will be discussed below. It is believed thatthis will augment critical subsets of the T-cells that are responding to a wider pool of endogenously released neoantigens and tumor-associated antigens (TAA).

[0161] While APC reprogramming by STING may suffice in generating a protective anti- PDAC immune response, without being bound to a particular theory, it is believed that the immune response at the primary cancer site can also be strengthened with the inclusion of neoantigens or TAAs in the cationic LNP. This could generate a pool of antigen-specific CTLs and memory T-cells that are available for recruitment to the metastatic immune landscape in the liver.

[0162] Among several choices, PDAC tumors express mutant KRAS epitopes that appear in over 90% of those cancers, one of which (the G12D epitope) is investigated and described herein. We demonstrate that an encapsulated G12D peptide can successfully generate antigen specific CTLs that participate in shrinking at the primary cancer site, including through the generation of tertiary lymphoid aggregates at the primary orthotopic PDAC site.

[0163] Additionally, we also provide for vaccination with a KRAS peptide to delivering a mRNA construct that encodes for multiple repeats of the same KRAS neoantigen, to be delivered by a cationic LPN that can also accommodate one or more STING agonists. Moreover, a nucleic acid approach also allows the inclusion of multiple KRAS epitopes, of widespread significance to PDAC and many other KRAS dependent cancers. Further, the use of LPN also allows the inclusion of STING agonists and a lipid tailed TLR7 agonist that can be accommodated by the same carrier. Also, the construction of cationic LPN is highly scalable and leads to the delivery of a degradable mRNA, lack of DNA integration, intracellular release at the site of action and decrease of systemic toxicity.

[0164] Successful design of a cationic LNP that can deliver a STING agonist, with and without nucleic acid KRAS epitopes, also facilitates the use of cationic LNP, in combination with ICD-inducing nanocarriers that can be used to treat primary as well as metastatic disease, through the intermingling of endogenous and exogenous vaccination. Combining chemo-immunotherapy with KRAS vaccination will generate additional response synergy through strengthening the cancer immunity cycle by enhancing the supply of additional antigen specific CTL. It is also possible to provide for the encapsulation of a TLR7 agonist that displays a lipid tail (e.g., 3M-052) in these particles, which would serve the function of providing an additional immunomodulator combination that strengthens APC activation and contributing to the anti-PDAC immune response. Illustrative, embodiments include, but are not limited to the following.

[0165] In one illustrative embodiment, the scope of the epitope and antigen delivery by cationic LPN, can be expanded, for example, by: (i) encapsulation of additional Kras mutant mRNA sequences or the synthesis of multi-epitope vaccines; (ii) encapsulation of epitope sequences from other PDAC TAA’s and neoantigens; (iii) endowing the particle surface with targeting ligands that facilitate uptake by lymphoid organs, including lymph nodes, spleen and tertiary lymphoid structures; (iv) endowing the LPN with STING agonists, including for STING agonists other than 2’,3’-cGAMP.

[0166] In another illustrative, but non-limiting embodiment, the combined use of nanocarriers that promote chemo-immunotherapy with cationic LPN can generate synergistic endogenous and exogenous vaccination responses. This includes the use of liposomes and silicasomes that deliver ICD-inducing chemotherapeutic agents plus other immunomodulators that can be creatively combined in a synergistic platform.

[0167] As noted above, KRAS mutations involved in PDAC generation and peptide selection for constructing vaccinating nanoparticles are shown below. The upper table outlines common Kras point mutations found in PDAC patients, from which we selected the G12D mutation for constructing mutant peptides for nanoparticle synthesis. Common KRAS oncogenic point mutations seen in PDAC patients KRAS mutant peptide Peptide sequenceModified and unmodified KRAS mutant peptides for vaccination KRAS mutant peptide (23 Peptide sequence

[0168] Owing to the hydrophobicity of the native mutant 23mer Kras peptide, termed M23, the modification of its terminal amino acids on either ends with aspartate residues enhanced its solubility, creating a hydrophilic 23mer designated as D23. Fig.20A further shows how the mutant Kras peptides (M23 or D23) are incorporated into the polymeric shell and aqueous core, respectively, along with the TLR7 / 8 agonist, R848. This was accomplished by a modified double emulsion solvent evaporation method. Briefly, a mixture of KrasG12D mutated peptides (23 mer) and R848 were added to PLGA, dissolved in dichloromethane (DCM), and sonicated to create a water in oil emulsion (w1 / o). This emulsion is added to a 1% solution of sodium cholate and sonicated again to form a water in oil in water double emulsion (w1 / o / w2). Vaccine particles were harvested after evaporating solvent overnight under continuous stirring at room temperature in 0.1% sodium cholate solution by ultracentrifugation.

[0169] A hemispleen injection liver metastasis model was developed. The hemispleen model is an experimental approach to investigate liver metastases by injecting KRAS pancreatic cancer (KPC) cells injected into a half-spleen section that contains a vascular pedicle in with venous training to the portal circulation. This approach requires a mini laparotomy in 8-week-old female B6129SF1 / J mice to mobilize the spleen for placement of two clips, allowing the organ to be sliced into two parts, one of which (the outer portion) is injected with 2 x106KPC-luciferase cells in 100 μL PBS. This splenic portion is resected a few minutes later, while the other half of the spleen is transposed into the abdominal cavity before closing with sutures. Animals were followed by IVIS imaging at regular intervals to observe the metastatic tumor growth and the impact of cationic LNP treatment of tumor growth by reprogramming the liver immune suppressive microenvironment. Hemispleen injection on day zero under isoflurane anesthesia, followed by the growth of KPC luc cells was visualized by IVIS imaging. Results show progressive tumor growth, as demonstrated by quantification of the average radiance intensity.

[0170] Since the immunogenicity of peptide vaccines is enhanced by lymph node (LN) delivery, we reasoned that the same approach could applied to the appearance of TLS in our murine model. Typically, the uptake of vaccinating nanoparticles in LNs are facilitated by a particle size of 25 to 200 nm. It has also been demonstrated that the immunogenicity of peptide vaccines can be enhanced by conjugating the peptides to TLR agonists or a carrier protein such as albumin. The strategy that we adopted was to use passive KRAS peptide encapsulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles that also include aTLR7 / 8 agonist, resiquimod (R848) (using e.g., SEQ ID Nos: 6 or 7; Fig.20A-C). PLGA nanoparticles are avidly taken up by LNs, allowing efficient encapsulation and cargo release for LN antigen presentation. Carrier synthesis was achieved by using a double-emulsion solvent evaporation technique to construct two distinct nanoparticles versions (Fig.20A). In the first embodiment, the unmodified M23 peptide, sparingly soluble in water, was added to the oil phase containing PLGA in dichloromethane, with R848 being added in the entrapped aqueous phase. These yielded particles encapsulating M23 peptide in the shell, with R848 in the aqueous core. In the second embodiment, the KrasG12D peptide was modified on both ends with aspartate residues for enhancing water solubility (SEQ ID NO: 7). This peptide was added to the inner aqueous phase, combined with R848, while the oil phase was comprised of PLGA solubilized in DCM (Fig.20A). Using the double emulsion approach, another nanoparticle was constructed where both the hydrophilic D23 peptide and R848 were both encapsulated in the aqueous core (Fig.20A). We also synthesized a DIiR-labeled (M23 / DiR) fluorescent nanoparticle with an empty core and a scrambled peptide (polyD) nanoparticle encapsulating the peptide with R848 in the aqueous core (Fig.20C). This yielded nanoparticles in the 150-200 nm size range with a zeta potential of -70mV, conducive to lymphatic spread. The SEM images further confirmed the size and polydispersity distribution (Fig.20B). Both D23 and M23 containing nanoparticles showed 23% loading efficiency of R848.

[0171] To demonstrate the agonist activity of the encapsulated R848, the nanoparticles were co-incubated with murine HEK-Blue cells to demonstrate a dose-dependent increase in TLR7-mediated reporter gene activity. We also employed the DiR-labelled nanoparticle to demonstrate lymphatic spread to regional lymph nodes when injected subcutaneously in B6 / 129J mice. Compared to the free DiR, the particle-encapsulated dye was retained for a prolonged period at the regional LN sites during live in vivo imaging (IVIS).

[0172] To determine vaccination impact on anti-tumor immunity, B6 / 129J mice were subcutaneously injected with M23+R848 PLGA and D23+R848 PLGA nanoparticles 13 days prior to subcutaneous challenge with Kras pancreatic cancer cells (KPC). As a control for the vaccinating nanoparticles impact, we also used subcutaneous injection of KPC cells treated with an ICD-inducing dose (300 mM) of irinotecan to demonstrate interference in live KPC growth.5×104KPC cells, undergoing ICD, were administered in one flank of the animal to induce a protective immune response, demonstrated by growth failure of live KPC cells, injected in the contralateral flank a few days later. The possible protective effect of the nanoparticles was tested by injecting the animals in one flank on three occasions, sevendays apart, with particles delivering peptide doses of 10 µg M23 or D23, plus 2 µg R848. Additional controls included animal groups vaccinated with an admix of free peptide plus adjuvant (i.e., M23+R848 admix and D23+R848 admix). A day prior to the final boost, the mice were challenged with 1.5×105live KPC cells subcutaneously on the contralateral flank and observed for tumor growth, as described in Fig.21A. The animals were monitored for 45 days during which tumor volumes and animal weights were recorded (Fig.21B-21C). This demonstrated that 50% of mice vaccinated with M23+R848 PLGA remained tumor- free for up to 45 days after challenge (Fig.21B). Further, in tumor-developing animals, there was significantly slower growth in the nanoparticle treated animals compared to unvaccinated or animals receiving the free (M23+R848) admix. All the animals vaccinated with irinotecan treated KPC cells remained tumor free, indicating the efficacy of the ICD effect. In contrast to the results obtained with the encapsulated M23 / R848 combination, animals vaccinated with D23+R848PLGA had higher instances of tumor formation where 5 out of 7 vaccinated mice (70%) developed tumors. However, like M23+R848 PLGA vaccination, the animals receiving D23 vaccine nanoparticles showed considerable slowing of tumor growth compared to admix vaccinated mice. (Fig.21C).

[0173] Mice remaining alive were sacrificed 45 days after challenge for tumor harvesting and IHC analysis. IHC staining to demonstrate the presence of CD8+ tumor infiltrating lymphocytes confirmed a significant increase in CTLs for tumors in all mice vaccinated with both M23+R848 PLGA and D23+R848PLGA nanoparticles, compared to the free admix or unvaccinated mice (Figs.21D-21E). We also observed the emergence of tertiary lymphoid structures (TLS), a feature of tumor immunity that has been more frequently recognized recently. Noteworthy, examination of the tumors in M23+R848 PLGA vaccinated mice, demonstrated the appearance of intra-tumoral TLS in 50% of tumors, with an illustrative example shown in (Fig.21F). This is illustrated by the H&E staining as well as the IHC analysis of CD8+ CTLs and CD21+ follicular dendritic cells in these structures (Fig.21F). In contrast to the results with the M23 encapsulated peptide, only 20% of tumors in the D23+R848 PLGA vaccine group developed lymphoid aggregates. In summary, the M23+R848 PLGA vaccine elicited a stronger and more persistent immune activation compared to D23+R848PLGA, evidenced by fewer instances of tumors, smaller tumor sizes and consistent formation of lymphoid aggregates.

[0174] To further assess the efficacy of the Kras vaccine in an orthotopic KPC-luciferase model, we selected the more robust M23+R848 PLGA carrier for prophylactic administration and histological analysis. Using the same vaccination timeline anddosimetry, tumor-bearing mice were vaccinated with the encapsulated M23+R848 PLGA combination (Fig.22A), prior to the implantation of 0.5×106KPC-luciferase cells in the pancreas, a day prior to the final boost. The control groups included animals that are not vaccinated or vaccinated with either M23 nanoparticles (without R848) or nanoparticles delivering a scrambled peptide (polyD) plus R848. The mice were monitored every 4 to 5 days for tumor progression using IVIS imaging (Figs.22B-22C). Mice were sacrificed 16 days post-challenge, followed by harvesting of tumors and other key organs for ex vivo imaging. This demonstrated the presence of smaller tumors with reduced luminescence intensity in mice immunized with M23+R848 PLGA, compared to unvaccinated or animals receiving the (polyD+R848) PLGA or M23 PLGA (no adjuvant) vaccines (Fig.22D). Subsequent performance of ex vivo IVIS analysis also demonstrated larger tumor masses with metastatic spread to surrounding organs in non-treated animals, while animals receiving M23+R848 PLGA showed significantly smaller tumors, judged by tumor luminescence and weight (Figs.22E-22F). This response was also significantly improved over animals vaccinated with (polyD+R848) PLGA or M23 PLGA. The observations suggest that control mice receiving injections with peptide or adjuvant only showed an inferior response compared to animals receiving nanoparticles encapsulating both M23 and R848. This reflects the known ability of TLR7 agonists to augment the immune response by the activation of innate immune cells in the LN or secondary lymphoid tissue.

[0175] Further demonstration of the immunogenicity of the M23+R848 PLGA vaccine was obtained by performing ELISpot assays on splenocytes retrieved from vaccinated and unvaccinated mice, followed by ex vivo stimulation with the corresponding Kras peptide. This demonstrated an increased number of IFN-γ+ and TNF-α producing cellular spots in mice vaccinated with M23+R848PLGA nanoparticles (Figs.23A-23B). In comparison, unvaccinated or animals treated with M23 PLGA showed fewer ELIspots. Interestingly, there was an increase in the number of IFNγ+ colonies in the spleens of mice receiving polyD+R848 PLGA, which is of uncertain significance considering that the same increase does not happen for TNFα. Tumor sections were also analyzed f and or T-cell infiltration, demonstrating an increased number of CD8+ and CD4+ T-lymphocytes in the M23+R848 PLGA vaccination group (Figs.23C-23D).

[0176] Moreover, like the results in the subcutaneous experiment, all the M23+R848 PLGA vaccinated mice showed development of lymphoid aggregates (Fig.23E). These immune clusters included positive staining for CD4, CD8 and CD21 cellular markers, which is characteristic of TLS. Despite their potential prognostic relevance, the actual mechanisminvolved in TLS generation at primary cancer sites and the precise contribution to antitumor immunity is incompletely understood. One possibility is that these structures provide an intra-tumoral reservoir of immune cells, available for antigen sampling and potential CTL recruitment to the tumor cores. This hypothesis is now being tested in the orthotopic model, where KRAS vaccination is combined with chemo-immunotherapy as well as other immunomodulators (see approach 3 below).

[0177] Accordingly, in certain embodiments, a drug delivery nanoparticle for enhancing an anti-tumor immune response is provided. In certain embodiments the drug delivery nanoparticle comprises: a lipid nanoparticle, a silicasome, and / or a biocompatible polymer nanoparticle comprising: one or more a tumor associated antigens or tumor-associated neoantigens; or a nucleic acid encoding one or more a tumor associated antigens or tumor- associated neoantigens. In certain embodiments the drug delivery nanoparticle comprises a lipid (lipidic nanoparticle). In certain embodiments, the drug delivery nanoparticle further comprises a TLR7 / 8 or a STING agonist. In certain embodiments, a method of enhancing an anti-tumor immune response is provided where the method comprises administering an effective amount of these nanoparticle to a subject in need thereof. Design principles for encapsulation of additional TAA and tumor neoantigen sequences to provide multi-epitope vaccines.

[0178] Utilizing multiple KRAS mutant epitopes for PDAC immunotherapy in one nanocarrier is an important consideration to address the potential heterogeneity of KRAS mutants that could develop in the same primary tumor type. By targeting a broader spectrum of KRAS epitopes, the prospect of more effective immunotherapy is increased by the immune recognition of a broader spectrum of cancer cells at the tumor and metastatic site, reducing the likelihood of immune escape. Also, this approach takes account of the diversity of KRAS mutations across different cancer types, which may benefit from the same therapeutic approach. As an example, the KRAS mutation at codon 12, presenting as G12D in 70% of PDAC tumors, is also seen in about 50% of colorectal carcinomas, while G12C appears more frequently in non–small cell lung carcinoma (NSCLC) and approximatively 40% of metastatic lung adenocarcinoma cases. Thus, the ease of multi-epitope incorporation into the nucleic acid vaccine allows the achievement of a more comprehensive and versatile immunotherapeutic strategy for cancer treatment, improving the complexity and cost of the synthesis of a comprehensive peptide episode series.

[0179] For illustrative purposes, we demonstrate multi-epitope vaccine design for a combination of mutant KRAS epitopes that are encoded by a series of linked mRNAsequences, leading to the expression of different length epitope sequences. Epitope specificity for T-cells is mediated by the T-cell receptor (TCR), which binds peptides presented in the peptide binding groove of class I or class II major histocompatibility complexes (MHCs), a.k.a. human leukocyte antigens (HLA’s) on APC. While class I MHC binds shorter (8-11 amino aa) sequences that are confined to the groove, MHC II can be loaded with peptide sequences of 11−30 aa in length, allowing some of these epitopes to protrude beyond the groove. This allows different peptide lengths to elicit distinct immune responses. Longer peptides are often associated with CD4+ T cell responses, which can help activate other immune cells and promote a robust and sustained immune response. Shorter peptides are better suited for eliciting CD8+ cytotoxic T cell responses, which can directly target cancerous cells. Interestingly, a variety of peptide lengths have been used with success in human clinical trials, including studies using 20- and 17-mer peptides. For illustrative purposes, we will show epitope selections for a mixture of 19 aa and 17 aa sequences for multi-epitope vaccine design. Illustration 1: Use of 19-mer top-ranking KRAS mutations (G12D, G12V, G12R, G12C, G13D, Q61H), appearing in human PDAC. Amino acid sequences of dominant KRAS mutations frequently appearing in PDAC KRAS mutation Location Sequence SEQ ID NO

[0180] As described in Figure 28, an important consideration for multi-epitope processing involves efficient epitope spacing and proteolytic cleavage in the cytosol for import into the MHC I complex by the TAP reporter. This is accomplishable by using natural flanking sequences, allowing proteolytic cleavage by the 26S proteosome in the cytosol for MHC lloading. The inclusion of flanking sequences and reverse translation of the peptide into cDNA sequences is shown below. Addition of natural flanking sequence and reverse translation for development of a mRNA vaccine KRAS Location Amino acid sequence cDNA sequence mutation Q Q g gg D ga

[0181] These epitope sequences are linked together into a single cDNA construct (SEQ ID NO:43): ACCGAATATAAACTGGTGGTGGTGGGCGCGGATGGCGTGGGCAAAAGC GCGCTGACCATTCAGCTGATTCAGACCGAATATAAACTGGTGGTGGTGG GCGCGGTGGGCGTGGGCAAAAGCGCGCTGACCATTCAGCTGATTCAGAC CGAATATAAACTGGTGGTGGTGGGCGCGCGCGGCGTGGGCAAAAGCGC GCTGACCATTCAGCTGATTCAGACCGAATATAAACTGGTGGTGGTGGGC GCGTGCGGCGTGGGCAAAAGCGCGCTGACCATTCAGCTGATTCAGACCG AATATAAACTGGTGGTGGTGGGCGCGGGCGATGTGGGCAAAAGCGCGCT GACCATTCAGCTGATTCAGGAAACCTGCCTGCTGGATATTCTGGATACCG CGGGCCATGAAGAATATAGCGCGATGCGCGATCAGTATATG (SEQ ID NO:43).

[0182] In addition to relying on peptide cleavage in the cytosolic proteosome for import into the MHC I compartment by the TAP reporter, there are several alternative processing pathways that can be used for antigen presentation by MHC I. This includes use of severalleader sequences that can facilitate TAP-independent access to the MHC I compartment. Some of these peptides are also referred to as TEIPP sequences, the abbreviation for “T-cell epitopes associated with impaired peptide processing”.

[0183] One example is the VLLQAGSLHA (SEQ ID NO:44) sequence that is derived from human preprocalcitonin16-25 (ppCT) signal peptide. This peptide, generated in the endoplasmic reticulum by signal peptidase and signal peptide peptidase, can serve as a lead sequence for accomplishing access of KRAS mutant sequences to the MHC-I compartment. Following the addition of KK flanking sequence, the reverse translation of VLLQAGSLHAKK (SEQ ID NO:2) yielded gtgctgctgcaggcgggcagcctgcatgcg (SEQ ID NO:3), which was added, as a leader sequence upstream of the 6 KRAS mutant sequences in SEQ ID NO:43.

[0184] Additional peptide sequences that can be used for MHC I targeting by upstream inclusion of the corresponding nucleotide sequence, as shown below. Peptides to promote MHC-1 loading for CD8 T-cell activation Source protein MHC class I Sequence Presented upon

[0185] Following the addition of start and stop codons and performance of codon optimization by the GenSmart Codon Optimization Tool, we obtained the cDNA sequence with a GC content of 52.26%, as shown in SEQ ID NO:87.

[0186] A codon-optimized cDNA construct is shown in SEQ ID NO:87:ATGGTGCTGCTGCAGGCTGGCAGCCTGCACGCCACCGAATATAAACTGG TGGTGGTGGGCGCGGATGGCGTGGGCAAAAGCGCGCTGACCATTCAGCT GATTCAGACCGAATATAAACTGGTGGTGGTGGGCGCGGTGGGCGTGGGC AAAAGCGCGCTGACCATTCAGCTGATTCAGACCGAATATAAACTGGTGG TGGTGGGCGCGCGCGGCGTGGGCAAAAGCGCGCTGACCATTCAGCTGAT TCAGACCGAATATAAACTGGTGGTGGTGGGCGCGTGCGGCGTGGGCAAA AGCGCGCTGACCATTCAGCTGATTCAGACCGAATATAAACTGGTGGTGG TGGGCGCGGGCGATGTGGGCAAAAGCGCGCTGACCATTCAGCTGATTCA GGAAACCTGCCTGCTGGATATTCTGGATACCGCGGGCCATGAAGAATAT AGCGCGATGCGCGATCAGTATATGTAA (SEQ ID NO:87). Illustration #2:

[0187] TG01, comprised of a mixture of 17-mer KRAS peptides emerged as the first cancer vaccine targeting KRAS oncogenic mutations in patients with resected pancreatic adenocarcinoma [. TG01 consists of a mixture of 7 synthetic RAS peptides representing the 7 most common codon 12 and 13 oncogenic mutations in KRAS. TG01 peptide sequences (Palmer, Br. J. Cancer 2020; 122(7):971-977). Target Peptide sequence cDNA sequence C T T T T T

[0188] These peptides are 17 aa long and designed to activate both MHC class II-restricted CD4 + helper T cells, as well as MHC class I-restricted CD8 + cytotoxic T cells, which arenecessary to sustain the CD8 + cytotoxic T-cell effect. Prior to the Palmer study, several studies have been performed with between 1-7 RAS peptides (same peptides as in the TG01-01 study), showing with 4 peptides, 20 / 34 (58%) of patients mounted an immune response, whereas in the 7-peptide study, 12 / 12 (100%) of patients mounted an immune response. Based on these findings, it was concluded that the use of the 7 peptides would account for most mutations in PDAC.

[0189] The 17 aa TG01 peptides can activate both major histocompatibility complex MHC class II-restricted CD4+ helper T cells as well as MHC class I-restricted CD8+ cytotoxic T cells, necessary to sustain the CD8 + cytotoxic T-cell effect. The activated CD4+ cells are also important for enhancing / facilitating cross-presentation of tumor neoantigens and tumor-associated antigens by dendritic cells (DC) at the tumor site and thus broadening the cytotoxic CD8+ anticancer activity. it was also demonstrated that the RAS-mutant-specific T-cell responses can be enhanced by co-administration of GM-CSF (Molgramostim). Reverse translation of the TG01 peptide sequences yields the cDNA sequences shown above, which can be combined into an integrated cDNA construct. For construction of the codon-optimized cDNA strand several options are available to us, including insertion of a nucleotide sequence for the preprocalcitonin16-25 (ppCT) signal peptide or the alternative, shown further above for accomplishing MHC I presentation. This will accomplish a multi- epitope mRNA vaccine for presentation to CD8+ T-cells, as illustrated in Fig.24A. It is also possible to design the cDNA strand by inserting the mutant KRAS epitopes downstream of an MHC II targeting sequences as shown in Fig.24B, using the transferrin receptor (TfR) sequence, aa 1-118, or the invariant (li) chain sequence, aa 1-80. This will allow epitope presentation to CD4+ T-cells that can make an additional contribution to their helper function for cytotoxic T cell responses, including cross-presentation. A third option is constructing a cDNA strand that encodes for the mutant KRAS epitopes under the instruction of both MHC I and MHC II leader sequences by inclusion of a T2A self- cleavage peptide that allows the two sets of epitopes to be separated (Fig.24C). T2A peptides are 18–22 aa viral oligopeptides, e.g., GSGEGRGSLLTCGDVEENPG (SEQ ID NO:64), mediating polypeptide cleavage during translation in eukaryotic cells. The same design applications can also be applied to the longer peptide sequences in Example #1. The rest of the procedure for ex vivo synthesis of a mRNA strand, with the necessary modifications, and synthesis of cationic lipid nanoparticles is like the procedures described herein. Encapsulation of epitope sequences from PDAC tumor-associated antigens (TAAs).

[0190] Based on their expression pattern, tumor antigens are classified into two main categories: (i) Tumor-specific antigens (TSA), including neoantigens, which are endogenously present only on tumor cells, (ii) tumor-associated antigens (TAAs), which are present on both tumor cells and some normal cells (albeit at different levels) and have been effectively targeted in solid malignancies. Tumor-associated antigens are proteins or molecules expressed by cancer cells, including pancreatic cancer cells, that are also present in normal cells, although often at lower levels. TAAs can be targeted by the immune system, but because they are not entirely foreign to the body, the immune response against them may be less potent compared to neoantigens. Thus, use of TAAs for cancer vaccination in comparison to neoantigens, could hold both advantages and disadvantages. Nonetheless, some of the advantages of considering this class of antigens include ease of identification, broad applicability, ease of combining with TSA, general stability that is not subject to rapid mutations in cancer cells and lower risk of autoimmunity.

[0191] Some key TAAs that can be considered for PDAC immunotherapy include, but are not limited to, mucin 1 (MUC1), carcinoembryonic antigen (CEA), mesothelin, survivin, VEGF receptor (VEGFR), human telomerase reverse transcriptase and (HTRT). TAA epitope Peptide sequence

[0192] Specifically, immunogenic peptide sequences, have been identified for these antigens, demonstrating effective response outcomes in human vaccination studies, for example: (i) MUC1 is a glycoprotein, often overexpressed in PDAC, that has been used as a frequent target for vaccine immunotherapy in cancer clinical trials. This includes vaccination with a 100-mer peptide of the MUC-1 extracellular tandem repeat in a human phase I clinical trial in pancreatic patients, with demonstration of mounting a MUC-1- specific T cell response, with 2 of 15 patients alive at 61 months; (ii) Use of the human VEGFR 2-169 epitope in patients with advanced pancreatic cancer, where in combination with gemcitabine, antigen-specific VEGFR2-specific CD8+ cells were generated in 61%patients, with an overall survival time of 8.7 months; (iii) Use of a telomerase-based vaccine, consisting of the human telomerase reverse transcriptase (GV1001) peptide, shown to induce a telomerase-specific immune response in 63% of unresectable pancreatic cancer patients, in which patients mounting a delayed immune response, showing longer survival, further augmented by the use of GM-CSF; (iv) Demonstration that a recombinant CRS-207 live-attenuated, double-deleted Listeria monocytogenes vaccine (CRS-207), engineered to secrete mesothelin for MHC presentation induced, increased survival in PDAC patients, in addition to demonstrating that a mesothelin-derived peptide (AFYPGYLCSL, SEQ ID NO:4) induce CTL clones in human peripheral blood mononuclear cells in PDAC patients. (v) CEA, which is expressed in >90% PDAC tumors is poorly immunogenic but through the use of an altered peptide ligand, CAP1-6D, it could be demonstrated that a more robust T- cell response could be generated in PDAC patients in a phase I clinical trial. (vi) One case has been reported in which vaccination with a modified HLA-A2-restricted survivin peptide could result in the remission of liver metastasis in a PC patient.

[0193] The above short list of TAA peptide sequences can serve as an illustrative, but not complete, list of immunogenic sequences that can be included in the design of mRNA-based PDAC vaccines, according to the same principles outlined for neoantigen epitopes. This could include use of individual or a combination of TAA epitopes or TAA epitopes that are combined with one or more KRAS mutant sequences for release and cleavage in the cytosol for MHC I loading. Alternatively, we could also add targeting sequence for specific loading of MHC I or MHC II as described in Figs.24A-C.

[0194] Because of the relatively low immunogenicity of TAA peptide vaccines, it has been demonstrated by some groups that the mutation of key anchor residues in the peptides can improve binding to MHC-I molecules for CD8+ T-cell presentation. For instance, a MUC-1 peptide vaccine modified in this way was shown to enhance production of IFN-γ from patient and normal donor T-cells and improve tumor cell killing. Endowing the particle surface with targeting ligands that facilitate uptake by lymphoid organs, including lymph nodes, spleen and tertiary lymphoid structures.

[0195] It was previously reported that the immunogenicity of peptide vaccines is increased by maximizing delivery to secondary lymphoid organs, where T-cell responses are generated. This includes the presence of 600–800 lymph nodes distributed across the body, the spleen, and the mucosa-associated lymphoid tissue. Moreover, our demonstration of the appearance of tertiary lymphoid structures during peptide vaccination introduces yet another possible target that can contribute. While traditional vaccines often act within a limitedradius to target a chain of lymph nodes that drain the injection site, this potentially limits immune orchestration to regional lymph nodes, leading to a competition for resources and limiting participation participate through the cancer immunity cycle. The solution is systemic administration of vaccinating peptides or nucleotide analogs to reach multiple lymph nodes and the spleen effectively to overcome resource constraints. Thus, despite demonstrating a vaccination response to subcutaneous administration of polymer nanoparticles delivering mutant KRAS peptides in Example 1, we disclose IV administration to be a more logical choice enhancing the cancer immunity cycle in PDAC. This is in keeping with the demonstration that IV administration of an RNA-based nanoparticle vaccine that links neoantigen peptides to a Toll-like receptor 7 / 8 agonist (SNP- 7 / 8a), is more immunogenic than direct lymph node injection. Because IV administration could be hampered by macrophage sequestration in the liver and the spleen, nanoparticle design is critical, including limiting nanoparticle size to 100-150 nm, providing a pegylated surface and co-delivering the antigen with a Toll-like receptor 7 / 8 (TLR7 / 8) agonist that ensures DC maturation in KPC acquiring the antigen. In addition, the lipid composition and surface charge of lipid nanoparticles to improve delivery to the spleen and lymph nodes is important, as demonstrated in clinical trials for melanoma immunotherapy and improving antigen delivery by nanoparticles with the vaccinating pathway in peptides are 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG (DSPE-PEG). It is also important to consider that injection of lipid nanoparticles for mRNA delivery only transfect 2% or less of immune cells, which can be improved by active targeting on the particle surface, to facilitate APC uptake.

[0196] It has been demonstrated that transmembrane mannose receptors, which allow particulate matter endocytosis, can assist in antigen capture and presentation by dendritic cells, including for purposes of vaccine development. Strikingly, mannosylated particles are capable of increasing antigen uptake by dendritic cells a 100-fold over antigen uptake from a fluid phase. Mannose targeting can be accomplished by DC expression of a Type II mannose membrane receptor (MMR), DC-SIGN (CD209), which is present in low abundance on the DC surface in the absence of a danger signal. This introduces the possibility of decorating lipid nanoparticle (LPN) surfaces with surface mannose ligands to improve nucleic acid uptake in lymph node and splenic APC, including by the addition of a TLR 7 / 8 agonist that increases DC-SIGN expression (Fig.25).

[0197] We have previously demonstrated that the surface of cationic LPN can be decorated with DSPE-PEG2000-Mannose that can enhance APC uptake in the liver and spleen. Figs.26A-C illustrate the comparison of non-decorated and decorated cationic LPN that includes either DMG-PEG2000 or DSPE-PEG2000-Mannose for particle synthesized to encapsulate and deliver GFP mRNA (mGFP) (Fig.26A). Non-decorated and decorated LNPs were synthesized by mixing of the ionizable cationic lipid, DLin-MC3-DMA, with DSPC, cholesterol and DMG-PEG-2K or DMG-PEG-2K / DSPE-PEG2000-mannose, dissolved in ethanol in the molar ratios of 50, 10, 38.5, 1.5 (Fig.26B). The chemical structures of the 4 lipids are listed in Fig.26B. The mRNA source (GFP-mRNA) was diluted in RNase-free sodium acetate buffer (pH 4.0). Following preparation of the aqueous and organic phases, a microfluidics mixer (NanoAssemblr Benchtop) was used to synthesize the LNPs, which were purified to determine particle sizes and surface potential by dynamic light scattering (DLS). The encapsulation efficiencies (EE %) of the mRNA, as determined by RiboGreen were > 97 %. Subsequently, the LNPs samples were imaged by cryogenic electron microscopy (Cryo-EM) to investigate a nano-precipitated core structure (Fig.26C). It was confirmed that, following IV injection, that both decorated and non-decorated particles could be seen to biodistribute to the liver (~70% of particles) and spleen (~20% of the particles). However, fluorescence microscopy examination of liver tissue demonstrated that while both carrier types induced widespread GFP expression in the liver, the relative fluorescence intensity of mGFP / LNP-Man was 1.7-fold higher than mGFP / LNP. Also, there was increased fluorescence overlap of GFP with tolerogenic APC in the liver for mannose- decorated particles.

[0198] While our experimentation has been limited to a simple receptor ligand, alternative approaches using more complex mannose ligands are also available to target DC-SIGN. Well-defined mannosides have been used (for example, by Li et al., Front Chem. 2019;7:650, based on the knowledge that multivalent nanostructures improve DC-SIGN binding. Thus, instead of using a monosaccharide, LPN can be constructed that include more complex oligomannosides. Several larger and more complex oligomannosides with intrinsic multivalence are available, including the oligomannose N-linked oligosaccharide, Nan 9, which displays 9 mannosyl residues.

[0199] Further improvement of the cationic LPN to further augment targeting to lymphoid structures include, but are not limited to, the use of TLR7 / 8 agonists and / or the use of alternative lipid components. For example, we have demonstrated inclusion of a commercially available TLR7 / 8 agonist, 3M-052, which contains a lipid tail allowing into the lipid bilayer of silicasomes.3M-052 (MEDI9197 (Singh, J Immunol 2014 Nov 1; 193(9) 4722-4731; Bhagchandani et al. (2021) Adv. Drug Deliv. Rev., 175: 113803) is aninjectable, tissue-retained agonist that forms a tissue depot with gradual, sustained release, allowing local TLR activation without systemic cytokine release; induces local innate immune activation as well as systemic, antigen-specific CD8+ T cell responses which suppress tumor growth; currently under clinical development for use in vaccines and cancer therapy. Design strategies for nanoparticles construction include self-assembly conjugate formulations, conjugation of protein antigens to copolymers or P(Man-TLR7) covalently conjugated onto synthetic polymer scaffolds.

[0200] Fig.27 illustrates one design of a decorated cationic LPN containing DLin-MC3, DSPC, cholesterol, DSPE-PEG2000-Mannose and 3M-052 into ethanol at molar ratios of 50.0: 9.0 : 36.5 : 1.5 : 3.0. Altering the cationic lipid component, e.g., DODAP, is another approach that can be used and described later. Similar designs can be used for encapsulating mRNA strands for mutant KRAS and TAA epitopes. The same particles can also be decorated with a mannose ligand, as demonstrated above.

[0201] Additionally, while DLin-MC3-containing cationic LPN favor liver biodistribution, we have shown that decorating the same type of LPN with a mannose ligand, allows 20% of particles to migrate to the spleen. While C12-200, DLin-MC3-DMA, SM-102, and ALC- 0315 are the dominant vectors for mRNA delivery by industry, a strategy has been reported for selective organ targeting (SORT) wherein multiple LPN classes can be systematically engineered by supplemental SORT lipids to allow access to extrahepatic tissues. One example of a SORT lipid component has been the use of dioleoyl-3-trimethylammonium propane (DOTAP), allowing the protein expression profile to be shifted dose-dependently from the liver to the spleen and the lung (Fig.27). This approach allows lung and spleen- targeting SORT nanoparticles to be developed for cargo delivery to relevant cell types, including epithelial cells, endothelial cells, B-cells, and T-cells. Another approach has been to use IV administration of plasmid DNA delivering nanoparticles using a lipid system based on the commercially available pH-sensitive lipid 1,2-dioleoyl-3-dimethylammonium propane (DODAP), one of the first recognized pH-sensitive lipids, not commonly used for in vivo gene delivery (Fig.27). However, DODAP is reported for use as an efficient and targeted gene delivery in vivo, particularly when combined with a specific helper lipid, dioleoylphosphatidylethanolamine (DOPE).

[0202] Against this background, cationic LPNs were constructed that include DOTAP and / or DODAP in particles for which the lipid composition can be optimized for uptake by the spleen and retroperitoneal lymph nodes adjacent the pancreas. The particles can be labeled with a fluorescent dye and biodistribution studies were performed to quantifyparticle uptake in the secondary lymphoid tissues, as well as in any tertiary lymphoid tissues that form at the primary tumor site. We can also determine whether encapsulation of 3M- 052 or surface decoration with the mannose ligand will impact the biodistribution as well as outcome of the antitumor immune response.

[0203] The presence of tumor-associated tertiary lymphoid structures is associated with prolonged patient survival, higher rates of disease-free survival, and a favorable response to current cancer therapies. However, much remains to be learned about the immune responses occurring in these structures, and how they contribute to improved clinical outcomes. It is also not known how the heterogeneity of TLS cellular composition, structural organization, and anatomical location influences their functionality and prognostic significance. Based on the preponderance of evidence, it seems desirable to induce and / or augment TLS development as a new aspect of cancer immunotherapy, either alone or in combination with immunotherapy or chemo-immunotherapy. Interestingly, the presence of TLS was initially associated with a favorable response to neoadjuvant chemotherapy in breast cancer. Immune checkpoint treatment of murine tumors increases the number and size of TLS, and promotes a classical organization, in association with diminished tumor outgrowth. Thus, it has been suggested that TLS may be the site at which these therapies interact, with the potential of great benefit if these structures can also be further exploited by cationic LPN design. Several therapeutics, including antibodies, STING agonists, recombinant cytokine / chemokines, and antibody-cytokine fusion proteins have been shown to induce the formation of TLS as well as exhibiting promising antitumor effects in various mouse cancer models. Based on our observation that vaccination with KRAS peptide mutants also induced TLS in PDAC (Figs.21 and 23), we propose to demonstrate similar effects with cationic LPN that deliver KRAS mRNA epitopes. If that notion is correct, it would constitute the basis for constructing cationic LPN that contain alternative lipid compositions, additional surface targeting receptors, TLR7 agonists, STING agonists and other immunomodulators for therapeutic impact at the level of TLS structures. Endowing the LPN with STING agonists, including STING agonists other than 2’ ,3’- cGAMP

[0204] In addition to the use of 2’,3’-cGAMP to reprogram liver APC for triggering an immune response against PDAC metastases in the liver, the activation of the type I interferon pathway could be advantageous to cationic LPNs that are used for targeting of lymphoid structures that communicate with the primary cancer site. Thus, in addition to encapsulation of antigenic epitopes and a TLR7 agonist, cationic LPN can also be constructed to include 2’ 3’ cGAMP as described in experiment #2. Because both immunemodulators activate the innate immune system, a combination of TLR and STING agonists may function synergistically in augmenting the cancer immunity cycle.

[0205] In addition to 2’ ,3’ -cGAMP, other natural STING agonists include, 3’ ,3’-cGAMP, c-di-GMP, and c-di-AMP, among which 2’ ,3’-cGAMP is produced by cGAS in mammalian cells and the latter three are produced by bacterial cGAS. While they may be little advantage for substituting 2’ ,3’ -cGAMP for the other natural analogs, the presence of a phosphodiester linkage has led to extensive efforts to generate hydrolysis-resistant derivatives by using phosphothioate analogues. Among the phosphothioate analogues, Rp,Rp-20,30-c-di-AMPSS (ADU-S100) has proven to be one of the most successful derivatives that have been has been evaluated in clinical trials, and can be encapsulated by cationic lipids.

[0206] The limited availability of natural STING agonists in the cytosol has prompted research into non-nucleotide alternatives. Particularly promising is the amidobenzimidazole (ABZI)-based compounds capable of activating both mSTING and hSTING, with di- amidobenzimidazole (diABZI) emerging as a highly efficient STING activator. It exerted superior potency to cGAMP and induced a robust immune response in mice. Co-crystal structure analysis revealed two ABZI molecules per STING dimer and an overlap of the non-protein binding part. Most interesting was the observation that STING maintained an open conformation upon ABZI binding, which contrasts with the closed conformation reported for other agonists. Dimerization of two ABZI maintained the same binding conformation but significantly increased affinity. Subsequent structure optimization yielded di-amidobenzimidazole (diABZI), a dimer of ABZI. diABZI is a highly efficient STING activator with a half maximum effective concentration of 130 nM, which is 400-fold more potent than cGAMP, dispiro diketopiperzine (DSDP), 6-bromo-N-(naphthalen-1-yl)- benzo[d][1,3]dioxole-5-carboxamide (BNBC), SR-001, SR-717, SR-301, and benzothiophene oxobutanoic acid (MSA-2). Design principles for the first approach.

[0207] The hypothesis is that low levels of endogenous TAA and neoantigens that are being released from dying cancer cells during ICD may not be effective for stimulating T-cell antigen receptors, leading to recruitment of CTLs that exhibit low cytotoxic potential for cancer cells. Thus, exogenous vaccination with PDAC antigens known to lead to effective MHC presentation, could populate the regional lymphoid structures, participating in the immunity cycle, with higher affinity CTL populations for recruitment to the primary cancer site. In addition to the spleen and regional lymph nodes, there is now growing awareness ofthe role of tertiary lymphoid structures (TLS), assembling at primary cancer sites for boosting antitumor immunity. TLS are organized structures, similar to lymphoid follicles that harbor T-cells, B-cells, and dendritic cells, with the ability to improve immune recruitment, activation, proliferation, and cytotoxic killing. Accordingly, the appearance of TLS in various cancers is regarded as predictive of an improved antitumor immune responses and patient outcome, including for pancreatic cancer.

[0208] We have collected considerable evidence that nano-enabled chemo-immunotherapy can significantly improve the PDAC immune response, including highly significant improvement in survival in the orthotopic PDAC cancer model. Most importantly, it has been demonstrated that the principle of chemo-immunotherapy is the generation of immunogenic cell death responses, which can be considered as a form of endogenous vaccination. We have exploited this principle by the development of a large number of liposome and silica silicasome nanocarriers that deliver immunogenic chemotherapy agents, in addition to allowing the incorporation of additional immune modulators into the lipid bilayer. This allows exploration of whether the best-performing cationic LNP can boost the endogenous immune responses by combination therapy, which also solicit exogenous vaccination against PDAC neoantigens and TAA as described herein.

[0209] Accordingly, in various embodiments, a drug delivery vehicle for enhancing an anti- tumor immune response by targeting tumor-associated lymphoid structures is provided. In certain embodiments the drug delivery vehicle comprises a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle that comprises a lipid composition and / or targeting molecules that facilitate uptake by lymphoid organs, including lymph nodes, spleen and / or tertiary lymphoid structures, where said drug delivery vehicle comprises one or more STING agonists; and one or more a tumor associated antigens or tumor-associated neoantigens or a nucleic acid encoding one or more a tumor associated antigens or tumor- associated neoantigens. TLR7 / 8 agonists for incorporation into the drug delivery nanoparticles described herein.

[0210] In certain embodiments, the drug delivery nanoparticles described herein comprise / include one or more TLR7 / 8 agonists. Lipid compatible TLR7 / 8 agonists are well known to those of skill in the art and include but are not limited to lipidated versions of known TLR7 / 8 agonists. Such lipidated TLR7 agonists include, but are not limited to lipidated imidazoquinoline derivatives (IMDs) such as a lipidated imiquimod, a lipidated resiquimod, a lipidated 852-A (PF-4878691), and the like, a lipidated pteridinone-basedTLR7 / 8 agonist such as lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD- 8848), and the like, and lipidated TLR8-specific benzazepine such as lipidated motolimod (VTX-2337), a lipidated and pyrimidine such as lipidated selgantolimod (GS-9688), and the like.

[0211] Thus, a TLR7 / 8 agonist may be selected from resiquimod (R848), imiquimod, 852A (PF-4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD- 8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX-2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD-8848) or lipidated selgantolimod (GS-9688).

[0212] Illustrative, but non-limiting, examples of lipidated TLR7 / TLR8 agonists are described by Miller et al. (2020) Front. Immunol., Vol.11, Art.406, doi: 10.3389 / fimmu.2020.00406. Illustrative TLR7 / 8 agonists described therein that can be used in the presently described drug delivery vehicles (drug carriers) include lipidated versions of UM-3001 shown below.

[0213] One example of such is shown by the formula: where n is 3-15 andis 3, 6, 9, or 12 and R is C(O)(CH2)14CH3. In certain embodiments the compound is UM-3003 (Miller et al. supra) in which n is 3 and R is C(O)(CH2)14CH3, or UM-3004 in which n is 6 and R is C(O)(CH2)14CH3.

[0214] In certain embodiments the TLR7 / TLR8 agonist is UM-3005 having the formula shown below.in which R is C(O)(CH2)7CH=CH(CH2)7CH3.

[0215] Other suitable TLR7 / 8 agonists are described, inter alia, in U.S. Patent Nos: 8,946,421 and / or 8,624,029 which are incorporated herein by reference in their entirety for all purposes. Typically, these compounds comprise an imidazoquinoline molecule that may be covalently linked to a phospho- or phosphonolipid group. In certain embodiments the agonists are broadly described by Formula I: I where R1is H, C1-66alkyl, C3-6cycloalkylC1-6alkylamino, C3-6cycloalkylC1-6 alkoxy, C1-6alkoxyC1-6alkyl, C1-6 alkoxyC1-6alkylamino, C1-6alkoxyC1-6alkoxy; branched or unbranched and optionally terminally substituted with a hydroxyl, amino, thio, hydrazino, hydrazido, azido, acetylenyl, carboxyl, or maleimido group, Z is C2-C6alkyl or alkenyl, unsubstituted or terminally substituted by - -(O--C2-C6 alkyl)1-6, Y is Y is O, or NH, X is X is O, CH2, or CF2, W is O or S, m is 1 or 2, and A is, straight / branched / unsaturated C4-C24 alkyl or acyl, R4 and R5 are independently H, C1-C6 alkyl, C1-C6 alkoxy, halogen, or trifluoromethyl; or taken together alternatively form a 6- membered aryl, heteroaryl containing one nitrogen atom, cycloalkyl, or heterocycloalkyl ring containing one nitrogen atom unsubstituted or substituted by one or more of C1-C6alkyl, C1-C6 alkoxy, halogen, or trifluoromethyl, or pharmaceutically acceptable salts thereof.

[0216] In one embodiment, these lipidated TLR7 / 8 agonists are more specifically described by Formula II: IIin which R1 6 6 6 6alkyl, C3-6cycloalkylC1-6alkylamino, C3-6cycloalkylC1-6 alkoxy, C1-6 alkoxyC1-6 alkyl, C1-6 alkoxyC1-6alkylamino, C1-6 alkoxyC1-6alkyl; branched or unbranched and optionally terminally substituted with a hydroxyl, amino, thio, hydrazino, hydrazido, azido, acetylenyl, carboxyl, or maleimido group, n is 1-6, Y is O or NH, X is O, CH2, or CF2, W is O or S, m is 1-2, R2 is H or straight / branched / unsaturated C4-C2alkyl or acyl, R3is straight / branched / unsaturated C4-C24 alkyl or acyl (e.g. phosphatidyl, lysophosphatidyl ether or ester, and when W is O, X is O and M is 1.

[0217] In certain embodiments, the compound is a compound of Formula II where R1 is H, n-butyl, or ethoxymethyl, n is 2-4, X is O, Y is O, W is o, m is 1-2, and both R2 and R3 are hexadecanoyl.

[0218] In one illustrative embodiment, the lipidated TLR7 / TLR8 agonist comprise compound L4 from U.S. Patent No: 8,624,029 B2 (4-amino-1-[2-(1,2-dipalmitoyl-sn- glycero-3-phospho)ethyl]-1H-imidazo[4,5-c]quinoline (Formula (I) where R1 is H, Y, W, and X are O, n is 2, m is 1, R2and R3 are n-C15H31CO): L1compound L4 from U.S. Patent No: 8,624,029 B2 (4-amino-1-[2-(1,2-dipalmitoyl-sn- glycero-3-phospho)ethyl]-2-butyl-1H-imidazo[4,5-c]quinoline (Formula I in which R1is n- C4H9, Y, W, and X are all O, n is 2, m is 1, and R2 and =R3 are both n-C15H31CO): L2

[0220] In one illustrative embodiment, the lipidated TLR7 / TLR8 agonist comprise compound L4 from U.S. Patent No: 8,624,029 B2 (4-Amino-1-[2-(1,2-dipalmitoyl-sn- glycero-3-phospho)ethyl]-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline (Formula I where R1 is CH2OCH2CH3, Y, W, and X are all O, n is 2, m is 1, and R2and R3are both n- C15H31CO): L3compound L4 from U.S. Patent No: 8,624,029 B2 (4-Amino-1-[2-(1,2-dipalmitoyl-sn- glycero-3-phospho)butyl]-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline (Formula 1 where R1 is H, Y, W, and X are all O, n is 4, m is 1, and R2 and R3 are both n-C15H31CO): L4compound L5 from U.S. Patent No: 8,624,029 B24-amino-1-[2-(1,2-dipalmitoyl-sn- glycero-3-diphospho)ethyl]-2-ethoxymethy- l-1H-imidazo[4,5-c]quinoline (Formula I wherein R1 is CH2OCH2CH3, Y, W, and X are all O, n is 2, m is 2, , and R2 and R3 are both n-C15H31CO):L5 exert adjuvant effects. Nanocarriers (drug delivery vehicles) that include these molecules can show activity at the level of lymph nodes and secondary lymphoid tissue, in addition to the action at the primary cancer site. For instance, Bhagchandani et al. (2021) Adv. Drug Deliv. Rev., 175: 113803 showed that particulate TLR7 / 8 conjugates exhibit enhanced lymph node cytokine production and uptake by migratory APCs. This is accompanied by an order of magnitude increase in the influx of CD11c+ DCs and monocytes in the draining LN compared with soluble forms. This allowed the draining lymph node to actively contribute to the immunogenic cell death effect leading to T-cell activation (by providing antigen ingested dendritic cells), before returning those T cells to the primary tumor site. Thus, it is believed the TLR7 agonists, and accordingly the drug delivery vehicles described herein comprising TLR7 / 8 agonists, can exert adjuvant effects at two separate but complementary localities in the so-called cancer immunity cycle, namely at the primary tumor site, as well as the participating draining lymph node.

[0224] Other suitable TLR agonists that can be included in a lipid bilayer are shown in the table below. Illustrative TLR agonists that can be included in the lipid bilayer of nanocarriers (e.g., liposomes and silicasomes) as additional component. Category DrugActivityReference r. l.3D-PHAD® (Monophosphoryl Pharmaceutics, 18(8): 3-Deacyl Lipid A): less 2867-2888 pyrogenic d J l. . ) et .Okusawa et al. (2004) Infect. Immun.72(3): 1657-1665 - r. n s.

[0225] The foregoing lipid compatible TLR agonists and other compounds are illustrative and non-limiting. Using the teaching provided herein, numerous drug delivery vehicles comprising other TLR agonists disposed in a lipid bilayer will be available to one of skill in the art. STING agonists for incorporation into the drug delivery nanoparticles described herein.

[0226] In certain embodiments, the drug delivery nanoparticles described herein comprise / include one or more Stimulator of Interferon Genes (STING) agonists. STING agonists are well known to those of skill and include, but are not limited toamidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'-Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'-cGAMP), 3',3'- Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), (DMXAA), CMA, MK-1454, CRD5500, cyclic di-nucleotide compounds as described in U.S. Patent Publication No. 2020 / 0062798 A1, which is incorporated herein by reference for the STING agonists described therein; tricyclic heteroaryl compounds as described in U.S. Patent Publication No: 2020 / 0040009 A1, which is incorporated herein by reference for the STING agonists described therein; and heteroaryl amide compounds as described in U.S. Patent Publication No: 2020 / 0039994 A1, which is incorporated herein by reference for the STING agonists described therein.

[0227] U.S. Patent Publication No.2020 / 0040009 describes compounds useful for the purposes disclosed herein, including 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14- (6-amino-9H-purin-9-yl)-15,16-difluoro-10-hydroxy-2-sulfanyl-2,10-disulfidooctahydro- 12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one; 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino- 9H-purin-9-yl)-15,16-difluoro-10-hydroxy-2-sulfanyl-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one (Diastereomer 2); 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(6-amino- 9H-purin-9-yl)-10-hydroxy- 2-sulfanyl-2,10-disulfidooctahydro-12H- 5,8- methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro- 6H-purin-6- one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(6-amino-9H-purin-9-yl)-10- hydroxy- 2-sulfanyl-2,10-disulfidooctahydro-12H- 5,8-methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6- one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)- 10-hydroxy-2-sulfanyl-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10]penta- oxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)- 10-hydroxy-2-sulfanyl-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10]penta- oxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7- yl)- 10-hydroxy-2-sulfanyl-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10]penta- oxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one;2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(4-aminoimidazo[2,1-f][1,2,4]triazin-7- yl)- 10-hydroxy-2-sulfanyl-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10]penta- oxadiphosphacyclotetradecin-7-yl]-1,9- dihydro-6H-purin-6-one; 2-amino-9-[(5R,7R,8S,12aR,14R,15R, 15aS,18S)-14-(6-amino-9H-purin-9-yl)- 18-fluoro- 10-hydroxy-2-sulfanyl-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7(12H)-yl]-1,9- dihydro-6H- purin-6-one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(6-amino-9H-purin-9-yl)-2-hydroxy-10- sulfanyl-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one; 2- amino-9-[(5S,7R,8R,12aR,14R,15aS)-14-(6-amino-9H-purin-9-yl)-2-hydroxy-10-sulfanyl- 2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one; 2- amino-9-[(5R,7R,8S,12aR,14R,15S, 15aR,16R)-14-(6-amino-9H-purin-9-yl)- 15,16- difluoro-2-hydroxy-10-sulfanyl- 2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one; 2-amino-9-[(5R,7R,8S,12aR,14R,15S, 15aR,16R)-14-(6-amino-9H-purin-9-yl)- 15,16- difluoro-2-hydroxy-10-sulfanyl- 2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(4-amino-7H-pyrrolo[2,3-d]pyrimidin- 7-yl)-2- hydroxy-10-sulfanyl-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl]- 1,9-dihydro-6H-purin-6-one; 2-amino-9-[(5R,7R,8S,12aR,14R,15R, 15aS,18S)-14-(6-amino-9H-purin-9-yl)- 18-fluoro- 2-hydroxy-10-sulfanyl-2,10- disulfidohexahydro-14H-15,12a- (epoxymethano)-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7(12H)-yl]-1,9- dihydro-6H- purin-6-one; 2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino- 9H-purin-9-yl)-15-fluoro-10,16-dihydroxy-2-sulfanyl-2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one; 2-amino-9-[(5R,7R,8S,12aR,14R,15R,15aS,18S)-14-(6-amino-9H-purin-9- yl)-18-fluoro-10-hydroxy-10-oxido-2-sulfanyl-2-sulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin- 7(12H)-yl]-1,9-dihydro-6H-purin-6-one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(6- amino-9H-purin-9-yl)-10-hydroxy- 10-oxido-2-sulfanyl-2-sulfidooctahydro- 12H-5,8- methanofuro[3,2-1][1,3,6,9,11, 2,10]pentaoxadiphosphacyclotetradecin-7- yl]-1,9-dihydro- 6H-purin-6-one; 2-amino-9-[(5R,7R,8S,12aR,14R,15S, 15aR,16R)-14-(6-amino-9H-purin-9-yl)- 15,16-difluoro-10-hydroxy-10-oxido-2- sulfanyl-2-sulfidooctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro- 6H-purin-6-one; 2-amino-9-[(5R,7R,8R,12aR,14R,15S,15aR,16S)-14-(6-amino-9H-purin- 9-yl)-15-fluoro-10,16-dihydroxy-10-oxido-2-sulfanyl-2-sulfidooctahydro-12H-5,8- methanofuro[3,2-l][1,3,9,11,6,2,10]tetraoxathiadiphosphacyclotetradecin-7-yl]-1,9-dihydro- 6H-purin-6-one; 2-amino-9-[(5R,7R,8R,12aR,14R,15R, 15aS,18S)-14-(6-amino-9H-purin- 9-yl)- 10,18-dihydroxy-10-oxido-2-sulfanyl-2- sulfidohexahydro-14H-15,12a- (epoxymethano)-5,8-methanofuro[3,2- 1][1,3,9,11,6,2,10] tetraoxathiadiphosphacyclotetradecin- 7(12H)-yl]-1,9-dihydro-6H- purin-6-one; 2-amino-9-[(5R,7R,8S,12aR,14R,15S,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16- difluoro-2-hydroxy-2-oxido-10-sulfanyl-10-sulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one; 2- amino-9-[(5S,7R,8R,12aR,14R,15aS)- 14-(6-amino-9H-purin-9-yl)-2-hydroxy-2- oxido-10- sulfanyl-10-sulfidooctahydro- 12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl]-1,9-dihydro-6H-purin-6-one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)-10-hydroxy-14-(6-oxo-1,6-dihydro-9H-purin-9-yl)- 2-sulfanyl-2,10-disulfidooctahydro-12H-5,8-methanofuro[3,2- l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one (Diastereomer 1); 2-amino-9-[(5S,7R,8R,12aR,14R, 15aS)-10-hydroxy-14-(6-oxo-1,6- dihydro-9H-purin-9-yl)-2-sulfanyl- 2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one; 2-amino-9-[(5S,7R,8R,12aR,14R,15aS)- 2-hydroxy-14-(6-oxo-1,6-dihydro-9H- purin-9-yl)- 10-sulfanyl-2,10- disulfidooctahydro-12H-5,8- methanofuro[3,2-1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one; and 2-amino-9- [(5R,7R,8S,12aR,14R,15S,15aR,16R)- 15,16-difluoro-10-hydroxy-14-(6-oxo- 1,6-dihydro- 9H-purin-9-yl)-2-sulfanyl- 2,10-disulfidooctahydro-12H-5,8- methanofuro[3,2- 1][1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7- yl]-1,9-dihydro-6H-purin-6-one.

[0228] US20200040009 describes compounds useful for the purposes disclosed herein, including (E)-9,9′-(but-2-ene-1,4-diyl)bis(2-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-8-methoxy- 9H-pyrimido[4,5-b]indole-6-carboxamide), (E)-9,9′-(but-2-ene-1,4-diyl)bis(2-(1-ethyl-3- methyl-1H-pyrazol-5-yl)-8-(3-hydroxypropoxy)-9H-pyrimido[4,5-b]indole-6-carboxamide), or a pharmaceutically acceptable salt of any of the foregoing.

[0229] US 20200039994 describes compounds useful for the purposes disclosed herein, including (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methyl-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5- carboxamido)-7,8-dihydro-1H-[1,4]dioxino[2′,3′:3,4]benzo[1,2-d]imidazole-5-carboxamide; (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methyl-1H- benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)- 1H-benzofuro[6,7-d]imidazole-5-carboxamide; and (E)-3-(((3-((5-carbamoyl-1-(4-(5- carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzofuro[6,7- d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H- benzo[d]imidazol-7-yl)oxy)propoxy)carbonyl)amino)propanoic acid.

[0230] Thus, as disclosed herein, nanoparticles are provided comprising the various components described herein and for the various purposes described herein. Some non- limiting examples of combinations comprising the components of such nanoparticles include the following. In one embodiment, a nanoparticle comprises Dlin-MC3-DMA, DSPC, cholesterol, DMG-PEG2000, 2’,3’-cGAMP and mRNA encoding a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises Dlin- MC3-DMA, DSPC, cholesterol, DMG-PEG2000, 2’,3’-cGAMP, resiquimod and mRNA encoding a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises Dlin-MC3-DMA, DSPC, cholesterol, DMG-PEG2000, 2’,3’- cGAMP, 3M-052 and mRNA encoding a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises Dlin-MC3-DMA, DSPC, cholesterol, DMG- PEG2000, 2’,3’-cGAMP and mRNA encoding multiple iterations of a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises Dlin- MC3-DMA, DSPC, cholesterol, DMG-PEG2000, 2’,3’-cGAMP, resiquimod and mRNA encoding multiple iterations of a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises Dlin-MC3-DMA, DSPC, cholesterol, DMG- PEG2000, 2’,3’-cGAMP, 3M-052 and mRNA encoding multiple iterations of a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises PLGA, resiquimod and a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises PLGA, 3M-052 and a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises Dlin-MC3-DMA, DSPC, cholesterol, DMG-PEG2000, mRNA encoding one or more iterations of a mutant KRAS peptide such as comprising G12D, and DSPE-PEG2000-mannose. In one embodiment, a nanoparticle comprises Dlin-MC3-DMA, DSPC, cholesterol, DMG-PEG2000, mRNA encoding one or more iterations of a mutant KRAS peptide such as comprising G12D, 3M- 052 and DSPE-PEG2000-mannose. In one embodiment, a nanoparticle comprises DODAP, DOPE, cholesterol, DMG-PEG2000 and mRNA encoding one or multiple iterations of amutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises DODAP, DOPE, cholesterol, DMG-PEG2000, 3M-052 and mRNA encoding one or multiple iterations of a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises DODAP, DOPE, cholesterol, DMG-PEG2000, resiquimod and mRNA encoding one or multiple iterations of a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises DODAP, DOPE, cholesterol, DMG-PEG2000, 3M-052, 2’,3’-cGAMP and mRNA encoding one or multiple iterations of a mutant KRAS peptide such as comprising G12D. In one embodiment, a nanoparticle comprises DODAP, DOPE, cholesterol, DMG-PEG2000, resiquimod, 2’,3’- cGAMP and mRNA encoding one or multiple iterations of a mutant KRAS peptide such as comprising G12D.

[0231] In any of the foregoing embodiments, the mutant KRAS peptide may be any one or more of SEQ ID Nos:2-13, 15-19, 22-23, 27, 29-36, 50, 52-56, 74, or 81-86, or a nucleic acid of any one or more SEQ ID NOs:37-43, 57, 59-63 or 77. In any of the embodiments disclosed herein, the KRAS peptide may be N-terminally acetylated, C-terminally amidated, or both acetylated and amidated. EXAMPLES

[0232] The following examples are offered to illustrate, but not to limit the claimed disclosure. Example 1 Reprogramming Immune Responses to Pancreas Cancer Metastases by Lipid Nanoparticles delivering a STING agonist plus Mutant KRAS mRNA Materials

[0233] The ionizable lipid, DLin-MC3-DMA (MC3), was purchased from MedKoo Bioscience, Inc. All other lipids, including DSPC, cholesterol and DMG-PEG2000, were purchased from Avanti Polar Lipids, Inc. The mRNA strands, described below were synthesized by TriLink Biotechnologies, using a 5’ Cap and 3’ PolyA modifications for stabilization. DiR, RiboGreen RNA Assay Kit were purchased from Thermo Fisher Scientific. Design of KRAS and wildtype RAS nucleic acid constructs

[0234] We chose the G12D mutation, corresponding to amino acids (aa) 5-21 in KRAS (SEQ ID NO: 16), to generate an anti-PDAC immune response by constructing a mRNA(mKRAS) strand that included 5 epitope repeats (Fig.1C). The epitopes were flanked by two natural amino acids on the 5’ and 3’ ends. As a control, five repeats of the corresponding wildtype RAS (aa 5-21; SEQ ID NO:16 sequence plus flanking aa were included in the mRASWT construct. Following the addition of start and stop codons, optimization of the codon sequence was performed using the GenScriptTM optimization tool. These sequences were shared with TriLink Biotechnologies to construct mKRAS and mRASWT strands, as demonstrated in Fig.2A. Preparation and characterization of lipid nanoparticles (LNP)

[0235] A microfluidics approach was used to synthesize a series of particles incorporating individual or combinations of cGAMP, mKRAS or mRASWT. For the synthesis of cGAMP / mKRAS / LNP, cGAMP and mKRAS were dissolved in 0.1 M NaAc (pH 4.0), serving as the aqueous phase, while the lipid components, MC3, DSPC, cholesterol and DSPE-PEG2000 were dissolved in ethanol, used as the organic phase. The molar ratio of MC3, DSPC, cholesterol and DSPE-PEG2000 was 50, 10, 38.5, 1.5, while the N / P ratio was 4. The aqueous and organic phases were blended in a NanoAssemblr at a 3:1 volume ratio and flow rate of 12 mL / min. More details about composition and Nanoassemblr settings appear in Fig.4. The nano-precipitated cGAMP / mKRAS / LNP were purified by dialysis against PBS (pH 7.4) at 4oC for 1 h, followed by pushing through a 0.2 μm cutoff filter. The particle size, polydispersity index (PDI) and surface charge were assessed in a ZetaPlus (Brookhaven) instrument. Particle morphology and size were also assessed by cryogenic electron microscopy (Cryo-EM, TF20 FEI Tecnai-G2). The particle encapsulation efficiency (EE) for cGAMP and mRNA was determined by cGAMP ELISA (Thermo) as well as RiboGreen assay kits (Thermo), respectively. A similar approach was used for synthesis of carriers that incorporate cGAMP only (cGAMP / LNP), mKRAS only (mKRAS / LNP), or a combination of cGAMP with mRASWT (cGAMP / mRASWT / LNP). To assess particle uptake and expression of GFP mRNA (mGFP) in the liver, a particle batch was synthesized in which the cGAMP and mKRAS were replaced by Cy5-cGAMP and mGFP respectively, yielding Cy5-cGAMP / mGFP / LNP. The in vitro stability of LNP was assessed by monitoring LNP size, PDI and EE during storage for 15 days at 4oC. Hemolysis assay to assess the hexagonal LNP lipid phase

[0236] A hemolysis assay was conducted to determine the efficacy of the cationic lipid component to disrupt the RBC bilayer. Briefly, purified murine red blood cells (RBC) suspended in PBS at pH 5.0 or 7.4. were mixed with mKRAS / LNP or cGAMP / mKRAS / LNP at concentrations of 0.5, 1.0, or 2.0 μg / mL for 1 h at 37 oC.0.1%Triton was used as positive control for expressing 100 % RBC lysis. After centrifugation (1000 rpm, 5 min), the sample plate was photographed, and absorbance of the supernatants assessed at 540 nm in a microplate reader (Molecular Devices, SpectraMax M5). In vitro transcriptional activation by cGAMP, a STING agonist

[0237] KRAS pancreatic cancer (KPC) cells were exposed to free cGAMP, cGAMP / LNP, mKRAS / LNP, cGAMP / mRASWT / LNP or cGAMP / mKRAS / LNP (1 μg / mL cGAMP, 4 μg / mL mRNA) for 24 h at 37oC. Total RNA was extracted by the TRIzol reagent (Thermo) and quantified by a NanoDrop Microvolume Spectrophotometer (Thermo). The RNA was used for cDNA synthesis after diluting in water (1:5, 1:25, 1:125, 1:625, 1:3125), mixed with SYBR green mix and a series of primer pairs for IFN-β1, CCL5, CXCL9, CXCL10). Real-time PCR (RT-PCR) analysis was performed in ViiA 7 Real-Time PCR System. mRNA expression was expressed as fold increase over the PBS-treated group. Bone marrow derived dendritic cell (BMDC) preparation and maturation

[0238] Bone marrow cells were isolated from the femur and tibia of 6 weeks old female B6129SF1 / J mice, followed by culture in RPMI 1640 medium (10% FBS), supplemented with GM-CSF (20 ng / ml) and IL-4 (10 ng / ml) for 6 days. BMDCs were then harvested and transferred to a new plate. The cells were treated with cGAMP / LNP, mKRAS / LNP, cGAMP / mRASWT / LNP or cGAMP / mKRAS / LNP (1 μg / mL cGAMP, 4 μg / mL mRNA) for 24 h at 37oC. Cells treated with LPS (1 μg / ml) served as positive control. Cells were stained with an antibody mixture (anti-CD11c, anti-CD80 and anti-CD86), followed by flow cytometry (Thermo Attnue NxT) to assess CD80 and CD86 expression on CD11c+ cells. LNP Biodistribution

[0239] Biodistribution was carried out in 8-week-old female B6129SF1 / J mice, using 3 animals per group. Six hours after IV injection of free Cy5-cGAMP or Cy5- cGAMP / mGFP / LNP (0.125 mg / kg Cy5-cGAMP, 0.5 mg / kg mGFP), IVIS imaging was carried out in a PerkinElmer Lumina II. Animals were sacrificed for organ harvesting and ex vivo imaging. We also collected paraffin-embedded liver tissues for sectioning and staining with DAPI, anti-GFP antibody and anti-CD146 antibody. The slides were scanned in a Vectra Polaris Quantitative Pathology Imaging System. Analysis of costimulatory and MHC expression on non-parenchymal liver cells

[0240] Mice (n = 3) were IV injected with two doses of PBS or cGAMP / mKRAS / LNP (0.3 mg / kg cGAMP and 1.25 mg / kg mRNA) on days 0 and 7, before being anesthetized for in vivo perfusion with a collagenase contained buffer. Animals were subsequently sacrificed,and liver tissue used for isolation of non-parenchymal cells (NPCs). NPCs were washed, counted, and stained with a mixture of antibodies recognizing CD80, CD86, MHC-I, and MHC-II molecules for flow cytometry analysis as described in the previous paragraph. IFN-γ ELISPOT assay and assessment of memory T-cells

[0241] Eight-week-old female B6129SF1 / J mice were randomly assigned to 2 groups, each including three animals. Four doses of PBS or cGAMP / mKRAS / LNP (0.3 mg / kg cGAMP and 1.25 mg / kg mRNA) were IV injected every 3 days. Following animal sacrifice 20 days after the last injection, spleens were harvested to isolate splenocytes. The cells were transferred into an ELISPOT plate, incubated in the presence of wild type KRAS peptide ([Acetyl]KLVVVGAGGVGKSALTI[Amide]) (SEQ ID NO:30) or KRAS G12D peptide ([Acetyl]KLVVVGADGVGKSALTI[Amide]) (SEQ ID NO: 16) for 24 h. The plate was washed and incubated with BCIP / NBT substrate for 5-15 min to obtain the desired color intensity. After drying, the plate was imaged in an ELISPOT reader (CTL Immunospot) to obtain the number of cytokine-producing spots by instrument software.

[0242] To assist the presence of memory T-cells, including for adoptive transfer, the same animal exposure conditions were used, followed by animal sacrifice 20 days after the last injection. Splenocytes were collected and stained with an antibody mixture (anti-CD3, anti- CD8, anti-CD44 and anti-CD62L), before the performance of flow cytometry (Attune NxT). Following the identification of single cells, gating was performed for the CD3+ / CD8+ subset before cross-gating on CD44 and CD62 to assess the presence of CD44+CD62L+ (central memory) and CD44-CD62L+ (effector memory) T-cell subsets (Fig.10B). Use of encapsulated cGAMP and mKRAS to treat animals with liver KPC metastases

[0243] To establish a liver metastasis model, a KRAS pancreatic cancer cell (KPC) line, derived from a transgenic KrasLSL G12D+ / Trp53LSL R172H+ / Pdx-1-Cre mouse model, was used for hemispleen injection, allowing spread to the liver via the portal vein (Fig. 11A). Briefly, murine spleens were surgically exposed under anesthesia, using two clips to divide the spleen in half (Fig.11A). This was followed by injecting 10,000-50,000 luciferase expressing KPC into the splenic portion draining to the liver, followed by removal of the other half. Metastatic spread to the liver was followed by d-luciferin injection into the peritoneum and IVIS imaging. The number of injected KPC cells were adjusted for the length of animal survival, established to be 16-22 days for 50,000 cells or 26-32 days for 10,000 cells. To study the therapeutic effect of cGAMP and mKRAS, 8- week-old female B6129SF1 / J mice were randomly assigned to 5 groups, each including 8 animals. Following injection of 50,000 KPC-luc cells per animal (day 1), 4 therapeuticdoses of cGAMP / LNP, mKRAS / LNP, cGAMP / mRASWT / LNP or cGAMP / mKRAS / LNP were IV injected on days 6, 9, 12, and 15 (Fig.13B). The dose of cGAMP was 0.3 mg / kg and that of mKRAS or mRASWT 1.25 mg / kg per administration. Tumor growth was monitored by IVIS imaging on days 5, 11, 14, 18, and body weights were recorded every 3 d. All mice were sacrificed on day 18 for blood collection and harvesting liver tissues for weighing and ex vivo IVIS imaging. Paraffin-embedded liver tissues were sectioned and stained with H&E or a fluorescent dye mixture (DAPI, anti-Ki67 antibody and anti-CD8 antibody) for confocal microscopy. The sections were scanned by Vectra Polaris Quantitative Pathology Imaging System. The blood was used for measuring serum IL-6 and IL-10 concentrations by ELISA according to the manufacturer’s instructions. Prophylactic administration of encapsulated cGAMP and mKRAS to determine the effect on KPC liver metastasis

[0244] Eight-week-old female B6129SF1 / J mice were randomly assigned to 2 groups, each including 8 animals. Four doses of saline or cGAMP / mKRAS / LNP (0.3 mg / kg of cGAMP and 1.25 mg / kg of mKRAS), were IV injected into mice on days 0, 3, 7, and 11. On day 14, 50,000 KPC-luc cells were injected into the hemispleen as described above. IVIS imaging system was used to monitor tumor growth on days 21, 28, 35, and 42. Survival and weight of the mice were also recorded. Adoptive cell transfer

[0245] Eight-week-old female B6129SF1 / J mice were randomly assigned among 2 groups, with 4 animals in each group. Four IV administrations of saline or cGAMP / mKRAS / LNP (0.3 mg / kg of cGAMP and 1.25 mg / kg of mRNA), were delivered on days 0, 3, 7, and 11. On day 14, all mice were euthanized to allow lymphocyte isolation from the spleen. The cells were used for adoptive transfer into recipient animals (n = 8), each receiving 3x106lymphocytes IV. Fifty thousand KPC-luc cells were injected into the recipient hemispleens on day 21, and IVIS imaging system used to monitor metastatic growth on days 28, 35, 42, and 49. The results were displayed in Kaplan-Meier plots. We also assessed animal weight every three days. Biosafety assessment

[0246] Mice were treated with four IV administrations of PBS, free cGAMP and cGAMP / mKRAS / LNP on days 1, 4.7, 10. The doses of cGAMP and mKRAS were 0.3 mg / kg and 1.25 mg / kg. Serum was collected for the quantification of IL-6, TNF-α, ALT, AST, BUN, creatinine on day 2, 11, and 24.Statistics

[0247] Comparative analysis of differences between groups was performed by 1-way ANOVA followed by Tukey’s multiple comparisons. Values were expressed as mean ± SD of multiple determinations. For all statistical analyses, a p value of < 0.05 was considered statistically significant. RESULTS LNP preparation for delivery of cGAMP and KRAS G12D mRNA combinations

[0248] Studies were undertaken to develop nanoparticles and methods for reprogramming tolerogenic effects of liver APCs using a STING agonist to induce a protective cytotoxic T- cell response against a KRAS neoepitope, thereby reversing the immune protective niche facilitating pancreatic cancer metastasis to the liver. In one embodiment, the KRAS G12D epitope was used, which is expressed in 35% of human PDAC cases and is also present in the robust transgenic KrasLSL G12D / +; Trp53LSL R172H / +; Pdx-1-Cre mouse model, enabling the development of an orthotopic KRAS pancreatic cancer (KPC) mouse model (Fig.1A). The KRAS G12D peptide sequence (aa 3-23, EYKLVVVGADGVGKSALTIQL, SEQ ID NO:30) was used to derive the corresponding cDNA sequence (Fig.1B), followed by insertion of five copies into an open reading frame, flanked by natural amino acid sequences (SEQ ID NO:77; Fig.1C). Additionally, a control cDNA sequence was constructed incorporating five repeats of a corresponding non-immunogenic peptide sequence from wild-type RAS (aa 5-21, EYKLVVVGAGGVGKSALTIQL, SEQ ID NO:29), also flanked by natural amino acid sequences (Fig.1B). Following the addition of start and stop codons, codon optimization was performed using the online GeneScript tool, yielding GC contents of 52.08% and 53.65% for the KRAS and RASWT strands, respectively (Fig.2A). These optimized cDNA sequences were used to order the corresponding mRNA strands from TriLink Biotechnologies, which delivered 582- nucleotide strands (Fig.2A: KRAS epitope SEQ ID NO:77; wildtype control, SEQ ID NO:78).

[0249] To construct an ionizable LNP that co-encapsulates a stimulator of interferon genes (STING) agonist with mRNA, a negatively charged 2’,3’-cGAMP dinucleotide sequence was selected (Fig.1D) for electrostatic binding to the cationic lipid, DLin-MC3-DMA (Fig. 1E). The LNP synthesis was carried out using a microfluidic mixer (Nanoassemblr), with the negatively charged mRNA and cGAMP introduced in a diluted acetic acid buffer in one channel, while the ethanol-diluted lipid mixture was infused into the second channel (Fig. 3A). Details of the analyte concentrations and volume calculations for microfluidicsynthesis appear in Fig.4. Following dialysis and particle filtration, dynamic light scattering (DLS) analysis showed average particle sizes of approximately 90 nm (Fig.3B) with polydispersity indexes (PDI) of 0.176. CryoEM imaging confirmed the synthesis of spherical particles, displaying nano-precipitated complexes. The concentrations of cGAMP and mKRAS were quantified using a cGAMP ELISA kit and a RiboGreen assay kit, respectively (Fig.3C). The encapsulation efficiency (EE) of the LNPs, defined as the % mKRAS entrapped inside the LNPs relative to total mRNA, demonstrated EE values of 92%. The stability of the cGAMP / mKRAS / LNP formulation was confirmed by monitoring size and EE continuously at 4°C for 15 days. The results showed a 10% increase in size (Fig.3C), with minimal impact on PDI and EE (Fig.3E).

[0250] The intracellular release of mRNA from the LNP is dependent on the ionizable lipid component forming a hexagonal lipid phase that is required for fusion with the APC endosomal membrane. To evaluate the preservation of this mechanism in the presence of cGAMP, we conducted red blood cell (RBC) lysis as a surrogate membrane for assessing the hexagonal phase. RBCs were suspended in PBS at pH 7.4 or 5.5, before incubation with cGAMP / mKRAS / LNP or mKRAS / LNP at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL. Triton X-100 was used as a positive control for total RBC lysis. As shown in Fig. 5A, both particle types induced concentration-dependent RBC lysis at pH 5.5, while no lysis occurred at pH 7.4. Fig.5B details the percent hemolysis compared to Triton (100%). These results demonstrate that the encapsulation of cGAMP does not affect hexagonal phase formation, safeguarding effective mRNA release from the LNP. Activation of the STING pathway by encapsulated cGAMP

[0251] The cGAS-STING signaling pathway, comprised of cyclic GMP-AMP synthase (cGAS), a transmembrane protein, STING, inserted in the endoplasmic reticulum membrane plus downstream signaling adapters, plays a crucial role in protective immune defense against microbial infections or internal damage-associated DNA (Fig.6A). In one embodiment, nanoparticles capable of overcoming the negative charge of dinucleotides that prevent membrane permeation, as well as protecting the cargo protection to allow liver uptake, are disclosed herein.

[0252] To investigate whether encapsulated cGAMP could activate the STING pathway, we examined the in vitro induction of interferon β-1 (IFN-β1), CCL5, CXCL9, and CXCL10 mRNA expression using RT-PCR (Figs.6B-6E). This analysis reflects the robust impact of the STING pathway on IFN-β1 production in the liver, capable of activating immune responses that combat viral infections and elimination of infected cells. Similarly, CCL5,CXCL9, and CXCL10 are chemokines that are involved in the recruitment and activation of innate and adaptive immune responses, playing key roles in inflammation and immune surveillance. RT-PCR expression analysis was conducted in KPC cells, derived from the transgenic KrasLSL G12D+ / Trp53LSL R172H+ / Pdx-1-Cre mouse model, exposed to the various stimuli shown in Fig.6 for 24 hours. Using a series of primers to quantify mRNA expression of IFN-β1, CCL5, CXCL9, and CXCL10, we observed the respective mRNA levels increasing by 201, 94, 22, and 43-fold, respectively, during cGAMP / mKRAS / LNP treatment compared to the untreated control (Figs.6B-6E). Similar increases were also noted in cells treated with cGAMP / LNP or cGAMP / mRASWT / LNP, while treatment with free cGAMP or mKRAS / LNP failed to show significant increases in mRNA expression. These data confirm that encapsulated cGAMP effectively stimulates the STING pathway in KPC cells, whereas mRASWT or mKRAS alone do not have effects.

[0253] To determine the impact of cGAMP / mKRAS / LNP on the maturation of DCs, we monitored the surface expression of CD80 and CD86 in mouse bone marrow-derived DCs, which were generated with GM-CSF and IL-4. CD80 and CD86 are crucial costimulatory molecules on APCs that interact with CD28 on T-cells, providing "signal 2", as depicted in Fig.7A. Exposure of DCs to cGAMP / LNP, cGAMP / mRASWT / LNP, and cGAMP / mKRAS / LNP significantly increased the percentage of CD80+ / CD86+ cells from 32% in the saline group to 62%, 57%, and 63%, respectively (Fig.7B, 7C). In contrast, mKRAS / LNP alone was ineffective. LPS treatment served as a positive control. These results confirm that encapsulated cGAMP effectively promotes DC maturation. Encapsulated cGAMP reprograms liver APC maturation markers and is accompanied by generation of IFN-γ producing effector and memory T-cells.

[0254] To demonstrate the effective delivery of cGAMP for reprogramming costimulatory receptor expression in liver non-parenchymal cell (NPC) populations, including LSECs, Kupffer cells, and DCs, LNP carriers were developed for Cy5-labeled cGAMP as well as a mRNA strand encoding green fluorescent protein (GFP) (mGFP) expression (Fig.8). This required the creation of Cy5-cGAMP / mGFP / LNP using a microfluidics approach with the components shown in Fig.8A. The resulting Cy5-cGAMP / mGFP / LNP had a size of 132 nm, with cryoEM features illustrated in Fig.8B. These particles were IV injected, followed by harvesting of major organs (including the heart, liver, spleen, lungs, and kidneys) 6 hours later for ex vivo IVIS imaging (Fig.8C). The Cy5 signal was discernible in the liver only of animals receiving encapsulated but not free Cy5-cGAMP (Fig.8D). Quantitative analysis of Cy5 radiance is shown in Fig.8E. No significant uptake of encapsulated particles was seenin other organs. Furthermore, harvested liver tissues were sectioned and subjected to immunofluorescence microscopy, which confirmed GFP expression throughout the liver, including uptake by fluorescent-labeled (red) sinusoidal endothelial cells (LSECs) (Fig.8F). LSECs are one of the principal tolerogenic antigen-presenting cell types in the liver, contributing to the tolerance and immune protection of cancer metastases.

[0255] To assess whether cGAMP / mKRAS / LNP activates and reprograms NPCs to express IFN-β1 and costimulatory receptors (CD80, CD86, MHC-I, and MHC-II), animals were treated with the particles on days 1 and 7. This was followed by in vivo liver perfusion under anesthesia with a collagenase IV buffer (Fig.9A). The digested livers were harvested and used to isolate the NPCs for evaluating IFN-β1 mRNA expression by RT-PCR and costimulatory receptor expression by flow cytometry (Fig.9B). RT-PCR analysis revealed a 12.5-fold increase in IFN-β1 mRNA expression following treatment with cGAMP / mKRAS / LNP (Fig.9C), indicating activation of the STING pathway in vivo. Flow cytometry analysis showed increased expression of CD80, CD86, and MHC-I on NPCs in response to cGAMP / mKRAS / LNP (Fig.9D). Conversely, the expression of MHC-II, which plays a role in tolerogenic effects, was decreased (Fig.9D). Overall, these phenotypic changes predict a favorable KRAS epitope response, dependent on MHC-I presentation plus the receipt CD28 costimulatory signals from CD80 and CD86 expressed on the APC surface.

[0256] To assess the ability of cGAMP / mKRAS / LNP to generate T-cell responses, female B6129SF1 / J mice (n = 3) received four IV administrations of saline or the nanocarrier. After 20 days, the mice were sacrificed, and splenocytes were harvested for IFN-γ ELISPOT assays (Fig.9E). Splenocytes from saline-treated animals showed few cytokine spots when incubated with the G12D or wildtype peptides. In contrast, a significant increase in the number of splenocyte IFN-γ producing spots was observed in the cGAMP / mKRAS / LNP-treated mice during treatment with the G12D but not with the wildtype peptide (Fig.9F, 9G). To demonstrate the presence of effector and memory CD8+ T-cells among the splenocytes harvested during the experiment, flow cytometry was performed to assess the changes in the abundance of CD44+CD62L+ (central memory) and CD44+CD62L- (effector memory) subsets within the CD3+ / CD8+ gated population (Fig. 10A-10C). Central (TCM) and effector memory (TEM) cells, which differentiate from effector T-cells (Fig.10A), are characterized by their development of CD44+ / CD62L+ and CD44+ / CD62L- phenotypes, respectively. Both TCM and TEM, were significantlyincreased (Fig.9I and 9J), indicating the possibility of an improved immune response upon secondary encounter and of importance to adoptive transfer. Use of a KPC metastasis model to demonstrate the therapeutic efficacy of cGAMP / mKRAS / LNP

[0257] A PDAC metastasis mouse model was established using hemispleen injection of KPC-luc cells, as detailed in Fig.11A. IVIS imaging confirmed that the KPC cells predominantly metastasize to the liver, without impacting the heart, lungs, kidneys, or remaining portion of the spleen. This liver-specific spread was further corroborated by photographing the harvested liver tissues. In the course of establishing this model, the number of injected KPC cells was adjusted to determine the duration of animal survival, with 50,000 cells resulting in animal demise within 16-22 days, while injecting 10,000 cells led to fatality within 26-32 days.

[0258] To assess the therapeutic impact of cGAMP / mKRAS / LNP in a PDAC metastasis mouse model, animals injected with 5×104KPC-luc cells on day zero received four IV injections of cGAMP / LNP, mKRAS / LNP, cGAMP / mRASWT / LNP, or cGAMP / mKRAS / LNP on days 6, 9, 12, and 15 (Fig.11B). Each injection delivered a cGAMP dose of 0.3 mg / kg and / or an mRNA dose of 1.25 mg / kg. Tumor growth was monitored by IVIS imaging, showing visible tumor growth on day 5, evolving to strong radiance on day 18 (Figs.11C, 12A). Among the treated groups, mice receiving cGAMP / mKRAS / LNP showed the least increase in radiance intensity, which was highly statistically different from animals treated with saline (p<0.001), cGAMP / LNP (p<0.05), mKRAS / LNP (p<0.05), or cGAMP / mRASWT / LNP (p<0.05) (Fig.11D). Body weight was recorded every three days (Fig.12B), and liver weight was measured at the time of animal sacrifice. This showed least liver weight gain in the dual cGAMP and mKRAS treatment group (Fig.11E). This is consistent with quantification of luciferase radiance during ex vivo IVIS imaging (Fig.12C), which showed a strong statistical deviation (p < 0.001) for cGAMP / mKRAS versus saline, with lesser impact (p<0.05) by other treatment groups (Fig. 12D). These results suggest that cGAMP / mKRAS / LNP can effectively interfere in metastatic growth.

[0259] To further evaluate the immune response in the liver, histology and immunohistochemistry were used to compare H&E with fluorescence staining, involving DAPI staining to detect nuclei (purple blue) and Ki67 fluorescence (green) to assess cellular proliferation. Collectively, these results showed a significant impact on KPC metastatic growth in the liver during LNP treatment (Fig.11F). Thus, H&E staining demonstratednoticeable shrinkage of the nuclear-dense area, also corresponding to the Ki-67-stained loci, in response to treatment, particularly prominent in the cGAMP / mKRAS / LNP group. These images were used for calculating metastatic burden, defined as the area occupied by metastatic foci relative to the total liver surface area (Fig.11G). The average metastatic burden in the cGAMP / mKRAS / LNP group was 6%, which was significantly lower than in other groups. This demonstrates the superior anti-tumor effect of cGAMP / mKRAS / LNP treatment.

[0260] To relate the decline in metastatic burden to anti-tumor immunity, immunohistochemistry was performed to assess CD8+ T-cell recruitment to the Ki-67-rich tumor loci (Fig.11H). While a paucity of T-cells was observed in the saline-treated group, a significant increase in CD8+ T-cell recruitment was observed in the other treatment groups. The most substantial increase was for the cGAMP / mKRAS / LNP-treated group, showing CD8+ T-cell levels 25 times higher than the saline group, while also deviating statistically from other treatment groups (Fig.11I). The increased number of cytotoxic T-cells was also accompanied by perforin deposition and IFN-γ immunohistochemistry staining, as demonstrated in Figs.11J and 11L. Survival impact of cGAMP / mKRAS / LNP as a therapeutic response model

[0261] To determine the survival impact of cGAMP / mKRAS / LNP administration in mice injected with 5×10⁴ KPC-luc cells, the same type of experiment was performed for comparing delivery of treatment on days 6, 9, 12, and 15 (Fig.13A). Mice in the saline group had a median survival of 19 days (ranging 15- 23 days). In contrast, the median survival for animals receiving cGAMP / LNP, mKRAS / LNP, cGAMP / mRASWT / LNP, and cGAMP / mKRAS / LNP was 24 days, 24 days, 25.5 days, and 31 days, respectively (Fig. 13B, Fig.14). The significant improvement in the median survival of the cGAMP / mKRAS / LNP group demonstrates the synergistic effect of cGAMP and mKRAS co-delivery.

[0262] Subsequently, we studied the survival rate for mice receiving a hemispleen injection of 1×10⁴ KPC cells, followed by treatment with saline, cGAMP / mRASWT / LNP, and cGAMP / mKRAS / LNP (Fig.13C). The Kaplan-Meier plot indicated that while animals treated with saline had a median survival of 29 days, those treated with cGAMP / mRASWT / LNP and cGAMP / mKRAS / LNP had median survival times of 33 days and 55 days, respectively (Fig.13D, Fig.15). Noteworthy, 50% of the animals treated with cGAMP / mKRAS / LNP remained alive up to day 63, when the animals were sacrificed.The impact of prophylactic cGAMP / mKRAS / LNP administration, including use of splenocytes for adoptive transfer to non-treated recipients

[0263] The development of a liver metastasis in PDAC is often fatal, irrespective of the treatment response at the primary cancer site. We therefore asked whether prophylactic administration could be used as a form of vaccination to prevent KPC metastatic growth in the liver (Fig.8A). Animals received the same dose of cGAMP / mKRAS / LNP in four IV administrations before splenic injection of 5×104KPC-luc cells for a survival experiment (Fig.16A). The resulting Kaplan-Meier plot showed that, in comparison to the saline control group (20 d), animal survival was highly significantly (p <0.001) extended by prior cGAMP / mKRAS / LNP treatment (Fig.16B, Fig.17).

[0264] The prophylactic model was subsequently used to determine whether the retrieval of T-cells from the spleen of LNP injected animals can be used for adoptive transfer, prior to recipients undergoing hemispleen injection of 5×104KPC-luc cells (Fig.16C). The results show that the median survival of mice receiving adoptively transfer of lymphocytes from cGAMP / mKRAS / LNP treated animals was 32.5 days, which was significantly higher (p <0.001) than 19 days for animals receiving lymphocytes from saline treated animals (Fig. 16D, Fig.18). Biosafety assessment

[0265] While therapeutic use of STING agonists holds great promise for cancer immunotherapy and infectious disease treatment, systemic administration can trigger significant off-target effects due to potent immune system activation. This includes the possibility of cGAMP generating a cytokine storm, which stands to be lessened by encapsulated delivery. To determine if the same holds true for the ionizable LNP, we undertook a biosafety experiment to assess the impact on cytokine levels in the serum, as well as assessment of biomarkers that reflect liver and renal toxicity. Four IV injections of cGAMP / mKRAS / LNP, each delivering a cGAMP dose of 0.3 mg / kg and an mKRAS dose of 1.25 mg / kg, were administered before blood collection and recording animal weight, as shown in Fig.19A. Saline and free cGAMP were used as controls. Noteworthy, no significant change in body weight or physical evidence of general health decline was observed during daily inspections over 24 days (Fig.19B).

[0266] Blood collected from the animals on days 2, 11, and 24 was used to study serum cytokine concentrations (IL-6 and TNF-α) as well as biomarkers that reflect liver (ALT and AST), or kidney (BUN and creatinine) damage. No significant changes in IL-6, TNF-α, ALT, AST, BUN, or creatinine levels were observed (Figs.19C-H). These resultsdemonstrate that four doses of cGAMP / mKRAS / LNP (0.3 mg / kg of cGAMP and 1.25 mg / kg of mKRAS) are relatively safe. We also did not observe evidence of animal toxicity in the studies described in Figs.6A-E to Figs.8A-G.

[0267] As shown herein, reprogramming of the tolerogenic immune environment in the liver generated an effective immune response against often-fatal PDAC metastases. In fact, a 50% survival rate was achieved in an experiment using 10,000 KPC (Fig.13D). This was attained by developing a lipid nanocarrier that delivers mRNA encoding a KRAS G12D neo-antigenic peptide along with cGAMP, a STING agonist. The LNPs were synthesized using a microfluidics process, enabling the nano-precipitation of mRNA and cGAMP by the ionizable lipid MC3, yielding 90 nm LNP. These carriers successfully activated the type I interferon pathway in vitro, enhanced effective particle uptake and triggered IFN-β1 expression in the liver. Moreover, this was accomplished by triggering IFN-γ producing T- cells in the spleen, demonstrated in ELISPOT assays. Additionally, cGAMP / mKRAS / LNP induced costimulatory receptor expression (CD80 and CD86) on non-parenchymal liver APCs, promoting the generation of cytotoxic and memory T-cells capable of mounting an effective immune response against a metastatic KRAS pancreatic cancer (KPC) mouse model. The efficacy of this approach was demonstrated by IV administration of cGAMP / mKRAS / LNPs, both prophylactically and following establishment of metastases. The immune response at the metastatic tumor site was characterized by the recruitment of CD8+ cytotoxic T-cells, leading to tumor shrinkage and prolonged survival. Moreover, the immune response could be adoptively transferred by injecting splenocytes into recipient animals.

[0268] The most impactful finding of this study is the demonstration of synergy between the KRAS mRNA and the STING agonist nanoparticles components in combating PDAC liver metastasis (Figs.11A-L, 13A-E, 16A-D). The liver is home to unique APCs for preventing an excessive immune response to foreign antigens from the gut. This tolerogenic immune environment also allows the liver to protect itself or co-transplanted organs against rejection, as well as hindering mounting of effective immune responses against metastatic cancer cells. Among the many pathways used by the liver for exerting immune suppressive effects is the generation of regulatory T-cells by Kupffer cells and LSECs. We demonstrate that it is possible to reprogram the APC effects, which are dependent differences on their unique antigen and processing effects, as well as the production of tolerogenic cytokines. When intracellularly delivered, cGAMP increases IFN-β1 production, as well as engaging in upregulation of costimulatory receptors (CD80, CD86) and MHC-I expression on non-parenchymal liver cells (Fig 9). These property changes favor MHC-I presentation of KRAS G12D to cytotoxic T-cells, also receiving additional costimulatory stimuli from surface expression of CD80 and CD86 on the APCs.

[0269] The ability to accomplish both vaccination as well as an adjuvant delivery with a single nanocarrier is highly advantageous, particularly when accomplishable with scalable carrier technology, which also enables incorporation of an ionizable lipid to promote liver targeting through the assembly of an ApoE protein corona. This approach also allows efficient nanoprecipitation of the negatively charged dinucleotide and mRNA by the cationic, ionizable lipid, e.g., MC3. It is notable that the synergy between these components applies to prophylactic as well as already established metastases models, allowing for a range of PDAC immunotherapy interventions (Fig.16). The first is the potential of using a vaccination approach to prevent new metastatic spread to the liver in recently diagnosed PDAC patients. This may help to strengthen the efforts of establishing therapeutic responses at the primary tumor site, more conducive to life extension in the absence of metastases. However, the demonstration of enhanced survival beyond the establishment of metastases, also show potential for improved immunotherapy, which may benefit the primary pancreas cancer site. We are currently investigating this possibility in a KRAS orthotopic model, allowing the development of spontaneous liver metastases from the primary cancer site. This could allow us to combine our LPN technology with our established use of lipid bilayer coated mesoporous silica nanoparticles (a.k.a. the silicasome nanocarrier), used for chemoimmunotherapy, capable of generating immunogenic cell death (e.g., irinotecan). Immunogenic cell death (ICD) releases multiple endogenous antigens, which can subsequently trigger a cancer immunity cycle that involves the spleen and regional lymph nodes to generate polyclonal immune responses. Thus, combining of a polyclonal ICD response with a strong vaccination response against neoantigens, could boost the cancer immunity cycle and accompanying immune response at the primary cancer site. This includes the generation of tertiary lymphoid follicles at the primary cancer site.

[0270] The breakthrough achieved with mRNA vaccine technology during the COVID-19 pandemic is making significant strides in cancer immunotherapy, targeting a range of neoantigens such as KRAS, EGFR, BRAF, TP53, and JAK2. Importantly, the mRNA approach for KRAS neoantigen delivery has been expanded to include mutations beyond G12D. For instance, Moderna's mRNA-5671 (V941) is a cancer vaccine candidate that targets a combination of KRAS mutations, including G12D, G12V, G13D, and G12C, and is currently being evaluated for various KRAS-expressing cancers such as PDAC, non-small cell lung cancer, and colorectal cancer. Our choice of the G12D mutation was dictated by the specific KRAS oncogene expressed in our animal KPC model, also widely expressed in several cancers, including those prone to metastatic spread to the liver. The LNP carrier disclosed herein could readily be augmented with additional KRAS mutants as well as other cancer neoantigens or tumor-associated antigens, such as EGFR, BRAF, or TP53. Notably, we have previously demonstrated the development of liver-targeting tolerogenic nanoparticles capable of generating multi-epitope mRNA constructs that can be routed to the MHC-II compartment for raising tolerogenic immune responses. The ability to accomplish similar outcomes in cytotoxic T-cells using MHC-I routing plus multi-epitope inclusion, could benefit from the use multiple neoantigens, potentially useful to treat a range of cancers.

[0271] It is important to emphasize the role of encapsulated delivery in terms of the safety of STING agonists. Negatively charged cyclic dinucleotides (CDNs), such as cGAMP, are unable to permeate cell membranes and, when systemically disseminated can lead to widespread innate immune activation, including the potential to generate a cytokine storm. However, restricted delivery to the liver reduces the risk of systemic toxicity, also allowing APCs uptake and reprogramming of the antitumor immune response. In addition to the use of an ionizable lipid, diacyl lipids have been employed for cGAMP conjugation and incorporation into nanodiscs, capable of inducing robust antitumor immunity.

[0272] In the realm of cancer immunotherapy, STING agonists co-delivered with mRNA vaccines have proven particularly potent in stimulating APCs through the STING pathway, leading to enhanced antitumor immunity with minimal systemic inflammation. A recent study also highlighted the use of STING agonists with mRNA in lymph node-targeted nanoparticles, which showed robust tumor-specific immune responses and long-term memory in murine models. As noted above, Moderna's mRNA-5671 (V941) is another cancer vaccine candidate that targets a combination of KRAS mutations, including G12D, G12V, G13D, and G12C, and is currently being evaluated for various KRAS-expressing cancers such as PDAC, non-small cell lung cancer, and colorectal cancer. These findings underline the potential of combining mRNA vaccines with STING agonists for both cancer and infectious diseases, but also point to the importance of additional unique formulations for combining adjuvants with a vaccine component that targets the liver to enhance efficacy. A major advance in the current study lies in addressing specific mechanistic limitations found in earlier designs, such as mRNA translation efficiency, while advancing targeted immunomodulatory responses not yet fully explored in existing literature.

[0273] Thus, reprogramming the immune-protective niche for metastatic pancreatic cancer can be achieved by delivering a STING agonist and mutant KRAS mRNA via ionizable LNPs, offering both prophylactic and therapeutic benefits. Example 2 Use of polymeric nanoparticles to boost chemo-immunotherapy

[0274] The aim of this section is to delineate is the use of polymeric nanoparticles for generating a vaccine response to the mutant KRASG12D epitope to boost chemo- immunotherapy. The potential advantage of such an approach would be to enhance the recruitment of high affinity, antigen-specific CTL clones from lymphoid organs (e.g., lymph nodes and spleen) or tertiary lymphoid structures that may participate in the cancer immunity cycle. The hypothesis is that low levels of endogenous TAA and neoantigens that are being released from dying cancer cells during ICD may not be effective for stimulating T-cell antigen receptors, leading to recruitment of CTLs that exhibit low cytotoxic potential for cancer cells. Thus, exogenous vaccination with PDAC antigens known to lead to effective MHC-I presentation, could populate the regional lymphoid structures with higher affinity CTL populations for recruitment to the primary cancer site. In addition to the spleen and regional lymph nodes, there is now growing awareness of the role of tertiary lymphoid structures (TLS), assembling at primary cancer sites for boosting antitumor immunity. TLS are organized structures, similar to lymphoid follicles that harbor T-cells, B-cells, and dendritic cells, with the ability to improve immune recruitment, activation, proliferation, and cytotoxic killing. Accordingly, the appearance of TLS in various cancers is regarded as predictive of improved of antitumor immune responses and patient outcome, including for pancreatic cancer. Thus, we hypothesize that exogenous vaccination will improve the impact of endogenous vaccination in PDAC by ICD.

[0275] KRAS frequently presents driver mutations in PDAC, colorectal and lung cancers, with mutation frequencies of 97.7, 44.7, and 30.9% respectively. Four mutations (G12, G13, Q61, G12D) cover more than 99% of the PDAC mutations, with KRAS G12D contributing up to 45%. Not only do these PDAC mutations drive the development of pancreatic intraepithelial neoplasia but prediction-making for selecting immunogenic KRAS peptides to perform vaccination, has also greatly improved. This includes evidence that KRAS G12D can be expressed on type I murine MHC and human HLA (e.g., HLA-A*11:01 or HLA- C*8:02) antigen presenting complexes, with the ability to induce PDAC targeting by CTL. This has resulted in demonstrating therapeutic responses to mutated KRAS peptides in human PDAC trials. Thus, while some phase I and II clinical trials have shown longersurvival in vaccine responders compared to non-responders, no therapeutic benefit was obtained in advanced-stage pancreatic cancer patients. We further propose that vaccine efficacy can be improved in combination with ICD therapy, thereby allowing higher affinity CTL to participate in the cancer immunity cycle. From a vaccination perspective, it is also relevant that the administration of an irradiated allogeneic PDAC vaccine (GVAX) could boost the generation of tertiary lymphoid structures in a human PDAC trials.

[0276] Since the immunogenicity of peptide vaccines is enhanced by lymph node (LN) delivery, we surmised that the same approach could be applicable to the generation of TLS in our murine model. Typically, the uptake of vaccinating nanoparticles in LNs are facilitated by a particle size of 25 to 200 nm. It has also been demonstrated that the immunogenicity of peptide vaccines can be enhanced by conjugating the peptides to TLR agonists or a carrier protein such as albumin. The strategy that we adopted was to use passive KRAS peptide encapsulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles that also include a TLR7 / 8 agonist, resiquimod (R848) (Figs.21A-F). PLGA nanoparticles are avidly taken up by LNs, allowing efficient encapsulation and cargo release for antigen presentation in LNs. Carrier synthesis was achieved by using a double-emulsion solvent evaporation technique to construct two distinct nanoparticles versions (Fig.21A). In the first embodiment, the unmodified M23 peptide, sparingly soluble in water, was added to the oil phase containing PLGA in dichloromethane, with R848 being added in the entrapped aqueous phase. This yielded particles that encapsulate M23 peptide in the shell, with R848 in the aqueous core. In the second embodiment, the KrasG12Dpeptide was modified on both ends with aspartate residues to enhance water solubility. This peptide was added to the inner aqueous phase, combined with R848, while the oil phase was comprised of PLGA solubilized in DCM (Figs.21A-F). Using the double emulsion approach, another nanoparticle was constructed where both the hydrophilic D23 peptide and R848 were both encapsulated in the aqueous core (Figs.21A-F). We also synthesized a M23 / DiR nanoparticle with an empty core and a scrambled peptide (polyD) nanoparticle encapsulating the peptide with R848 in the aqueous core (Fig.21C). This yielded nanoparticles in the 150-200 nm size range with a zeta potential of -70mV, conducive to lymphatic spread. The SEM images further confirmed the size and polydispersity distribution (Fig.21B). Both D23 and M23 containing nanoparticles showed 23% loading efficiency of R848.

[0277] To demonstrate the agonist activity of the encapsulated R848, the nanoparticles were co-incubated with murine HEK-Blue cells to demonstrate a dose-dependent increase inTLR7-mediated reporter gene activity. We also employed the DiR-labelled nanoparticle to demonstrate its lymphatic spread to regional lymph nodes when injected subcutaneously in B6 / 129J mice. Compared to the free DiR, the particle-encapsulated dye was retained for a prolonged period during live in vivo imaging (IVIS).

[0278] To determine vaccination impact on anti-tumor immunity, B6 / 129J mice were subcutaneously injected with (M23+R848) PLGA and (D23+R848)PLGA nanoparticles 13 days prior to subcutaneous challenge with Kras pancreatic cancer cells (KPC). As a control for the impact of the nanoparticles, we also used subcutaneous injection of KPC cells treated with an ICD-inducing dose (300mM) of irinotecan to demonstrate interference in live KPC growth.5×104KPC cells, undergoing ICD, were administered in one flank of the animal to induce a protective immune response, demonstrated by growth failure of live KPC cells, injected in the contralateral flank a few days later.

[0279] The possible protective effect of the nanoparticles was tested by injecting the animals in one flank on three occasions, seven days apart, with particles delivering peptide doses of 10µg M23 or D23, plus 2µg R848. Additional controls included animal groups vaccinated with an admix of free peptide plus adjuvant (i.e., M23+R848 admix and D23+R848 admix). A day prior to the final boost, the mice were challenged with 1.5×105live KPC cells subcutaneously on the contralateral flank and observed for tumor growth, as described in Fig.21A. The animals were monitored for 45 days during which tumor volumes and animal weights were recorded (Figs.21B-21C). This demonstrated that 50% of mice vaccinated with (M23+R848)PLGA remained tumor-free for up to 45 days after challenge (Fig.21B). Further, in tumor developing animals, there was significantly slower growth in the nanoparticle treated animals compared to unvaccinated or animals receiving the free (M23+R848) admix. All the animals vaccinated with irinotecan treated KPC cells remained tumor free, indicating the efficacy of the ICD effect. In contrast to the results obtained with the encapsulated (M23+R848)PLGAcombination, animals vaccinated with (D23+R848)PLGA had higher instances of tumor formation where 5 out of 7 vaccinated mice (70%) developed tumors. However, like (M23+R848)PLGAvaccination, the animals receiving D23 vaccine nanoparticles showed considerable slowing of tumor growth compared to admix vaccinated mice (Fig.21C).

[0280] Mice remaining alive were sacrificed 45 days after challenge for tumor harvesting and IHC analysis. IHC staining to demonstrate the presence of CD8+tumor infiltrating lymphocytes confirmed a significant increase in CTLs in tumors from mice vaccinated with both (M23+R848) PLGA and (D23+R848)PLGA nanoparticles compared to the free admix orunvaccinated mice (Figs.21D-21E). We also observed the emergence of tertiary lymphoid structures (TLS), a feature of tumor immunity that has been more frequently recognized recently. Noteworthy, examination of the tumors in (M23+R848)PLGA vaccinated mice, demonstrated the appearance of intra-tumoral lymphoid aggregates in 50% of tumors, with an illustrative example shown in (Fig.21F). This is illustrated by the H&E staining as well as the IHC analysis of CD8+CTLs and CD21+follicular dendritic cells in these structures (Fig.21 F). In contrast to the results with the M23 encapsulated peptide, only 20% of tumors in the (D23+R848)PLGAvaccine group developed lymphoid aggregates. In summary, (M23+R848)PLGA vaccine elicited a stronger and more persistent immune activation compared to (D23+R848)PLGA, evidenced by fewer instances of tumors, smaller tumor sizes and consistent formation of lymphoid aggregates.

[0281] To further assess the efficacy of the Kras vaccine in an orthotopic KPC-luciferase model, we selected the more robust (M23+R848)PLGAnanoparticle for prophylactic administration and histological analysis. Using the same vaccination timeline and dosimetry as in Fig.21A, tumor-bearing mice were vaccinated with the encapsulated (M23+R848)PLGAcombination (Fig.22A), prior to the implantation of 0.5×106KPC-luciferase cells in the pancreas, a day prior to the final boost. The control groups included animals that are not vaccinated or vaccinated with either M23 nanoparticles (without R848) or nanoparticles delivering a scrambled peptide (polyD) plus R848. The mice were monitored every 4 to 5 days for tumor progression using IVIS imaging (Figs.22B-22C). Mice were sacrificed 16 days post-challenge, followed by harvesting of tumors and other key organs for ex vivo imaging. This demonstrated the presence of smaller tumors with reduced luminescence intensity in mice immunized with (M23+R848)PLGA, compared to unvaccinated or animals receiving the (polyD+R848)PLGA or M23PLGA (no adjuvant) vaccines (Fig.22D). Subsequent performance of ex vivo IVIS analysis also demonstrated larger tumor masses with metastatic spread to surrounding organs in non-treated animals, while animals receiving (M23+R848)PLGAshowed significantly smaller tumors, judged by tumor luminescence and weight (Figs.22E-22F). This response was also significantly improved over animals vaccinated with (polyD+R848)PLGAor M23PLGA. The observations suggest that control mice receiving injections with peptide or adjuvant showed an inferior response compared to animals receiving nanoparticles encapsulating both M23 and R848.

[0282] Further demonstration of the immunogenicity of the (M23+R848)PLGAvaccine was obtained by performing ELISpot assays on splenocytes retrieved from vaccinated and unvaccinated mice, followed by ex vivo stimulation with the corresponding Kras peptide.This demonstrated an increased number of IFNγ+and TNFα+producing cellular spots in mice vaccinated with (M23+R848)PLGAnanoparticles (Figs.23A-23B). In comparison, unvaccinated or animals treated with M23PLGA showed fewer ELISpots. Interestingly, there was an increase in the number of IFNγ+colonies in the spleens of mice receiving (polyD+R848)PLGA, which is of uncertain significance considering that the same does not happen for TNFα. Tumor sections were also analyzed for T-cell infiltration, demonstrating an increased number of CD8+and CD4+T lymphocytes in the (M23+R848)PLGA vaccination group (Fig.23C-23D). Moreover, like the results in the subcutaneous experiment, all the (M23+R848)PLGA vaccinated mice showed development of lymphoid aggregates (Fig.23E). These immune clusters included positive staining for CD4, CD8 and CD21 cellular markers, as is characteristic of TLS. Despite their potential prognostic relevance, the actual mechanism involved in TLS generation at primary cancer sites and the precise contribution to antitumor immunity is incompletely understood. One possibility is that these structures provide an intra-tumoral reservoir of immune cells, available for antigen sampling and potential CTL recruitment to the tumor cores. This hypothesis is now being tested in the orthotopic model, where KRAS vaccination is combined with chemo-immunotherapy as well as other immunomodulators.

[0283] Despite the formidable treatment challenges of pancreatic ductal adenocarcinoma (PDAC), considerable progress has been made improving drug delivery through pioneering nanocarriers. These innovations are geared towards overcoming the obstacles presented by dysplastic stroma and fostering anti-PDAC immune reactions. We are currently conducting research aimed at enhancing chemotherapy to stimulate anti-tumor immunity by inducing immunogenic cell death (ICD). This is accomplished using lipid bilayer-coated nanocarriers, which enable the attainment of synergistic results. Noteworthy examples include liposomes and lipid-coated mesoporous silica nanoparticles, known as "silicasomes". These nanocarriers facilitate remote chemotherapy loading as well as seamless integration of immunomodulators into the lipid bilayer. In this communication, we elucidate innovative ways for further improving chemo-immunotherapy. The first is the development of a liposome platform engineered by remote loading of irinotecan, while incorporating a pro-resolving lipoxin in the lipid bilayer. This carrier interfered in stromal collagen deposition as well as boosting the irinotecan-induced ICD response. The second approach was to synthesize polymer nanoparticles for the delivery of mutated Kras peptides, in conjunction with a TLR7 / 8 agonist. The dual delivery vaccine particle boosted the generation of antigen-specific cytotoxic T-cells that are recruited to lymphoid structures atthe cancer site, with a view to strengthening the endogenous vaccination response achieved by chemo-immunotherapy.

[0284] Pancreatic ductal adenocarcinoma (PDAC) represents a significant challenge in the field of oncology, ranking as the twelfth most common cancer worldwide with 495,000 new cases in 2020. In the United States, it’s the fourth leading cause of cancer-related deaths where patients diagnosed with PDAC face an alarming median survival of less than 6 months, and the five-year survival rate remains distressingly in the single-digit range. One of the most challenging aspects of PDAC is its tendency to be detected at an advanced stage, often limiting the feasibility of surgical intervention, which is crucial for successful treatment in many other cancer types. Consequently, patients are frequently left with limited treatment options. Despite extensive research efforts, the response to traditional chemotherapy or immunotherapy, including the use of checkpoint blocking antibodies, has been disappointing. Over the years, we have introduced several nanotechnology-enabled strategies for the treatment of PDAC, aimed at improving drug delivery by nanocarriers that provide improved pharmacokinetics and the use of single or synergistic drug combinations that trigger cytotoxic cell death and / or immunogenic cell death (ICD) for chemo- immunotherapy strategies. The purpose of this communication is to discuss new ground- breaking studies recently undertaken as we continue to develop nano-enabled chemo- immunotherapy in PDAC.

[0285] Cancer's intricate relationship with peripheral tolerance and imperfect adaptive response is well-established. The efficacy of treatment varies according to the tumor mutational burden, which is often low enough that even with the addition of checkpoint blocking antibodies only 20-30% of tumors respond. Notably, pancreatic tumors exhibit heightened ICI resistance due to the development of an immune suppressive TME. Within this context, this example introduces two innovative approaches: stroma-ablative chemo- immunotherapy and the application of exogenous KRAS vaccine nanoparticles. These strategies offer distinct mechanisms that hold promising translational prospects. They have the potential to transform aggressive, stroma-rich, and immune-evading tumors into entities that can be recognized by the immune system.

[0286] It is also believed that it is possible to combine nano-enabled chemo-immunotherapy with exogenous vaccination, provided by nanocarriers that deliver PDAC-specific neoantigens and tumor-associated antigens (TAAs). We believe that this will lead to strengthening of the cancer immunity cycle. The exogenous vaccination strategy can benefit from a variety of additional smart-design features, including selection of multiple mutantKRAS epitopes that can be assembled into a single vaccine (e.g., as described herein) to cover the heterogeneous tumor landscape that may include more than one KRAS mutant. This vaccine will also be valuable for treatment of cancers, other than PDAC. Moreover, neoantigen epitopes can be combined with TAAs (e.g., mucin 1, mesothelin) that, although less immunogenic, are more stably expressed. A multi-epitope strategy will increase the number of antigen specific T-cell clones that can be recruited from adjacent lymphoid structures (including TLS) to participate in the polyclonal immune response triggered by ICD-inducing chemotherapeutic agents. One of the approaches for facilitating multi-epitope delivery includes, but is not limited to the use of nucleic acid analogs that can be linked in a single mRNA strand that can be delivered by cationic lipid nanoparticles. These epitope- delivering nanocarriers can also be endowed with surface ligands that target lymphoid structures such as the mannose receptor, widely expressed in antigen presenting cells in lymph nodes and the spleen. In addition, the particles can be endowed with a TLR7 agonist (e.g., as described herein) that also serves to strengthen antigen presentation and immune recruitment in lymphoid tissue, which possibly could be extended to TLS developing at the primary cancer site. Thus, an array of vaccination carriers can be provided for use in combination with the liposomes and silicasomes that deliver chemo-immunotherapy agents plus a number of co-packaged immunomodulators. EXAMPLE 3 Spleen targeting of KRAS nanoparticles to boost the generation of immunotherapy by the induction of immunogenic cell death and strengthening the cancer immunity cycle

[0287] The endogenous vaccination response triggered by an irinotecan silicasome can be boosted by combining the ICD effect with an exogenous vaccination response to a KRAS delivering lipid nanoparticle, providing a potential synergistic immune response. Furthermore, endowing the cationic LPN with a TLR7 agonist such as 3M-052 would boost the cancer immunity cycle by activating the participating APC and cytotoxic T cells at multiple sites in the immunity cycle. Since 3M-052 has a lipid tail, this agonist should also be incorporated into the LPN. The LPN composition could be changed to achieve spleen rather than liver targeting, with a view to boosting the cancer immunity cycle.

[0288] The cancer immunity cycle describes the process by which the immune system detects, targets, and destroys cancer cells. It involves several key steps: 1. Release of cancer antigens: cancer cells die and release antigens, which are recognized as foreign by the immune system.2. Antigen presentation – Dendritic cells in the draining lymph nodes and spleen capture these antigens and present them to T-cells in the lymph nodes and spleen.3.Priming and activation – T-cells become activated upon recognizing the cancer antigens and proliferate to prepare for attacking cancer cells.4. Trafficking of T-cells – Activated T-cells migrate through lymph vessels and the bloodstream to the tumor site.5. Infiltration of T- cells – T-cells infiltrate the tumor microenvironment.6. Recognition of cancer cells – T- cells recognize cancer cells through specific interactions with cancer antigens presented on their surface.7. Killing cancer cells – T-cells release cytotoxic substances, leading to cancer cell death and the release of more cancer antigens, restarting the cycle. Steps 2-6 are enhanced by TLR7 and similar immune agonists.

[0289] In this example, we show the development of spleen-targeting LNP's, which were designed by experimenting with different lipid compositions, including the use of a lipid mix comprised of DODAP / DOPE / cholesterol and DMG-PEG2000. The in vivo biodistribution to the liver and spleen is described, using a fluorescent marker. This demonstrated successful redirection of approximately 50% of the particles to the spleen, compared to predominant liver targeting by the previously described MC3 nanoparticles used for treating liver metastases. Noteworthy, the encapsulation of luciferase mRNA in the DODAP nanoparticles, showed that that the mRNA was predominantly expressed in the spleen but not the liver. A comparable cationic LPN was constructed that includes a TLR7 agonist, 3M-052. This agonist has a lipid tail that embeds in the particle surface. These particles maintain their distribution to the liver and spleen, with predominant luciferase mRNA expression in the spleen.

[0290] Cationic LPN delivering a mRNA strand were constructed that encodes five copies of the KRAS epitope (as in a prior example). These particles were constructed with or without embedding of 3M-052, as well as nanoparticles control such as encapsulating a mRNA strand that encodes for the wildtype Ras sequence; these particles were administered IV in an orthotopic KPC mouse model for demonstrating the impact on tumor cell growth and survival. This showed a significant improvement by the particle designated as 3M- LNP / mKRAS epitope. This exogenous vaccinating particle was used in combination with the administration on irinotecan delivering silicasomes (Si-IR). Using an IV administration protocol in which there was overlap in the days of administration, it could be seen that the combination of the 3M-LNP / mKRAS epitope plus Si-IR yielded the best tumor response and survival. Since the response to Si-IR was modest, this experiment was repeated where 3M-Si-IR was used prior to injecting 3M-LNP / mKRAS epitope and comparative controls. This demonstrated further improvement in tumor shrinking and survival. Development of spleen-targeting LNPs.

[0291] Fig.29A depicts the microfluidics mixing for the construction of spleen targeting LNPs. In brief, spleen-targeting LNPs were synthesized by the NanoGeneratorTM Flex-M system (Precigenome LLC) to mix the lipid-containing organic phase with an aqueous phase containing mRNA constructs. The organic phase was comprised of a lipid mix (DODAP / DOPE / cholesterol / DMG-PEG2000) dissolved in ethanol in a molar ratio of 18.5: 60: 20: 1.5. The aqueous phase was comprised of RNase-free sodium acetate buffer (0.1M, pH 4.0) containing the mRNA constructs (polyA, mLuc, mRASwt, or mKRAS). The flow rate ratio of the aqueous vs. organic phase was 3:1. LNPs in the effluent phase were dialyzed against PBS (pH 7.4) to remove ethanol before use. The LNPs were characterized for hydrodynamic size, polydispersity index (PDI), and zeta potential by a ZETAPALS instrument (Brookhaven Instruments Corporation). The encapsulation efficiency (EE%) of mRNA constructs was performed through the RiboGreen assay and determined by UV spectroscopy. Fig.29B depicts the characterization of LNP / polyA for hydrodynamic size, PDI, zeta potential, and EE%. Demonstration of the biodistribution and spleen-targeting function of LNPs.

[0292] For the animal imaging studies, we developed fluorescently labeled LNPs to deliver mLuc (DiR-LNP / mLuc) by adding DiIC18(7) (1,1′-Dioctadecyl3,3,3′,3′-Tetramethylindo- tricarbocyanine Iodide (DiR dye) into the lipid mix as described above. The weight ratio of DiR to lipid mix was 0.5%. The schematic in Fig.30A depicts the experimental timeline for conducting the biodistribution and luciferase expression of DiR-LNP / mLuc. In brief, healthy B6129SF1 / J mice received a single IV administration of DiR-LNP / mLuc to deliver 0.3 mg / kg luciferase mRNA (n=3). Mice were intraperitoneally injected with D-Luciferin 24 h after LNP injection. Mice were euthanized within 10 min after administration. Ex vivo major organs (heart, lungs, kidneys, spleen and liver) were collected for the bioluminescent imaging of luciferase expression and the fluorescent imaging of DiR dye. Fig.30B depicts the DiR fluorescent imaging and normalized DiR fluorescence intensity of the organs, 24 h after IV injection through the IVIS imaging system. Fig.30C depicts the luciferase expression and normalized luciferase intensity of the same organs described above.

[0293] DiR fluorescence intensity was used to quantify the biodistribution of LNPs. The data show that most of the LNPs located in the spleen and liver after 24 h, accounting for 46% and 43%, to each organ, respectively. Luciferase expression intensity was used to quantify the mRNA expression of LNPs. The data showed that the LNPs prominently expressed mRNA at the spleen site accounting for 90% of the expressed luciferase. This indicates the spleen-targeting LNPs were successfully synthesized and suitable fordelivering tumor antigen mRNA at the spleen to boost the vaccination response. The reason for predominant mRNA expression in the spleen, in spite of approximately half of the particles distributing to the liver is unknown, but likely due to the impact DODAP on the protein corona and possible impact on transcriptional activation or suppression. Development of 3M-LNPs.

[0294] Fig.31A depicts the loading of the lipophilic TLR7 / 8 agonist, 3M-052, into the LNP structure by substituting a small fraction of DOPE with this lipid tailed agonist. Fig. 31B depicts that 3M-LNPs were formulated as indicated to produce a series of LNPs with 0% to 4.5% 3M-052 (fraction of total lipids). The rest of the synthesis procedure for 3M- LNP / polyA was the same as the LNP / polyA described above. The 3M-LNPs were characterized for size, PDI, zeta potential, EE%, and concentration ratio of 3M / PolyA.

[0295] The data show that a series of 3M-LNPs could be successfully synthesized. The sizes of 3M-LNPs were between 75 to 95 nm with PDI below 0.21 and EE% above 95%. Considering the amount of 3M-052 needed in subsequent animal experiments, LNPs with 4.5% 3M-053 were chosen for use in in vivo experimentation. Demonstration of the biodistribution and spleen-targeting function of 3M-LNPs.

[0296] For the animal imaging studies, we developed DiR fluorescently labeled LNPs with 4.5% 3M-053 to deliver mLuc (4.5% 3M DiR-LNP / mLuc). The weight ratio of DiR to lipid mix was 0.5%. The schematic in Fig.32A depicts the experimental timeline for conducting the biodistribution and luciferase expression of DiR-LNP / mLuc and 4.5% 3M DiR- LNP / mLuc. In brief, healthy B6129SF1 / J mice received a single IV administration of DiR- LNP / mLuc or 4.5% 3M DiR-LNP / mLuc to deliver 0.3 mg / kg luciferase mRNA (n=3). The rest of the procedure, including intraperitoneal injection with D-Luciferin and euthanasia, was the same as described in Fig.30A. Fig.32B depicts the DiR fluorescent imaging and normalized DiR fluorescence intensity of the organs 24 h after IV injection of DiR- LNP / mLuc or 4.5% 3M DiR-LNP / mLuc. Fig.32C depicts the luciferase expression, normalized luciferase intensity, and bioluminescent radiance of the same organs from DiR- LNP / mLuc or 4.5% 3M DiR-LNP / mLuc groups.

[0297] The DiR fluorescence IVIS imaging data showed that most of the LNPs localized in the spleen and liver after 24 h, as demonstrated for both the DiR-LNP / mLuc and 4.5% 3M DiR-LNP / mLuc treated animals (Fig.32B). There were no significant differences in the organ distribution for the DiR-LNP / mLuc and 4.5% 3M DiR-LNP / mLuc groups, indicating that 4.5% 3M did not impact the biodistribution of LNPs. The luciferase bioluminescenceIVIS imaging data showed that almost all of the luciferase expression (≥ 90%) occurred at the spleen for both the DiR-LNP / mLuc and 4.5% 3M DiR-LNP / mLuc groups. There was no significant difference in luciferase expression between these two groups, confirming that 4.5% 3M did not impact the spleen-targeting function and mRNA expression of LNPs. Overall, 4.5% 3M-LNPs appeared to be suitable for mRNA delivery to spleen site, with the potential of allowing the TLR 7 / 8 pathway to strengthen the vaccination response at intersection of the primary cancer site with the immunity cycle. The 4.5% 3M-LNPs were used for further experiments. Development of LNP / mRNA and 3M-LNP / mRNA carriers.

[0298] Fig.33A depicts the lipid composition of LNP / mRNA and 3M-LNP / mRNA. The microfluidics mixing procedures were the same as described in Fig.29A. The spleen- targeting LNP / mKRAS, 3M-LNP / mRASwt, and 3M-LNP / mKRAS were synthesized by the NanoGeneratorTM Flex-M system as described in Fig.1A. The obtained LNPs were characterized for hydrodynamic size, PDI, zeta potential, and EE% by a ZETAPALS instrument and UV spectroscopy (Fig.33B).

[0299] The data show that a series of spleen-targeting LNPs, with or without 3M, were successfully synthesized to encapsulate mKRAS or mRASwt, in the size range of 100- 120 nm. The encapsulation efficiencies of these LNPs were > 95%. These LNPs were earmarked for performing further animal experiments. Study of the therapeutic effects of the spleen-targeting LNP system in an orthotopic KPC pancreatic cancer model.

[0300] Fig.34A depicts the experimental outline of the study conducted by orthotopic implantation of KPC-luc tumor cells in the pancreatic tail of B6129SF1 / J mice. In brief, 50 μL of PBS / Matrigel (6:4 v / v), containing 0.8 × 106 KPC-luc cells, was used for injection into the tail of the pancreas of female B6129SF1 / J mice 6 days before the treatment. Once establishing the KPC-luc tumor model was confirmed by IVIS bioluminescence imaging, 1 day before drug injection, tumor-bearing mice were randomly divided into 4 groups (n=6), namely: saline, 3M-LNP / mRASwt, LNP / mKRAS, and 3M-LNP / mKRAS. These animals received IV injections of the different treatment modalities every 3 days on 4 occasions. The different dose equivalents for mRNA and 3M-052 amounted to 1.25 mg / kg and 1.75 mg / kg respectively. Animals were monitored daily until reaching moribund status or spontaneous death. Fig.34B depicts the IVIS imaging intensity for each orthotopic tumor in each of the animal groups, recorded on days 5, 11, 14, 18, 25, and 33. Fig.34C depicts the average quantitative tumor bioluminescence in each group on days 14 and 18, as calculated by IVISsoftware. Fig.34D depicts the Kaplan–Meier plots to display the change in survival for the different animal groups. A summary of the median survival time (MST) and percentage increase in life span (%ILS) for each group are presented in Fig.34D. Data represent mean ± SEM. #p<0.05; ##p<0.01; ###p<0.001; *p<0.05; **p<0.01.

[0301] The results demonstrate the efficacy of mKRAS delivery by spleen-targeting LNPs, either as a single treatment or in combination with 3M. Each animal displayed roughly the same level of tumor bioluminescence before introducing different treatments (day 5). The tumors in the saline group (control) group continued to grow rapidly, achieving the highest bioluminescence intensity on day 18. Mice treated with 3M-LNP / mRASwt, LNP / mKRAS, or 3M-LNP / mKRAS showed significant reduction in bioluminescence intensity compared to those treated with saline, emphasizing the efficacy of encapsulated 3M and mKRAS delivery. Furthermore, the best outcome was achieved after the administration of 3M- LNP / mKRAS, indicating the significantly synergistic effect of dual delivery spleen- targeting LNPs (Fig.34C). These results were also in agreement with the significant improvement in survival rate. The results of Kaplan–Meier analysis show that the mean survival time (MST) of the saline group was 19.5 days, demonstrating a significant improvement in the survival of LNP-treated mice compared to the saline control. While the effects of 3M-LNP / mRASwt (p<0.01) and LNP / mKRAS (p<0.05) were significantly improved compared to the saline control, best survival was obtained with the 3M- LNP / mKRAS (p<0.001). The MST of 39 days and %ILS of 100% were significantly better than other treatment groups. All considered, the data in Figure 6 demonstrate that the strategy for mKRAS expression at the spleen site trigger an exogenous vaccination response, with the TLR7 agonist strengthening the immune response, most likely by recruiting APC and cytotoxic T cells that strengthen the cancer immune cycle. Demonstration of a synergistic therapeutic effect by combining 3M-LNP / mKRAS with an Irinotecan delivering silicasome (Si-IR) in an orthotopic KPC pancreatic cancer model.

[0302] Fig.35A depicts the experimental outline of the study conducted in an orthotopic KPC pancreatic cancer model. In brief, 50 μL of PBS / Matrigel (6:4 v / v), containing 0.8 × 106 KPC-luc cells, was orthotopically injected into the tail of the pancreas of female B6129SF1 / J mice 5 days before administration of treatment. Once establishment of the KPC-luc tumor growth was confirmed by IVIS bioluminescence imaging on day 5, tumor- bearing mice were randomly divided into 5 groups (n=6), namely: saline, Si-IR, 3M- LNP / mRASwt + Si-IR, 3M-LNP / mKRAS, and 3M-LNP / mKRAS + Si-IR. Mice receivedIV injection of 3M-LNP / mRNA every 3 or 4 days on 4 occasions, starting on day 5. Mice were treated with the Si-IR every 4 days on 4 occasions, starting on day 9. The different dose equivalents for mRNA, 3M-052, and irinotecan amounted to 1.25 mg / kg, 1.75 mg / kg, and 40 mg / kg, respectively. Animals were monitored daily until reaching moribund status or spontaneous death. Fig.35B depicts the IVIS imaging intensity for each orthotopic tumor in each of the animal groups, recorded on days 5, 11, 15, 19, and 28. Fig.35C depicts the average quantitative tumor bioluminescence in each group on days 15, 19, and 28, as calculated by IVIS software. Fig.35D depicts the Kaplan–Meier plots displaying the survival rate of the different animal groups. A summary of the MST and %ILS for each group was also presented in Fig.35D. The tumors from the animals reaching moribund status or spontaneous death were harvested for immunohistochemistry (IHC) analysis. Fig. 35E depicts the representative IHC images and quantification of CD8+ cells in each group. Bar is 200 μm. Data represent mean ± SEM. #p<0.05; ##p<0.01; ###p<0.001; *p<0.05; **p<0.01; ***p<0.001.

[0303] The bioluminescence results, depicting tumor growth, show that while the tumors in the saline (control) group continued to expand over time and achieving the highest bioluminescence intensity on day 19, the various treatments had different impacts. While the impact of Si-IR on the inhibition of tumor growth did not reach statistical significance compared to the saline control (Fig.35C), Si-IR treatment significantly prolonged the animal survival with the %ILS of 25.6% (Fig.35D) and increased CD8+ T cell accumulation at the primary tumor site (Fig.35E). While 3M-LNP / mRASwt + Si-IR or 3M- LNP / mKRAS showed inhibition of tumor growth, the most significant growth interference was achieved by the combination of 3M-LNP / mKRAS and Si-IR (Fig.35C). These results were also in agreement with the significant improvement in animal survival rate and CD8+ T cell accumulation. The results of Kaplan–Meier plots demonstrated that while the effects of 3M-LNP / mRASwt + Si-IR (p<0.001) and 3M-LNP / mKRAS (p<0.001) achieved significant results compared to the saline control, the best survival was obtained with the 3M-LNP / mKRAS+ Si-IR (p<0.001), with MST of 42.5 days and %ILS of 117.9%. The IHC analysis in Fig.35E for demonstrating the presence of CD8+ cytotoxic T cells at the primary tumor site, show increased recruitment of cytotoxic T cells in response to monotherapy with Si-IR or 3M-LNP / mKRAS. This effect was synergistically enhanced by combination therapy. These results confirm the hypothesis that combining endogenous and exogenous vaccination responses could synergistically induce the anti-PDAC immune response by strengthening of the PDAC immunity cycle.Combining 3M-LNP / mKRAS administration with 3M-Si-IR administration further boosts the treatment outcome in an orthotopic model.

[0304] Fig.36A depicts the experimental outline of the study conducted in an orthotopic KPC pancreatic cancer model, which looked at the impact of triggering the endogenous ICD pathway before administration of the exogenous LNP vaccine. In brief, 50 μL of PBS / Matrigel (6:4 v / v), containing 0.8 × 106 KPC-luc cells, was injected into the tail of the pancreas of female B6129SF1 / J mice 5 days before the treatment. After establishing KPC- luc tumor growth, as confirmed by IVIS bioluminescence imaging on day 5, tumor-bearing mice were randomly divided into 4 groups (n=6), namely: saline, 3M-Si-IR, 3M- LNP / mKRAS, and 3M-LNP / mKRAS + 3M-Si-IR. Mice received IV injections of 3M-Si-IR for priming of subsequent vaccination on day 5 and 9. Mice were treated with 3M- LNP / mRNA as booster vaccination every 3 or 4 days on 4 occasions starting on day 13. The different dose equivalents for irinotecan and 3M-052 amounted to 40 mg / kg and 2 mg / kg respectively for 3M-Si-IR treatment, while the different dose equivalents for mRNA and 3M-052 amounted to 1.25 mg / kg and 1.75 mg / kg respectively for 3M-LNP / mKRAS treatment. Animals were monitored daily until reaching moribund status or spontaneous death. Fig.36B depicts the IVIS imaging intensity for each orthotopic tumor in each animal group, recorded on days 5, 10, 15, 19, 26, 33, and 40. Fig.36C depicts the average quantitative tumor bioluminescence in each group on days 10, 15, 19, and 26, as calculated by IVIS software. Fig.36D depicts the Kaplan–Meier plots to display the different animal groups' survival rates, MST, and %ILS. Data represent mean ± SEM. #p<0.05; ###p<0.001; *p<0.05; **p<0.01.

[0305] Quantitative analysis of tumor bioluminescence intensity demonstrated significant luminescence reduction in mice treated with 3M-Si-IR (with or without 3M-LNP / mKRAS), compared to saline or 3M-LNP / mKRAS (Fig.36C). Moreover, the combination therapy showed a statistically significant effect compared to monotherapy, indicating synergistic benefit from combining endogenous vaccination and exogenous vaccination. These findings were further confirmed by assessment of the survival rate (Fig.36D). The results demonstrated a significant improvement in the survival (p < 0.001) of 3M-Si-IR (with or without 3M-LNP / mKRAS) compared to the saline control. The best survival was obtained with the 3M-Si-IR plus 3M-LNP / mKRAS, which was significantly better than 3M-Si-IR alone (p < 0.01) or the use of 3M-LNP / mKRAS alone (p < 0.05). The MST of 43 days and %ILS of 126.3% in the combination group were significantly better than other treatment groups. All considered, these data strongly support the ability of a combination of 3M-LNP / mKRAS and 3M-Si-IR to synergistically boost anti-PDAC immunity cycle, with 3M- Si-IR priming the endogenous vaccination response before boosting of that response by the exogenous vaccination approach, using the KRAS delivering LNP. The clonality of the T cell responses to irinotecan versus KRAS vaccination still needs to be determined. Most likely the ICD response will be polyclonal in nature while the KRAS immune response will be oligoclonal in nature. Example 4 Combining 3M-LNP / mKRAS with a STING agonist in the same LNP provides further boosting of the treatment outcome in an orthotopic model.

[0306] Spleen-targeting LNP were prepared, delivering a cargo comprised of KRAS G12D encoding mRNA, the TLR7 / 8 agonist, 3M-052, and the STING agonist 2’,3’-cGAMP (Fig. 37). The particles were synthesized by microfluidic synthesis of DODAP -containing LNP as described in our recent disclosure. Preparation of the stock solution containing cGAMP and mRNA was carried by dissolving these components in 0.1 M NaAc medium at 5 mg / mL. The stock solution containing the lipid phase was prepared by dissolving the DOTAP, DOPE, cholesterol, DMG-PEG2000 , and 3M-052 in ethanol at 20 mg / ml, yielding molar ratio of 18.5:57.0:20.0:1.5:3.0. The resulting N:P ratio was 4, while the volume ratio of the aqueous phase vs. organic phase was 3.

[0307] LNPs described above can target the spleen and the liver, while mRNA encoding for luciferase is only expressed in the spleen. Fig.38A shows the experimental planning involved IV injection into female B6129SF1 / J mice for in vivo IVIS imaging, followed by animal sacrifice and harvesting the major organs (heart, liver, spleen, lung and kidneys) for ex vivo IVIS imaging. Photograph suspensions of mLuc / DiR-3M-LNP and mLuc / cGAMP / DiR-3M-LNP are shown in Fig.38B, and the physiochemical characterizations of mLuc / DiR-3M-LNP and mLuc / cGAMP / DiR-3M-LNP in Fig.38C. Representative ex vivo images of main organs from mice, prior injected with mLuc / DiR- 3M-LNP and mLuc / cGAMP / DiR-3M-LNP are shown in Fig.39D. The images at the top reflect the DiR Ex / Em wavelength. The lower images reflect GFP luminescence in the spleen. The average radiance efficiency of DiR from main organs is shown in Fig.38E, the percentage distribution of DiR in main organs in Fig.38F, and the average radiance of luminescence from main organs in Fig.38G. The percentage of luminescence in main organs is shown in Fig.38H. In brief, these results demonstrate that the DiR labeled particles localizing both the liver and spleen, but that the expression of luciferase mRNA only occurs in the spleen.

[0308] An experiment was performed to adjust the number of KPC cells for examining growth and survival of orthotopic implant in the pancreas mice. A schematic describing the investigation of the influence of number of KPC cells inoculated to the survival of mice is shown in Fig.39A. Mice were divided into three groups and each group received tumor inoculation with 1000k, 100k and 10 k KPC-Luc cells. Tumor growth was monitored by IVIS imaging (Fig.39B). All mice were sacrificed on day 30. The average radiance of luminescence of all mice. based on these results, it was decided to use a dose of 100,000 KPC cells to establish an orthotopic model with the survival of the animal as sufficient for doing comparative analysis of the impact of LNP’s (Fig.39C).

[0309] An ELISpot Assay for IFN-γ was used to demonstrate synergy between cGAMP and 3M-052 in generating cytotoxic T cells triggered by mRNA expression of the KRAS epitope. A schematic to describe the in vivo assessment of immune activation parameters in mice receiving IV injection of 2 doses of saline, mKRAS / LNP, cGAMP / mKRAS / LNP, mKRAS / 3M-LNP or cGAMP / mKRAS / 3M-LNP is shown in Fig.40A. Animals (n = 3 in each group) were sacrificed to separate splenocytes for conducting ELISPOT assays on the splenocytes seven days later. ELISPOT assays were conducted by treating the splenocytes with the G12D or wildtype peptide for 24 h and assessing IFN-γ producing colonies (Fig. 40B). Quantitative analysis of ELISPOT colonies (n=3) is shown in Fig.40C.

[0310] The therapeutic effect in an orthotopic PDAC, established by injecting 100,000 KPC cells into the pancreas was assessed. A schematic describing the investigation of the therapeutic effect in mice receiving IV injection of 4 doses of saline or cGAMP / LNP, mKRAS / LNP, cGAMP / mKRAS / LNP, mKRAS / 3M-LNP or cGAMP / mKRAS / 3M-LNP (0.3 mg / kg cGAMP, 0.3 mg / kg 3M and 1.25 mg / kg mKRAS) (Fig.41A). Tumor growth was monitored by IVIS imaging and body weight was recorded every 7 d. Cell number inoculated was 100k. Characterization of different LNPs is shown in Fig.41B. In vivo IVIS images of the animals on Day 6 and Day 13 (experiment still ongoing (Fig.41C) and a summary of average radiance from liver area of each mouse (n=6) is in Fig.41D. A statistical analysis of the radiance efficiency for luciferase expression by the growing KPC cells four days 6 and 13 is shown in Fig.41E. These data demonstrate a statistically significant reduction in orthotopic tumor cell growth by a combination of cGAMP plus 3M- 052, compared to the individual agonists.

[0311] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview ofthis application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

What is claimed is:

1. A drug delivery vehicle for reprogramming the tolerogenic liver immune landscape thereby reducing or eliminating tumor antigen tolerance and / or enhancing immune defense against metastatic spread, said vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle configured for preferential liver uptake; one or more STING agonists; and a nucleic acid encoding one or more tumor neoantigens or encoding one or more tumor associated antigens, or one or more a tumor neoantigens or one or more tumor-associated antigens.

2. The drug delivery vehicle of claim 1, wherein said nanoparticle comprises a cationic lipid nanoparticle.

3. The drug delivery vehicle of claim 2, wherein said cationic lipid is selected from the group consisting of dilinoleylmethyl-4 dimethyl aminobutyrate (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), didodecyl-dimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), C12-200 (Lipid 5), 3- (dimethylamino) propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa- 12,15-dienoate (DMAP-BLP), and (2Z)-non-2-en-1-yl 10-[(Z)-(1-methylpiperidin-4 yl)carbonyloxy]nonadecanoate (L101), dicetylphosphate-tetraethylenepentaamine- based polycation lipid, YSK05, YSK12-C4, YSK13-C4, and YSK15-C4, or any combination thereof.

4. The drug delivery vehicle according to any one of claims 1-3, wherein said lipid comprises a lipid composition that favors hepatic biodistribution.

5. The drug delivery vehicle according to any one of claims 1-4, wherein said lipid comprises MC3, DSPC, Cholesterol, and PEG-DMG2000.

6. The drug delivery vehicle of claim 5, wherein said lipid comprises in mol percent: MC350%, DSPC 10%, Cholesterol, 38.5%, and PEG-DMG20001.5%.

7. The drug delivery vehicle according to any one of claims 1-6, wherein the one or more STING agonists are selected from the group consisting of 2′,3′-cGAMP, 3′,3′- cGAMP, c-di-GMP, and c-di-AMP, Rp,Rp-2′,3′-c-di-AMPSS (ADU-S100), amidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'-Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'- Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'- cGAMP), 3',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), CMA, MK-1454 and CRD5500, or any combination thereof.

8. The drug delivery vehicle of claim 7, wherein said STING agonist is 2’,3’-cGAMP.

9. The drug delivery vehicle according to any one of claims 1-8, wherein said tumor neoantigen is selected from the group consisting of a mutant KRAS peptide, a mutant P53, or an immunogenic fragment thereof.

10. The drug delivery vehicle of claim 9, wherein said tumor neoantigen comprises one or more KRAS mutants.

11. The drug delivery vehicle of claim 10, wherein said tumor neoantigens comprises one or more KRAS mutants selected from among SEQ ID Nos:2-13, 15-19, 22-24, 27, 29, 30-36, 50, 52-56, 74, or 81-86.

12. The drug delivery vehicle of claim 10, wherein said one or more KRAS mutants comprise G12D.

13. The drug delivery vehicle according to any one of claims 1-8, wherein said drug delivery vehicle comprises a nucleic acid encoding said one or more tumor neoantigens such as SEQ ID Nos:37-43, 57, 59-63 or 77.

14. The drug delivery vehicle of claim 13, wherein said nucleic acid encodes an MHC-I targeting signal.

15. The drug delivery vehicle according to any one of claims 13-14, wherein said nucleic acid encodes an MHC-II targeting signal.

16. The drug delivery vehicle according to any one of claims 1-8, wherein said drug delivery vehicle comprises a tumor associated antigen comprising MUC1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof,ERBB2 or an immunogenic fragment therein, carcinoembryonic antigen (CEA) or an immunogenic fragment thereof, surviving or an immunogenic fragment thereof, VEGF receptor (VEGFR) or an immunogenic fragment thereof, and human telomerase reverse transcriptase and (HTRT) or an immunogenic fragment thereof.

17. The drug delivery vehicle of claim 16, wherein said drug delivery vehicle comprises a tumor associated antigen that comprises or consists of an amino acid sequence of SEQ ID Nos:44-49, 65-71 or 73.

18. The drug delivery vehicle according to any one of claims 1-8, wherein said drug delivery vehicle comprises a nucleic acid encoding said one or more tumor associated antigens.

19. The drug delivery vehicle of claim 18, wherein said nucleic acid encodes an MHC-I targeting signal.

20. The drug delivery vehicle according to any one of claims 18-19, wherein said nucleic acid encodes an MHC-II targeting signal.

21. The drug delivery vehicle according to any one of claims 1-20, wherein said drug delivery vehicle further comprises a TLR agonist.

22. The drug delivery vehicle according to claim 21, wherein the TLR agonist is a TLR7 / 8 agonist.

23. The drug delivery vehicle according to claim 22, wherein said TLR7 / 8 agonist is a lipid compatible or lipidated TLR7 / 8 agonist.

24. The drug delivery vehicle of claim 22, wherein said drug delivery vehicle comprises a TLR7 / 8 agonist selected from the group consisting of resiquimod (R848), imiquimod, 852A (PF-4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD-8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX-2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD-8848) and lipidated selgantolimod (GS-9688).

25. The drug delivery vehicle according to any one of claims 1-24, wherein said nanoparticle further comprises an inducer of immunogenic cell death (ICD inducer).

26. The drug delivery vehicle of claim 25, wherein said ICD inducer is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

27. The drug delivery vehicle of claim 1, comprising a STING agonist and a TLR7 / 8 agonist.

28. The drug delivery vehicle of claim 27, comprising 2’,3’-cGAMP and resiquimod.

29. A drug delivery vehicle for enhancing an anti-tumor immune response by targeting tumor-associated lymphoid structures, said drug delivery vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle that comprises a lipid composition and / or targeting molecules that facilitate uptake by lymphoid organs, including lymph nodes, spleen and / or tertiary lymphoid structures; one or more STING agonists; and one or more a tumor associated antigens or tumor neoantigens or a nucleic acid encoding one or more a tumor associated antigens or tumor neoantigens.

30. The drug delivery vehicle of claim 29, wherein said nanoparticle comprises a cationic lipid nanoparticle.

31. The drug delivery vehicle of claim 30, wherein said cationic lipid is selected from the group consisting of dilinoleylmethyl-4 dimethyl aminobutyrate (DLin-MC3- DMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), didodecyl- dimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-3- dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4-dimethylaminomethyl-[1,3]- dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]- dioxolane (DLin-KC2-DMA), C12-200 (Lipid 5), 3-(dimethylamino) propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa-12,15-dienoate (DMAP-BLP), and (2Z)-non-2-en-1-yl 10-[(Z)-(1-methylpiperidin-4 yl)carbonyloxy]nonadecanoate (L101), dicetylphosphate-tetraethylenepentaamine- based polycation lipid, YSK05, YSK12-C4, YSK13-C4, and YSK15-C4, or any combination thereof.

32. The drug delivery vehicle according to any one of claims 29-31, wherein said lipid comprises MC3, DSPC, Cholesterol, and PEG-DMG2000.

33. The drug delivery vehicle of claim 32, wherein said lipid comprises in mol percent: MC350%, DSPC 10%, Cholesterol, 38.5%, PEG-DMG20001.5%.

34. The drug delivery vehicle according to any one of claims 29-31, wherein said lipid comprises DODAP, DOPE, Cholesterol, and PEG-DMG2000.

35. The drug delivery vehicle of claim 34, wherein said lipid comprises in mol percent: DODAP 18.5%, DOPE 60%, Cholesterol, 20%, and PEG-DMG20001.5%.

36. The drug delivery vehicle according to any one of claims 29-35, wherein said one or more STING agonists are selected from the group consisting of 2′,3′-cGAMP, 3′,3′- cGAMP, c-di-GMP, and c-di-AMP, Rp,Rp-2′,3′-c-di-AMPSS (ADU-S100), amidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'-Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'- Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'- cGAMP), 3',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), CMA, MK-1454 and CRD5500, or any combination thereof.

37. The drug delivery vehicle of claim 36 wherein said delivery vehicle comprises the STING agonist 2’,3’-cGAMP.

38. The drug delivery vehicle according to any one of claims 29-37, wherein said drug delivery vehicle comprises a tumor neoantigen selected from the group consisting of a mutant KRAS peptide, a mutant P53, or an immunogenic fragment thereof.

39. The drug delivery vehicle of claim 38, wherein said neoantigens comprise one or more KRAS mutants.

40. The drug delivery vehicle of claim 39, wherein said KRAS mutant is a peptide selected from among SEQ ID Nos: 2-13, 15-19, 22-24, 27, 29, 30-36, 50, 52-56, 74, or 81-86.

41. The drug delivery vehicle of claim 39, wherein said one or more KRAS mutants comprise G12D.

42. The drug delivery vehicle according to any one of claims 29-37, wherein said nanoparticle comprises a nucleic acid encoding said one or more neoantigens such as SEQ ID NOs:37-43, 57, 59-63 or 77.

43. The drug delivery vehicle of claim 42, wherein said nucleic acid encodes an MHC-I targeting signal.

44. The drug delivery vehicle according to any one of claims 42-43, wherein said nucleic acid encodes an MHC-II targeting signal.

45. The drug delivery vehicle according to any one of claims 29-37, wherein said drug delivery vehicle comprises a tumor associated antigen comprising MUC1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof, ERBB2 or an immunogenic fragment therein, carcinoembryonic antigen (CEA) or an immunogenic fragment thereof, surviving or an immunogenic fragment thereof, VEGF receptor (VEGFR) or an immunogenic fragment thereof, and human telomerase reverse transcriptase and (HTRT) or an immunogenic fragment thereof.

46. The drug delivery vehicle according to any one of claims 29-37, wherein said drug delivery vehicle comprises a tumor associated antigen that comprises or consists of an amino acid sequence of SEQ ID Nos:44-49, 65-71 or 73.

47. The drug delivery vehicle according to any one of claims 29-37, wherein said drug delivery vehicle further comprises targeting ligands that facilitate uptake by lymphoid organs, including lymph nodes, spleen and tertiary lymphoid structures.

48. The drug delivery vehicle of claim 47, wherein said delivery vehicle comprises a targeting ligand that binds to a mannose receptor.

49. The drug delivery vehicle of claim 47, wherein said targeting ligand comprises a mannose targeting ligand or a complex oligomannoside.

50. The drug delivery vehicle according to any one of claims 29-49, wherein said drug delivery vehicle comprises a cationic lipid that shows preferential distribution to a lymphoid organ.

51. The drug delivery vehicle of claim 50, wherein said nanoparticle comprises dioleoyl-3-trimethylammonium propane (DOTAP) and / or dioleoyl-dimethylammonium propane (DODAP), and optionally dioleoylphosphatidylethanolamine (DOPE).

52. The drug delivery vehicle according to any one of claims 29-51, wherein said drug delivery vehicle further comprises a TLR agonist.

53. The drug delivery vehicle according to claim 52, wherein said TLR agonist is a TLR7 / 8 agonist.

54. The drug delivery vehicle according to claim 53, wherein said drug delivery vehicle comprises a lipid compatible TLR7 / 8 agonist.

55. The drug delivery vehicle of claim 53, wherein said drug delivery vehicle comprises a TLR7 / 8 agonist selected from the group consisting of resiquimod (R848), imiquimod, 852A (PF-4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD-8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX-2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD-8848) and lipidated selgantolimod (GS-9688).

56. The drug delivery vehicle according to any one of claims 29-55, wherein said nanoparticle further comprises an inducer of immunogenic cell death (ICD inducer).

57. The drug delivery vehicle of claim 56, wherein said ICD inducer is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

58. The drug delivery vehicle of claim 29 comprising a STING agonist and a TLR7 / 8 agonist.

59. The drug delivery vehicle of claim 58 comprising 2’,3’-cGAMP and resiquimod.

60. A drug delivery vehicle for enhancing an anti-tumor immune response by targeting tumor-associated lymphoid structures, said drug delivery vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle that comprises a lipid composition and / or targeting molecules that facilitate uptake by lymphoid organs, including lymph nodes, spleen and / or tertiary lymphoid structures;one or more TLR7 / 8 agonists; and one or more a tumor associated antigens or tumor neoantigens, or a nucleic acid encoding one or more a tumor associated antigens or tumor neoantigens.

61. The drug delivery vehicle of claim 60, wherein said nanoparticle comprises a cationic lipid nanoparticle.

62. The drug delivery vehicle of claim 61, wherein said cationic lipid is selected from the group consisting of cationic lipids selected from the group consisting of dilinoleylmethyl-4 dimethyl aminobutyrate (DLin-MC3-DMA), 1,2-dioleoyl-3- dimethylaminopropane (DODAP), didodecyl-dimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), (N- [1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2- dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), C12-200 (Lipid 5), 3- (dimethylamino) propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa- 12,15-dienoate (DMAP-BLP), and (2Z)-non-2-en-1-yl 10-[(Z)-(1-methylpiperidin-4 yl)carbonyloxy]nonadecanoate (L101), dicetylphosphate-tetraethylenepentaamine- based polycation lipid, YSK05, YSK12-C4, YSK13-C4, and YSK15-C4, or any combination thereof.

63. The drug delivery vehicle according to any one of claims 60-62, wherein said lipid comprises MC3, DSPC, Cholesterol, and PEG-DMG2000.

64. The drug delivery vehicle of claim 63, wherein said lipid comprises in mol percent: MC350%, DSPC 10%, Cholesterol, 38.5%, PEG-DMG20001.5%.

65. The drug delivery vehicle according to any one of claims 60-62, wherein said lipid comprises DODAP, DOPE, Cholesterol, and PEG-DMG2000.

66. The drug delivery vehicle of claim 65, wherein said lipid comprises in mol percent: DODAP 18.5%, DOPE 60%, Cholesterol, 20%, and PEG-DMG20001.5%.

67. The drug delivery vehicle according to any one of claims 60-66, wherein said TLR7 / 8 agonist is a lipid compatible TLR7 / 8 agonist.

68. The drug delivery vehicle of any one of claims 60-66, wherein said TLR7 / 8 agonist is selected from the group consisting of resiquimod (R848), imiquimod, 852A (PF- 4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD- 8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX-2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD-8848) and lipidated selgantolimod (GS-9688).

69. The drug delivery vehicle according to any one of claims 60-68, wherein said drug delivery vehicle comprises a tumor-associated neoantigen selected from the group consisting of a mutant KRAS peptide, a mutant P53, or an immunogenic fragment from one or more of these neoantigens.

70. The drug delivery vehicle of claim 69, wherein said neoantigens comprise one or more KRAS mutants.

71. The drug delivery vehicle according to claim 69, wherein said one or more KRAS mutants are selected from SEQ ID Nos: 2-13, 15-19, 22-24, 27, 29, 30-36, 50, 52- 56, 74, or 81-86.

72. The drug delivery vehicle of claim 70, wherein said one or more KRAS mutants comprise G12D.

73. The drug delivery vehicle according to any one of claims 60-68, wherein said nanoparticle comprises a nucleic acid encoding said one or more neoantigens such as SEQ ID Nos:37-43, 57, 59-63 or 77.

74. The drug delivery vehicle of claim 73, wherein said nucleic acid encodes an MHC-I targeting signal.

75. The drug delivery vehicle according to any one of claims 73-74, wherein said nucleic acid encodes an MHC-II targeting signal.

76. The drug delivery vehicle according to any one of claims 60-68, wherein said drug delivery vehicle comprises a tumor associated antigen comprising MUC1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof, ERBB2 or an immunogenic fragment therein, carcinoembryonic antigen (CEA) or an immunogenic fragment thereof, surviving or an immunogenic fragment thereof,VEGF receptor (VEGFR) or an immunogenic fragment thereof, and human telomerase reverse transcriptase and (HTRT) or an immunogenic fragment thereof.

77. The drug delivery vehicle according to any one of claims 60-68, wherein said drug delivery vehicle comprises a tumor associated antigen that comprises or consists of an amino acid sequence of SEQ ID Nos:44-49, 65-71 or 73.

78. The drug delivery vehicle according to any one of claims 60-77, wherein said drug delivery vehicle comprises targeting ligands that facilitate uptake by lymphoid organs, including lymph nodes, spleen and tertiary lymphoid structures.

79. The drug delivery vehicle of claim 78, wherein said delivery vehicle comprises a targeting ligand that binds to a mannose receptor.

80. The drug delivery vehicle of claim 78, wherein said targeting ligand comprises a mannose targeting ligand or a complex oligomannoside.

81. The drug delivery vehicle according to any one of claims 60-80, wherein said drug delivery vehicle comprises a cationic lipid that shows preferential uptake by a lymphoid organ.

82. The drug delivery vehicle of claim 81, wherein said nanoparticle comprises dioleoyl-3-trimethylammonium propane (DOTAP) and / or dioleoyl- dimethylammonium propane (DODAP), and optionally dioleoylphosphatidylethanolamine (DOPE).

83. The drug delivery vehicle according to any one of claims 60-82, further comprising one or more STING agonists.

84. The drug delivery vehicle according to claim 83, wherein said one or more STING agonists are selected from the group consisting of 2′,3′-cGAMP, 3′,3′-cGAMP, c-di- GMP, and c-di-AMP, Rp,Rp-2′,3′-c-di-AMPSS (ADU-S100), amidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'- Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'-cGAMP), 3',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), CMA, MK-1454 and CRD5500, or any combination thereof.

85. The drug delivery vehicle of claim 83 wherein said delivery vehicle comprises the STING agonist 2’,3’-cGAMP.

86. The drug delivery vehicle according to any one of claims 60-85, wherein said nanoparticle further comprises an inducer of immunogenic cell death (ICD inducer).

87. The drug delivery vehicle of claim 86, wherein said ICD inducer is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

88. The drug delivery vehicle according to any one of claims 60-87, further comprising a TLR agonist.

89. The drug delivery vehicle of claim 60 comprising a STING agonist and a TLR7 / 8 agonist.

90. The drug delivery vehicle of claim 89 comprising 2’,3’-cGAMP and resiquimod.

91. A drug delivery vehicle for enhancing an anti-tumor immune response by targeting tumor-associated lymphoid structures, said drug delivery vehicle comprising: a lipid nanoparticle, a silicasome, or a biocompatible polymer nanoparticle that comprises a lipid composition and / or targeting molecules that facilitate uptake by lymphoid organs, including lymph nodes, spleen and / or tertiary lymphoid structures; one or more a tumor associated antigens or tumor-associated neoantigens or a nucleic acid encoding one or more a tumor associated antigens or tumor- associated neoantigens.

92. The drug delivery vehicle of claim 91, wherein said nanoparticle comprises a cationic lipid nanoparticle.

93. The drug delivery vehicle of claim 92, wherein said cationic lipid nanoparticle comprises a cationic lipid selected from the group consisting of cationic lipids selected from the group consisting of dilinoleylmethyl-4 dimethyl aminobutyrate (DLin-MC3-DMA), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), didodecyl- dimethylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-3- dimethylaminopropane (DLinDMA), 2,2-dilinoleyl- 4-dimethylaminomethyl-[1,3]- dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]- dioxolane (DLin-KC2-DMA), C12-200 (Lipid 5), 3-(dimethylamino) propyl(12Z,15Z)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl]henicosa-12,15-dienoate (DMAP-BLP), and (2Z)-non-2-en-1-yl 10-[(Z)-(1-methylpiperidin-4 yl)carbonyloxy]nonadecanoate (L101), dicetylphosphate-tetraethylenepentaamine- based polycation lipid, YSK05, YSK12-C4, YSK13-C4, and YSK15-C4, or any combination thereof.

94. The drug delivery vehicle according to any one of claims 91-93, wherein said lipid comprises MC3, DSPC, Cholesterol, and PEG-DMG2000.

95. The drug delivery vehicle of claim 94, wherein said lipid comprises in mol percent: MC350%, DSPC 10%, Cholesterol, 38.5%, PEG-DMG20001.5%.

96. The drug delivery vehicle according to any one of claims 91-93, wherein said lipid comprises DODAP, DOPE, Cholesterol, and PEG-DMG2000.

97. The drug delivery vehicle of claim 96, wherein said lipid comprises in mol percent: DODAP 18.5%, DOPE 60%, Cholesterol, 20%, and PEG-DMG20001.5%.

98. The drug delivery vehicle according to any one of claims 91-97, wherein said drug delivery vehicle further comprises a TLR agonist.

99. The drug delivery vehicle according to claim 98, wherein the TLR agonist is a TLR7 / 8 agonist.

100. The drug delivery vehicle according to claim 98, wherein the TLR7 / 8 agonist is a lipid compatible TLR7 / 8 agonist.

101. The drug delivery vehicle of claim 99, wherein said TLR7 / 8 agonist is selected from the group consisting of resiquimod (R848), imiquimod, 852A (PF-4878691), motolimod (VTX-2337), vesatolimod (GS-9620), 8-oxoadenine (AZD-8848), selgantolimod (GS-9688), 3M-052, lipidated resiquimod (R848), lipidated imiquimod, lipidated 852A (PF-4878691), lipidated motolimod (VTX-2337), lipidated vesatolimod (GS-9620), lipidated 8-oxoadenine (AZD-8848) and lipidated selgantolimod (GS-9688).

102. The drug delivery vehicle according to any one of claims 91-101, wherein said drug delivery vehicle further comprises one or more STING agonists.

103. The drug delivery vehicle according to claim 102, wherein said one or more STING agonists are selected from the group consisting of 2′,3′-cGAMP, 3′,3′-cGAMP, c-di- GMP, and c-di-AMP, Rp,Rp-2′,3′-c-di-AMPSS (ADU-S100), amidobenzimidazole (diABZI), 3',5'-Cyclic diadenylic acid sodium salt (c-DI-AMP sodium salt), 3',5'- Cyclic diguanylic acid sodium salt (c-Di-GMP sodium salt), 2',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (2',3'-cGAMP), 3',3'-Cyclic guanosine monophosphate-adenosine monophosphate sodium salt (3',3'-cGAMP), 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), CMA, MK-1454 and CRD5500, or any combination thereof.

104. The drug delivery vehicle of claim 103, wherein said delivery vehicle comprises the STING agonist 2’,3’-cGAMP.

105. The drug delivery vehicle according to any one of claims 91-104, wherein said nanoparticle further comprises an inducer of immunogenic cell death (ICD inducer).

106. The drug delivery vehicle of claim 105, wherein said ICD inducer is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin, daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

107. The drug delivery vehicle according to any one of claims 91-106, wherein said drug delivery vehicle comprises a tumor-associated neoantigen selected from the group consisting of a mutant KRAS peptide, a mutant P53, or an immunogenic fragment from one or more of these neoantigens.

108. The drug delivery vehicle of claim 107, wherein said neoantigens comprise one or more KRAS mutants.

109. The drug delivery vehicle of claim 108, wherein the mutant KRAS peptide is a peptide selected from among SEQ ID Nos: 2-13, 15-19, 22-24, 27, 29, 30-36, 50, 52-56, 74, or 81-86.

110. The drug delivery vehicle of claim 108, wherein said one or more KRAS mutants comprise G12D.

111. The drug delivery vehicle according to any one of claims 91-106, wherein said nanoparticle comprises a nucleic acid encoding said one or more neoantigens such as SEQ ID Nos:37-43, 57, 59-63 or 77.

112. The drug delivery vehicle of claim 111, wherein said nucleic acid encodes an MHC- I targeting signal.

113. The drug delivery vehicle according to any one of claims 111-112, wherein said nucleic acid encodes an MHC-II targeting signal.

114. The drug delivery vehicle according to any one of claims 91-106, wherein said drug delivery vehicle comprises a tumor associated antigen comprising MUC1 or an immunogenic fragment thereof, mesothelin or an immunogenic fragment thereof, ERBB2 or an immunogenic fragment therein, carcinoembryonic antigen (CEA) or an immunogenic fragment thereof, surviving or an immunogenic fragment thereof, VEGF receptor (VEGFR) or an immunogenic fragment thereof, and human telomerase reverse transcriptase and (HTRT) or an immunogenic fragment thereof.

115. The drug delivery vehicle of claim 91-106, wherein said drug delivery vehicle comprises a tumor associated antigen that comprises or consists of an amino acid sequence of SEQ ID Nos: 44-49, 65-71 or 73.

116. The drug delivery vehicle according to any one of claims 91-115, wherein said drug delivery vehicle comprises targeting ligands that facilitate uptake by lymphoid organs, including lymph nodes, spleen and tertiary lymphoid structures.

117. The drug delivery vehicle of claim 116, wherein said delivery vehicle comprises a targeting ligand that binds to a mannose receptor.

118. The drug delivery vehicle of claim 116, wherein said targeting ligand comprises a mannose targeting ligand or a complex oligomannoside.

119. The drug delivery vehicle according to any one of claims 91-118, wherein said drug delivery vehicle comprises a cationic lipid that shows preferential uptake by a lymphoid organ.

120. The drug delivery vehicle of claim 119, wherein said nanoparticle comprises dioleoyl-3-trimethylammonium propane (DOTAP) and / or dioleoyl-dimethylammonium propane (DODAP), and optionally dioleoylphosphatidylethanolamine (DOPE).

121. A pharmaceutical formulation comprising: a drug delivery vehicle according to any one of claims 1-120; and a pharmaceutically acceptable carrier.

122. The pharmaceutical formulation of claim 120, wherein said formulation is formulated for administration via a route selected from the group consisting of intravenous administration, intraarterial administration, intracerebral administration, intrathecal administration, oral administration, aerosol administration, administration via inhalation, intracranial administration via a cannula, intratumoral administration and subcutaneous or intramuscular depot deposition.

123. A method of treating a cancer said method comprising administering to a subject in need thereof a drug delivery vehicle according to any one of claims 1-120 or a pharmaceutical formulation thereof.

124. A method of for enhancing an anti-tumor immune response in a subject in need thereof, said method comprising administering to said subject a drug delivery vehicle according to any one of claims 29-120.

125. A method of reprogramming the tolerogenic liver immune landscape to reduce or eliminate tumor antigen tolerance, leading to lack if immune prevention against metastatic spread, said method comprising administering to a subject in need thereof a drug delivery vehicle according to any one of claims 1-28.

126. A method of treating cancer in a subject, said method comprising administering to a subject that is receiving or that has received a chemotherapeutic agent, a drug delivery vehicle according to any one of claims 1-120.

127. The method of claim 126, wherein said chemotherapeutic agent is an immunogenic cell death (ICD) inducing chemotherapeutic agent.

128. The method according to any one of claims 126-127, wherein said chemotherapeutic agent is selected from the group consisting of mitoxantrone (MTX), doxorubicin (DOX), oxaliplatin, anthracenedione, bleomycin, bortezomib, cisplatin,daunorubicin, docetaxel, epirubicin, idarubicin, paclitaxel, R2016, cyclophosphamide, and irinotecan.

129. The method according to any one of claims 123-126, wherein said method is an adjunct therapy to a primary chemotherapy regime.

130. The method according to any one of claims 123-129, wherein said subject is a human or a non-human mammal that has a KRAS dependent cancer.

131. The method of claim 130, wherein said KRAS dependent cancer comprises a cancer selected from the group consisting of cancer of the pancreas, lung, breast, colon, rectum, prostate, stomach, esophagus, cervix, uterus, thyroid, lung and bladder.

132. The method of claim 131, wherein said KRAS dependent cancer comprises pancreatic ductal adenocarcinoma (PDAC).

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