Methods and compositions for lipid nanoparticle (LNP) mediated delivery of mRNA encoded synthetic antigens for universal tumor targeting by car t cells

Abstract

Nanoparticles for engineering cells to express target antigens and methods of treating / preventing cancer or solid tumors using the same.

Description

METHODS AND COMPOSITIONS FOR LIPID NANOPARTICLE (LNP) MEDIATED DELIVERY OF MRNA ENCODED SYNTHETIC ANTIGENS FOR UNIVERSAL TUMOR TARGETING BY CAR T CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 589,804, filed October 12, 2023, which is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. R01 EB032822, GR00018410, and GR00018260 awarded by the National Institutes of Health. The Government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on October 15, 2024, as an .XML file entitled “10034-319WO1 2024_10_15 ST26” created on October 15, 2024, and having a file size of 3,637 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0004] Chimeric antigen receptor (CAR) T cell therapy for solid tumors remains limited. CAR targeting of tumor-associated antigens (TAA) is particularly challenging as heterogenous antigen expression, low antigen density, and antigen loss results in poor efficacy and antigen escape. Expression of CAR antigens by healthy tissues also pose safety concerns from on- target off-tumor toxicities. For example, CD19 is expressed by healthy B cells and brain mural cells, and patients that receive aCD19 CAR T cells require lifelong IgG replacement therapy and are at risk for neurotoxicity. Similarly, CAR T cells designed to target solid tumor TAAs (e.g., HER2, CAIX) show cross-reactivity with healthy cells in vital organs, limiting their clinical translation. Emerging strategies to address scarcity of tumor-antigens include genome wide activation of endogenous genes using CRISPRa, combinatorial or universal antigen sensing circuits to improve discrimination of malignant cells from normal tissue, or spatially controlled redirection of CAR T cells against tumor cells that lack the CAR antigen. To alleviate the need to validate CARs for each target antigen candidate, direct delivery of CD 19 via oncolytic virus, GFP by tumor-colonizing bacteria, or the fluorescent small-molecule FITCby membrane-inserting ligands, enabled tumor recognition by a single CAR T cell construct. These efforts underscore the need to reduce the bottleneck of antigen selection and enable CAR T cell recognition of tumors that otherwise lack targetable antigens.SUMMARY

[0005] In one aspect, disclosed herein are nanoparticles (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle) comprising one or more lipids comprising an ionizable lipid (such as, for example, cKK-E12, SM102, MC3, Lipid-5, Alc- 0315, LP01, Lipid A9, or any combination thereof), a cholesterol, a phospholipid (such as, for example, DSPC, DOPE, or any combination thereof), a PEGylated lipid (such as, for example, ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof), or any combination thereof; and an encapsulated nucleic acid (such as, for example, DNA, mRNA, cell free DNA (cfDNA), long noncoding DNA (IncDNA), or microRNA (miRNA)) encoding a target antigen (including, but not limited to non-naturally occurring antigens such as, for example a target antigen that comprises 80% similarity or more to SEQ ID NO: 2). In some aspects, the nanoparticle comprises from about 30 mol% to about 60 mol% of the ionizable lipid, from about 35 mol% to about 50 mol% of the cholesterol, from about 5 mol% to about 20 mol% of the phospholipid, and / or from about 0.5 mol% to about 5 mol% of the PEGylated lipid. In some aspects the ratio of PEGylated lipid to total lipids is from about 0.5: 1 to about 2: 1.

[0006] Also disclosed herein are nanoparticles of any preceding aspect wherein the target antigen comprises a detectable moiety.

[0007] Also disclosed herein are methods of engineering a cell (such as, for example an abnormal or cancerous cell) to express a target antigen, the method comprising exposing the cell to the nanoparticle of any preceding aspect. For example, disclosed herein are methods of engineering a cell to express a target antigen the method comprising exposing the cell to a nanoparticle (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), the nanoparticle comprising one or more lipids comprising an ionizable lipid (such as, for example, cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof), a cholesterol, a phospholipid (such as, for example, DSPC, DOPE, or any combination thereof), a PEGylated lipid (such as, for example, ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof), or any combination thereof; and an encapsulated nucleic acid (such as, for example, DNA, mRNA, cell free DNA (cfDNA), long noncoding DNA (IncDNA), or microRNA (miRNA)) encoding a target antigen (including, butnot limited to non-naturally occurring antigens such as, for example a target antigen that comprises 80% similarity or more to SEQ ID NO: 2). In some aspects, the cell comprises a breast cancer cell, a colon cancer cell, a brain cancer cell, a lung cancer cell, a prostate cancer cell, or a melanoma cell.

[0008] In one aspect, disclosed herein are methods of engineering a cell of any preceding aspect wherein the method is carried out in vitro, in vivo, or ex vivo.

[0009] Also disclosed herein are engineered cells produced by the method of engineering a cell of any proceeding aspect.

[0010] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer, solid tumor, and or metastasis (including, but not limited to breast cancer, colon cancer, brain cancer, lung cancer, prostate cancer, or melanoma) in a subject, the method comprising a) providing to the subject the nanoparticle of any preceding aspect and b) subsequence providing to the subject an immunotherapy (such as, for example, antibodies that block PD-1, PD-L1, CTLA-4, PD-L2 IDO, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT) B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), TIM3, or LAG-3) and adoptive cell therapies such as, for example chimeric antigen receptor (CAR) T cells, CAR Natural Killer (NK) (CAR NK cells), CAR Macrophage (CARMAC) CAR NK T cells, Tumor Infiltrating Lymphocytes (TILs), and / or Marrow Infiltrating Lymphocytes (MILs)). For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer, solid tumor, and or metastasis (including, but not limited to breast cancer, colon cancer, brain cancer, lung cancer, prostate cancer, or melanoma) in a subject, the method comprising a) providing to the subject (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), the nanoparticle comprising one or more lipids comprising an ionizable lipid (such as, for example, cKK-E12, SM102, MC3, Lipid-5, Alc- 0315, LP01, Lipid A9, or any combination thereof), a cholesterol, a phospholipid (such as, for example, DSPC, DOPE, or any combination thereof), a PEGylated lipid (such as, for example, ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof), or any combination thereof; and an encapsulated nucleic acid (such as, for example, DNA, mRNA, cell free DNA (cfDNA), long noncoding DNA (IncDNA), or microRNA (miRNA)) encoding a target antigen (including, but not limited to non-naturally occurring antigens such as, for example a target antigen that comprises 80% similarity or more to SEQ ID NO: 2); and b) subsequently providing to the subject an immunotherapy (including, but not limited to immune checkpoint inhibitors (such as, for example, antibodies that block PD-1, PD-L1, CTLA-4, PD-L2 IDO, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT) B- and T- lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), TIM3, or LAG-3) and adoptive cell therapies such as, for example, tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (NILs), chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) (CAR NK) cells, CAR macrophage (CARMACs), or CAR NK T cells expressing a CAR, T cell receptor, or NK receptor which recognizes the target antigen). In some aspects, at least about 15% of the cancer or tumor cells express the target antigen. In some aspects, step b) is carried out from about 24 hours to about 72 hours after step a).

[0011] Also disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer, solid tumor, and or metastasis of any preceding aspect, comprising administering from about 0.05 mg / kg to about 1 mg / kg nanoparticle.

[0012] In one aspect, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer, solid tumor, and or metastasis of any preceding aspect, wherein the nanoparticle is administered intratum orally.

[0013] In one aspect, disclosed herein are synthetic antigens comprising at least 80, 85, 90, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 similarity or more to SEQ ID NO: 2.

[0014] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0015] FIGURE 1 depicts LNP sizes 1 day and 365 days after production as measured by dynamic light scattering.

[0016] FIGURES 2A-2E depict the design of synthetic antigen constructs. FIG. 2A shows that the mRNA constructs tested include a membrane anchor, a linker, and a recognition domain. These constructs are transfected into tumor cells using the lipoplex jetMESSENGER to achieve synthetic antigen expression (FIG. 2B). FIG. 2C shows expression kinetics of GPL anchored and CD4TM-anchored synthetic antigens including a lx G4S linker and the RSV-F VHH recognition domain on the surface of indicated tumor cells. Mean ± s.d. n=3 independently transfected wells. FIG. 2D shows expression kinetics of mRNA constructs with a GPI-anchor, SunTag recognition domain, and indicated linker domains. Two-way ANOVA, mean ± s.d., n=3 independently transfected wells (for lx G4S vs 3x G4S: *p=0.0123 [6 hr],*p=0.0280 [12 hr], *p=0.0107 [24 hr]; for lx G4S vs VHH: ffp= 0.0063 [6 hr], ffp= 0.0068 [12 hr], fp= 0.0124 [24 hr]). FIG. 2E shows expression kinetics of GPI-anchored SunTag or VHH on the surface of indicated tumor cell lines. n=3 independently transfected wells.

[0017] FIGURE 3 depicts a general gating strategy for flow cytometry analysis of VHH and SunTag synthetic antigen expression on the surface of tumor cells. Flue mRNA transfected cells were used as negative controls for gating.

[0018] FIGURE 4 depicts flow cytometry staining for VHH and SunTag in A549 cells following transfection of GPI-anchored, VHH-linked SunTag. Analysis was performed 12 hours and 5 days post transfection.

[0019] FIGURE 5 depicts surface expression kinetics of the Kb-SIINFEKL pMHC complex on the surface of MC38 tumor cells following peptide pulsing with the SIINFEKL peptide for one hour at room temperature. n=3 independently peptide pulsed wells, mean ± s.d.

[0020] FIGURE 6A is a schematic showing the four types of lipid components used to form LNPs and encapsulate mRNA for delivery. FIGURE 6B shows detailed molar ratios of each lipid component used to formulate LNPs tested in FIGS. 7A-7D.

[0021] FIGURES 7A-7D depict that mRNA-loaded LNPs deliver synthetic antigens to the surface of tumor cells. FIG. 7A shows that various LNP formulations were loaded with mRNA encoding for GPI-anchored nLuc and co-incubated with MDA-MB-468 tumor cells for 18 hours and nLuc expression quantified. FIG. 7B shows nLuc expression on MDA-MB-468s after co-incubation with various 1 pg / mL of LNP formulations with or without 1 pg / mL ApoE. Luminesence quantified by IVIS 18 hours post transfection. One-way ANOVA; mean ± s.e.m., n = 4 independently transfected wells (**p=0.0029 for Alc-0315, ***p=0.0003 for Lipid A9, **p=0.0041 for Lipid 5, *p=0.0135 for SM-102, ***p=0.0010 for MC3, ****p<0.0001.) FIG. 7C shows that VHH-mRNA loaded cKK-E12 LNPs (VHH-LNPs) were screened against various murine and human tumor cell lines. FIG. 7D shows that VHH expression was quantified by flow cytometry 16-18 hours after transfection at the indicated concentrations of VHH-LNP with or without 1 pg / mL ApoE. Two-way ANOVA; mean ± s.e.m., n=4 independently transfected wells; n.s. = not significant, *p=0.0107; **p=0.0043; ***p=0.0006; ****p<0.0001.

[0022] FIGURE 8 depicts the kinetics of expresssion of LNP mediated delivery of VHH synthetic antigen to tumor cells. VHH expression on MDA-MB-468 (left) and E0771 (right) following VHH-LNP treatment at the indicated doses. VHH mean fluorescent intensity (MFI) quantified by flow cytometry at indicated days after transfection. Mean ± s.d. n=4 independently transfected wells.

[0023] FIGURES 9A-9F depict aVHH CAR T cells recognize and kill tumor cells expressing their cognate synthetic antigens in murine and human models. FIGS. 9A-9B show a schematic of murine and human CAR constructs for targeting synthetic antigens (SyntAg) expressed on the surface of tumor cells. FIG. 9C shows representative surface expression of aSunTag or aVHH CARs on primary murine or human T cells following viral transduction. E0771 tumor cells were transfected with VHH or SunTag mRNA and co-incubated with either murine aSunTag (S) or aVHH (V) CARs. FIGS. 9D-9E show IFN-y secretion (FIG. 9D) and killing (FIG. 9E) by indicated murine T cells was measured following a 24 hr coculture at a 2: 1 effectortarget (E:T) ratio with transfected tumor cells. One-way ANOVA; mean ± s.d., n = 4 independently transfected and cocultured wells (****p<0.0001). FIG. 9F shows killing of untransfected, SunTag- or VHH-transfected A549 tumor cells following co-incubation with WT T cells, aSunTag, or aVHH CAR T cells for 24 hours quantified by LDH assay. Two-way ANOVA; mean ± s.d., n=3 (untransfected and SunTag mRNA), n=4 (VHH mRNA) independently transfected and cocultured wells; ****p<0.0001.

[0024] FIGURE 10A shows a representative gating strategy used to identify CD4 and CD8 murine T cell populations. FIGURE 10B shows that the CD4 and CD8 composition of the transduced T cell population was evaluated following activation of either Pmel-1 splenocytes by gplOO peptide pulsing or C57BL / 6J derived CD3 isolated splenocytes by polyclonal DynabeadTM expansion. FIGURE 10C shows CAR expression by direct biotinylated VHH (b-VHH) and Streptavidin-APC (s-APC) staining of bulk populations, CD8 population, or CD4 populations for either monoclonal Pmel-1 derived T cells or polyclonal C57BL / 6J derived T cells.

[0025] FIGURE 11 depicts representative flow cytometry data showing the general gating strategy for flow CD69 and CD25 on the surface of primary murine CAR T cells following coincubation with tumor cells expressing their cognate synthetic antigen.

[0026] FIGURES 12A-12C depict characterization of murine CAR T cells sourced from either pmel-1 or C57BL6 / J mice. FIG. 12A shows staining of indicated murine T cell population with activation markers CD25 and CD69 following co-incubation with ST- or VHH-expressing E0771 tumor cells. FIG. 12B shows IFN-y secretion by Pmel-1 or C57BL / 6J derived aVHH CAR T cells quantified following a 24 hr coculture with MC38-VHH tumor cells at a 1 : 1 E:T ratio. n=3 independently cocultured wells. FIG. 12C shows cytotoxicity of Pmel-1 and C57BL / 6 derived aVHH CAR T cells were assessed following a 24 hr coculture with MC38-VHH at a 1 : 1 E:T ratio. Two-way ANOVA, mean ± s.e.m, n=3 independently cocultured wells; n.s. = not significant, **p=0.0058; ****p<0.0001.

[0027] FIGURES 13A-13D depict that aVHH CAR T cells are well tolerated by immunocompetent mice and isolated human PBMCs. FIGS. 13A-13B show that systemic toxicity of aVHH CAR T cells was assessed by the measure of liver enzyme alanine transaminase (ALT) and aspartate aminotransferase (AST) levels in serum following systemic administration (i.v.) of indicated T cells, one-way ANOVA, mean ± s.e.m., n= 5 mice. FIG. 13C shows that human aVHH CAR T cells were cocultured human PBMCs from the matching donor. FIG. 13D shows IFN-y, TNF-a and IL-6 secretion by human aVHH CAR T or aCD19 CAR T cells following a 48 hr coculture at a 1 : 1 E:T ratio with donor-matched PBMCs. Oneway ANOVA; mean ± s.d. n = 3 independently cocultured wells; IFN-y (UTD v aCD19 CAR **p=0.0021, aVHH CAR v aCD19 CAR **p=0.0033), TNF-a (UTD v aCD19 CAR **p=0.0082, aVHH CAR v aCD19 CAR *p=0.0281), IL-6 (UTD v aCD19 CAR *p=0.0192, aVHH CAR v aCD19 CAR **p=0.0213).

[0028] FIGURES 14A-14D depict that murine aVHH CAR T cells are well tolerated by immunocompetent mice. FIG. 14A shows blood serum analysis 7d post i.v. administration of aVHH CAR T cells, untransduced T cells, or saline into naive C57BL6 / J mice. One-way ANOVA, mean ± s.d., n = 4 mice.; n.s. = not significant. FIG. 14B shows body weight measurements following i.v. administration of aVHH CAR T cells, wild-type T cells, or saline into naive C57BL6 / J mouse. Two-way ANOVA, mean ± s.d., n = 5 mice per cohort; n.s. = not significant. FIG. 14C shows PBMC analysis before and 3 days after ACT of saline, WT, aVHH CAR, or aCD19 CAR T cells into C57BL6 / J mice. One-way ANOVA; mean ± s.e.m., n = 4 mice for aCD19 CAR treated mice, n = 5 mice in all other cohorts; n.s. = not significant ***p=0.0002. FIG. 14D shows blood serum analysis 3 d post i.v. administration of saline, WT, aVHH CAR, or ahHER2 CAR T cells into naive B6-HER2 mice. One-way ANOVA; mean ± s.e.m., n = 3 mice for hHER2 and VHH treatments, n=4 mice for Saline and WT treatments.; *p=0.01, **p=0.0065.

[0029] FIGURES 15A-15C depict that VHH expression alone does not alter tumor growth. FIG. 15A shows VHH expression on wildtype or transduced MC38 and E0771 tumor cells. FIG. 15B shows tumor growth curves of wildtype MC38 (MC38-WT) or MC38 cells transduced to stably express VHH (MC38-VHH) without adoptive cell transfer of aVHH CAR T cells, n = 4 mice inoculated with MC38-WT and n=3 mice inoculated with MC38-VHH. Two-way ANOVA, mean ± s.e.m. is depicted; n.s. = not significant. FIG. 15C shows tumor growth curves of wildtype E0771 (E0771-WT) or E0771 cells transduced to stably express VHH (E0771-VHH) without adoptive cell transfer of aVHH CAR T cells n=4 mice inoculatedwith E0771-WT and n=6 mice inoculated with E0771-VHH; Two-way ANOVA, mean ± s.e.m. is depicted; n.s. = not significant.

[0030] FIGURE 16 shows that adoptive transfer of aVHH CAR T cells to mice bearing wildtype tumors does not alter tumor growth. Tumor growth curves of mice bearing wiltdype E0771 tumors treated intravenously with either saline, 5 * 106untransduced (UTD) or aVHH CAR T cells. Two-way ANOVA, mean ± s.e.m., n=5 mice; n.s. = not significant.

[0031] FIGURES 17A-17C depict that the antitumor activity aVHH CAR T cells is comparable to aHER2 CAR T cells against VHH+ HER2+ tumors. FIG. 17A shows representative flow plots of HER2 and VHH expression on either wildtype or 468-HV tumor cells. FIG. 17B shows that 468-HV tumor cells were cocultured with either human untransduced (UTD), aHER2 CAR, or aVHH CAR T cells at a 2: 1 E:T ratio and assessed for cytotixicity after 24 hrs (One-way ANOVA, mean ± s.e.m.; n=3 independently cocultured wells, ****p<0.0001). FIG. 17C shows growth curves of 468-HV tumors following treatment with the UTD, aHER2 CAR, or aVHH CAR T cells. (Two-way ANOVA, mean ± s.e.m., n=6 mice, aVHH (**p=0.0080 on day 6, **p=0.0015 on day 13, ***p=0.0003 on day 18, ***p=0.0005 on day 25, ***p=0.0003 on day 33) and aHER2 (ffp=0.0054 on day 6, ffp=0.0016 on day 13, fffp=0.0003 on day 18, fffp=0.0005 on day 25, fffp=0.0003 on day 33) CAR T cell treatments are compared to UTD.

[0032] FIGURES 18A-18H depict that adoptive transfer of aVHH CAR T cells into mice with VHH-expressing tumors delays tumor growth, enhances infiltration of tumor-reactive T cells, and promotes endogenous antitumor immunity. FIGS. 18A-18B show that MC38-VHH tumors were treated with murine aVHH CAR T cells and monitored for tumor growth. Two- way ANOVA, mean ± s.e.m.; n=4 mice; **p=0.0043, ****p<0.0001. FIG. 18C shows count and frequency of indicated T cell populations isolated from the tumor. Unpaired one-sided Student’s t-test, mean displayed, n=4 tumors (Ctrl CAR T cells) and n=5 tumors (aVHH CAR T cells); left **p=0.0013, middle **p=0.0016, and right *p=0.0492. FIG. 18D shows a representative flow plot of endogenous (CAR-) T cell expression of the Reps-1 TCR in tdLNs and the frequency of this population in the ipsilateral and contralateral draining lymph nodes. Unpaired two-sided student’s t-test, mean displayed, n=3 independent lymph nodes (contralateral, Ctrl CAR T cell), n=4 independent lymph nodes (ipsilateral, Ctrl CAR T cell and contralateral aVHH CAR T cell), and n=5 independent lymph nodes (ipsilateral, aVHH CAR T cell); *p=0.0374, ns= nonsignificant. FIG. 18E shows that E0771-VHH tumors were treated with murine aVHH CAR T cells. FIG. 18F shows individual traces of tumor growth curves of E0771-VHH tumor-bearing mice treated with control CAR T cells (black) or aVHHCAR T cells (blue) (CR = complete responders) (n=4 mice). FIG. 18G shows that 45 days after initial treatment, cured mice (n=3) were rechallenged with wildtype E0771 tumor cells. Two-way ANOVA, mean ± s.e.m. (n=5 mice); ****p<0.0001. FIG. 18H shows survival curves of tumor-bearing mice following treatment, log-rank (Mantel-Cox) test; **p=0.0019.

[0033] FIGURE 19 depicts a representative flow cytometry gating strategy for identifying Reps 1 -reactive T cells isolated from tumor-infiltrating lymphocytes isolated from Thy 1.1+ MC38 tumors.

[0034] FIGURES 20A-20H depict that LNP-mediated synthetic antigen delivery sensitizes tumors to CAR-mediated cytoxicity in models of triple negative breast cancer. FIG. 20A shows that murine and human tumor cells were transfected with VHH mRNA-loaded cKK-E12 LNPs (VHH-LNPs) for 18 hrs before co-incubation with aVHH CAR T cells. FIG. 20B shows cytotoxicity of E0771s and MDA-MB-468 tumor cells quantified by LDH assay following 20-hr co-incubation with either murine or human untransduced (UTD) or aVHH CAR T cells respectively at various VHH-LNP doses. Multiple unpaired t-tests, mean ± s.d., n=4 independently transfected and cocultured wells (for E0771 : ****p<0.0001 [0.1-100 ng]; for MDA-MB-468: **p=0.0040 [1 ng], ***p=0.0005 [10 ng], **p=0.0039 [100 ng]). FIG. 20C shows that mice bearing wt E0771 tumors were treated intratumorally with saline, Fluc-LNP, or VHH-LNP followed by adoptive transfer of aVHH CAR T cells. FIG. 20D shows) the MFI of VHH expression on E0771 tumor cells 24 hrs following LNP treatment (left) and a representative flow plot of the same population (right). Unpaired two-sided Student’s t-test, mean ± s.e.m., n= 3 mice; *p=0.0480. FIG. 20E shows tumor growth curves of wildtype E0771 tumor-bearing mice treated with intratumorally with either saline, Fluc-LNP or VHH-LNP and aVHH CAR T cells on days indicated by a dashed line (DO, D7). Mixed-effects analysis with Tukey correction for multiple comparisons, mean ± s.e.m., n=5 mice for Fluc-LNP and VHH- LNP treatments, n=6 mice for saline treatment; *p=0.0204, ****p<0.0001. FIG. 20F shows survival curves of tumor-bearing mice following 2 (n=5 mice for Fluc-LNP and VHH-LNP treatments, n=6 mice for saline treatment) or 5 doses of indicated LNPs and CARs (n=5 mice). FIG. 20G shows that mice bearing wildtype MDA-MB-468 (468-wt) tumors were treated intratumorally with either saline or VHH-LNP followed by adoptive transfer of aVHH CAR T cells. FIG. 20H shows tumor growth curves of wildtype MDA-MB-468 tumor-bearing mice treated with saline, VHH-LNP or VHH-LNP with aVHH CAR T cells on days indicated by a dashed line (DO, D7). Two-way ANOVA, mean ± s.e.m., n=4 mice for VHH-LNP + CAR T cells, n=5 mice for all other cohorts; ****p<0.0001.

[0035] FIGURES 21A-21B depict in vivo biodistribution of functional mRNA delivery mediated by CKK-LNPs following intratumoral injection. FIG. 21 A shows in vivo biodistribution following saline or a 2 pg intratumoral injection of nLuc mRNA-loaded LNPs (nLuc-LNPs) as measured by IVIS 24 hrs following injection. Luminesence in organs quantified (left) and representative images are displayed on the right, mean ± s.d., multiple unpaired t test, n=3 organs; *p=0.0397, ***p=0.00047, ****p<0.0001. FIG. 21B shows in vivo kinetics of nLuc expression following one (light blue) or two (dark blue) intratumoral injections of 5 pg nLuc-LNPs. Transfection kinetics quantified by IVIS. Two-way ANOVA, mean ± s.e.m., n=4 mice; *p=0.0196, **p=0.0080.

[0036] FIGURE 22A shows tumor growth curves of wildtype E0771 (E0771-wt) tumors transfected with VHH-LNPs (CKK-E12) by intratumoral injection of 5 pg mRNA-loaded LNPs without adoptive cell transfer of aVHH CAR T cells. Two-way ANOVA, mean ± s.e.m. is depicted; n=6 saline injected mice and n= 5 for VHH-LNP injected mice.; n.s. = not significant. FIGURE 22B shows tumor growth curves of wildtype E0771 tumor-bearing mice treated with intratum orally with either saline, Fluc-LNP or VHH-LNP and aVHH CAR T cells on days indicated by a dashed line (DO, D7, D14, D21, and D28). Two-way ANOVA, mean ± s.e.m., n = 5 mice. Saline vs. VHH-LNP: *p=0.0176, ***p=0.0006, ****p<0.0001.

[0037] FIGURES 23A-23F depict that LNP-mediated synthetic antigen treatment augments antitumor immunity in a preclinical model of heterogenous glioblastoma tumors. FIG. 23A shows that EGFRvIII- (vIIL) or EGFRvIII+ (vIII+) SB28 tumor cells were transfected with 100 ng of VHH-LNP and evaluated for VHH expression by flow cytometry after 18 hrs, n=5 independent wells, two-tailed unpaired t test; n.s .= not significant. FIG. 23B shows that mice bearing a heterogenous tumor mixture including 90% vIIL and 10% vIII+ SB28 tumor cells were treated with saline or VHH-LNP followed by adoptive transfer of indicated CAR T cells. FIG. 23C shows that 24 hrs following intratumoral injection VHH- LNP (5 pg), SB28 tumors were dissociated and analyzed by flow cytometry for EGFRvIII (left) and VHH (right) expression, one-way unpaired T test, mean ± s.d., n=3 dissociated tumors; ***p=0.0008, n.s.=nonsignficiant. FIG. 23D shows representative flow plots of VHH expression on SB28 tumors treated with either saline or VHH-LNP. FIG. 23E shows tumor growth curves of heterogenous SB28 tumor-bearing mice treated with intratumorally with saline or VHH-LNP and systemically with either WT, aVHH, or aEGFRvIII CAR T cells days indicated days by dashed lines (DO, D7, & D14) n = 5 untreated and VHH-LNP + aVHH CAR treated mice, n=6 mice with aEGFRvIII CAR T cell treatment, n = 7 mice with VHH-LNP + saline treatment. Two-way ANOVA, mean ± s.e.m. FIG. 23F shows survival curves from FIG.23E of heterogenous SB28 tumor-bearing mice following synthetic antigen treatment, log-rank (Mantel-Cox) test; * *p=0.0019 comparing EGFRvIII CAR treatment with LNP + aVHH CAR.

[0038] FIGURES 24A-24E depict that LNP-mediated synthetic antigen treatment augments antitumor immunity in preclinical models of heterogenous tumors and mitigates outgrowth of antigen negative tumor cells. FIG. 24A shows that mice bearing a heterogenous tumor mixture including 85% HER2- and 15% HER2+ MDA-MB-468 tumor cells were first treated with intratumorally with either saline or VHH-LNP followed by adoptive transfer of aVHH CAR T cells or aHER2 CAR T cells. FIGS. 24B-24C show tumor growth curves (FIG. 24B) and individual spider plots (FIG. 24C) of heterogenous MDA-MB-468 tumors treated intratumorally with saline or VHH-LNP and systemically with either indicated T cells on days indicated by a dashed line (DO, D7, D14). Mixed-effect analysis with Tukey correction for multiple comparisons, mean ± s.e.m., n= 4 mice for WT and aHER2 CAR T cell treatments, n=5 mice for LNP + WT and LNP + aVHH CAR T cell treatments. Statistical comparisons between Saline + WT and LNP + aVHH CAR T cell (**p=0.0050 on day 16, ***p=0.0008 on day 20, ****p<0.0001 for days 25-34) and between LNP + WT and aHER2 CAR T cell (*p=0.0337 for day 27, **p=0.0010 for day 31, ****p<0.0001 for day 34). FIGS. 24D-24E show representative flow plots (FIG. 24D) and quantification of endogenous antigen (HER2) tumor composition at endpoint (FIG. 24E) of tumors that were harvested and evaluated for HER2 expression by flow cytometry. Multiple, unpaired, one-sided T test, mean ± s.e.m., n=4 tumors; *p=0.0427 for HER2 CAR vs LNP + WT, *p=0.0267 for HER2 CAR vs saline + WT.DETAILED DESCRIPTION

[0039] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0040] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0041] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0042] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0043] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0044] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0045] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0046] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0047] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0048] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptomsbut is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0049] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0050] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0051] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0052] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0053] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0054] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0055] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

[0056] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0057] Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to thecomplete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0058] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0059] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0060] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0061] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.

[0062] The terms “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand).

[0063] Reference also is made herein to peptides, polypeptides, proteins and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110).

[0064] A “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability. In this case, the functional fragment would retain the ability to perform as a telomerase.

[0065] As disclosed herein, exemplary peptides, polypeptides, proteins may comprise, consist essentially of, or consist of any reference amino acid sequence disclosed herein, or variants of the peptides, polypeptides, and proteins may comprise, consist essentially of, or consist of an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any amino acid sequence disclosed herein. Variant peptides, polypeptides, and proteins may include peptides, polypeptides, and proteins having one or more amino acid substitutions, deletions, additions and / or amino acid insertions relative to a reference peptide, polypeptide, or protein. Also disclosed are nucleic acid molecules that encode the disclosed peptides, polypeptides, and proteins (e.g., polynucleotides that encode any of the peptides, polypeptides, and proteins disclosed herein and variants thereof).

[0066] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consistingof homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3 -Aminoadipic acid, Hydroxylysine, P-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine, 3 -Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0067] The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C- terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine).

[0068] Variants comprising deletions relative to a reference amino acid sequence or nucleotide sequence are contemplated herein. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20,50, 100, or 200 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation or both of a reference polypeptide or a 5 '-terminal or 3 '-terminal truncation or both of a reference polynucleotide).

[0069] Variants comprising a fragment of a reference amino acid sequence or nucleotide sequence are contemplated herein. A “fragment” is a portion of an amino acid sequence or a nucleotide sequence which is identical in sequence to but shorter in length than the reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide / amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C-terminal region of a polypeptide or the 5 '-terminal region and / or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.

[0070] Variants comprising insertions or additions relative to a reference sequence are contemplated herein. The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues or nucleotides.

[0071] Fusion proteins and fusion polynucleotides also are contemplated herein. A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N- terminus, the C-terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.

[0072] A fusion polynucleotide refers to the fusion of the nucleotide sequence of a first polynucleotide to the nucleotide sequence of a second heterologous polynucleotide (e.g., the 3' end of a first polynucleotide to a 5' end of the second polynucleotide). Where the first and second polynucleotides encode proteins, the fusion may be such that the encoded proteins are in-frame and results in a fusion protein. The first and second polynucleotide may be fused such that the first and second polynucleotide are operably linked (e.g., as a promoter and a gene expressed by the promoter as discussed below).

[0073] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0074] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0075] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0076] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0077] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1 3, Cold Spring Harbor Press, Plainview N.Y. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0078] Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0079] “Substantially isolated or purified” nucleic acid or amino acid sequences are contemplated herein. The term “substantially isolated or purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment, and are at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more preferably at least 95% free from other components with which they are naturally associated.NANOPARTICLES

[0080] In an aspect, provided is a nanoparticle including: one or more lipids including an ionizable lipid, a cholesterol, a phospholipid, a PEGylated lipid, or any combination thereof; and an encapsulated nucleic acid encoding a target antigen.

[0081] As used herein, the term “ionizable lipid” refers to a lipid, e.g., cationic lipid or anionic lipid, having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 toabout 7. In some aspects, the ionizable lipid can include cKK-E12, SM102, MC3, Lipid-5, Alc- 0315, LP01, Lipid A9, or any combination thereof.

[0082] In some aspects, the nanoparticle can include at least about 30 mol% (e.g., at least about 32 mol%, at least about 34 mol%, at least about 36 mol%, at least about 38 mol%, at least about 40 mol%, at least about 42 mol%, at least about 44 mol%, at least about 46 mol%, at least about 48 mol%, at least about 50 mol%, at least about 52 mol%, at least about 54 mol%, at least about 56 mol%, at least about 58 mol%, at least about 60 mol%) of the ionizable lipid. In some aspects, the nanoparticle can include up to about 60 mol% (e.g., up to about 58 mol%, up to about 56 mol%, up to about 54 mol%, up to about 52 mol%, up to about 50 mol%, up to about 48 mol%, up to about 46 mol%, up to about 44 mol%, up to about 42 mol%, up to about 40 mol%, up to about 38 mol%, up to about 36 mol%, up to about 34 mol%, up to about 32 mol%, up to about 30 mol%) of the ionizable lipid.

[0083] It is considered that the nanoparticle can include an amount of the ionizable lipid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 30 mol% to about 60 mol% (e.g., from about 32 mol% to about 58 mol%, from about 34 mol% to about 56 mol%, from about 36 mol% to about 54 mol%, from about 38 mol% to about 52 mol%, from about 40 mol% to about 50 mol%, from about 42 mol% to about 48 mol%, from about 44 mol% to about 46 mol%, from about 30 mol% to about 46 mol%, from about 32 mol% to about 44 mol%, from about 34 mol% to about 42 mol%, from about 36 mol% to about 40 mol%, from about 44 mol% to about 60 mol%, from about 46 mol% to about 58 mol%, from about 48 mol% to about 56 mol%, from about 50 mol% to about 54 mol%) of the ionizable lipid.

[0084] In some aspects, the nanoparticle can include at least about 35 mol% (e.g., at least about 36 mol%, at least about 37 mol%, at least about 38 mol%, at least about 39 mol%, at least about 40 mol%, at least about 41 mol%, at least about 42 mol%, at least about 43 mol%, at least about 44 mol%, at least about 45 mol%, at least about 46 mol%, at least about 47 mol%, at least about 48 mol%, at least about 49 mol%, at least about 50 mol%) of the cholesterol. In some aspects, the nanoparticle can include up to about 50 mol% (e.g., up to about 49 mol%, up to about 48 mol%, up to about 47 mol%, up to about 46 mol%, up to about 45 mol%, up to about 44 mol%, up to about 43 mol%, up to about 42 mol%, up to about 41 mol%, up to about 40 mol%, up to about 39 mol%, up to about 38 mol%, up to about 37 mol%, up to about 36 mol%, up to about 35 mol%) of the cholesterol.

[0085] It is considered that the nanoparticle can include an amount of the cholesterol ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 35 mol% to about 50 mol% (e.g., from about 36 mol% to about 49 mol%, from about 37 mol% to about 48 mol%, from about 38 mol% to about 47 mol%, from about 39 mol% to about 46 mol%, from about 40 mol% to about 45 mol%, from about 41 mol% to about 44 mol%, from about 42 mol% to about 43 mol%, from about 35 mol% to about 43 mol%, from about 36 mol% to about 42 mol%, from about 37 mol% to about 41 mol%, from about 38 mol% to about 40 mol%, from about 42 mol% to about 50 mol%, from about 43 mol% to about 49 mol%, from about 44 mol% to about 48 mol%, from about 45 mol% to about 47 mol%) of the cholesterol.

[0086] In some aspects, the phospholipid can include DSPC, DOPE, or any combination thereof.

[0087] In some aspects, the nanoparticle can include at least about 5 mol% (e.g., at least about 6 mol%, at least about 7 mol%, at least about 8 mol%, at least about 9 mol%, at least about 10 mol%, at least about 11 mol%, at least about 12 mol%, at least about 13 mol%, at least about 14 mol%, at least about 15 mol%, at least about 16 mol%, at least about 17 mol%, at least about 18 mol%, at least about 19 mol%, at least about 20 mol%) of the phospholipid. In some aspects, the nanoparticle can include up to about 20 mol% (e.g., up to about 19 mol%, up to about 18 mol%, up to about 17 mol%, up to about 16 mol%, up to about 15 mol%, up to about 14 mol%, up to about 13 mol%, up to about 12 mol%, up to about 11 mol%, up to about 10 mol%, up to about 9 mol%, up to about 8 mol%, up to about 7 mol%, up to about 6 mol%, up to about 5 mol%) of the phospholipid.

[0088] It is considered that the nanoparticle can include an amount of the phospholipid ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the nanoparticle can include from about 5 mol% to about 20 mol% (e.g., from about 6 mol% to about 19 mol%, from about 7 mol% to about 18 mol%, from about 8 mol% to about 17 mol%, from about 9 mol% to about 16 mol%, from about 10 mol% to about 15 mol%, from about 11 mol% to about 14 mol%, from about 12 mol% to about 13 mol%, from about 5 mol% to about 13 mol%, from about 6 mol% to about 12 mol%, from about 7 mol% to about 11 mol%, from about 8 mol% to about 10 mol%, from about 12 mol% to about 20 mol%, from about 13 mol% to about 19 mol%, from about 14 mol% to about 18 mol%, from about 15 mol% to about 17 mol%) of the phospholipid.

[0089] In some aspects, the PEGylated lipid can include ALC-0159, DMG-PEG, DSPE- PEG, PEG14-2000, or any combination thereof.

[0090] In some aspects, the nanoparticle can include at least about 0.5 mol% (e.g., at least about 1 mol%, at least about 1.5 mol%, at least about 2 mol%, at least about 2.5 mol%, at least about 3 mol%, at least about 3.5 mol%, at least about 4 mol%, at least about 4.5 mol%, at least about 5 mol%) of the PEGylated lipid. In some aspects, the nanoparticle can include up to about 5 mol% (e.g., up to about 4.5 mol%, up to about 4 mol%, up to about 3.5 mol%, up to about 3 mol%, up to about 2.5 mol%, up to about 2 mol%, up to about 1.5 mol%, up to about 1 mol%, up to about 0.5 mol%) of the PEGylated lipid.

[0091] It is considered that the nanoparticle can include an amount of the PEGylated lipid ranging from any of the minimum values described above. For example, in some aspects, the nanoparticle can include from about 0.5 mol% to about 5 mol% (e.g. from about 1 mol% to about 4.5 mol%, from about 1.5 mol% to about 4 mol%, from about 2 mol% to about 3.5 mol%, from about 2.5 mol% to about 3 mol%, from about 0.5 mol% to about 3 mol%, from about 1 mol% to about 2.5 mol%, from about 1.5 mol% to about 2 mol%, from about 2.5 mol% to about 5 mol%, from about 3 mol% to about 4.5 mol%, from about 3.5 mol% to about 4 mol%) of the PEGylated lipid.

[0092] In some aspects, the ratio of PEGylated lipid to total lipids can be at least about 0.5: 1 (e.g., at least about 0.6: 1, at least about 0.7: 1, at least about 0.8: 1, at least about 0.9: 1, at least about 1 : 1, at least about 1.1 : 1, at least about 1.2: 1, at least about 1.3 : 1, at least about 1.4: 1, at least about 1.5: 1, at least about 1.6: 1, at least about 1.7: 1, at least about 1.8: 1, at least about 1.9:1, at least about 2: 1). In some aspects, the ratio of PEGylated lipid to total lipids can be up to about 2: 1 (e.g., up to about 1.9: 1, up to about 1.8: 1, up to about 1.7: 1, up to about 1.6: 1, up to about 1.5: 1, up to about 1.4: 1, up to about 1.3: 1, up to about 1.2: 1, up to about 1.1 : 1, up to about 1 : 1, up to about 0.9:1, up to about 0.8: 1, up to about 0.7: 1, up to about 0.6: 1, up to about 0.5: 1)

[0093] It is considered that the ratio of PEGylated lipid to total lipids can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the ratio of PEGylated lipid to total lipids can be from about 0.5: 1 to about 2: 1 (e.g., from about 0.6: 1 to about 1.9:1, from about 0.7:1 to about 1.8: 1, from about 0.8:1 to about 1.7:1, from about 0.9: 1 to about 1.6: 1, from about 1 : 1 to about 1.5:1, from about 1.1 :1 to about 1.4: 1, from about 1.2:1 to about 1.3: 1, from about 0.5: 1 to about 1.3: 1, from about 0.6: 1 to about 1.2: 1, from about 0.7: 1 to about 1.1 :1, from about 0.8: 1 to about 1 : 1, from about 1.2: 1 to about 2: 1, from about 1.3 : 1 to about 1.9: 1, from about 1.4: 1 to about 1.8: 1, from about 1.5: 1 to about 1.7: 1).

[0094] In some aspects, the nanoparticle can be a lipid nanoparticle, a liposome, or a polymeric nanoparticle. As used herein, “lipid nanoparticles” refer to particles having at least one dimension on the order of nanometers (e.g., 1-1000 nm) and including one or more lipids. In the context of the present invention, a lipid nanoparticle typically serves to transport a desired nucleic acid to a target cell or tissue. The process of incorporation of a desired nucleic acid into a lipid nanoparticle is often referred to as “loading”. The lipids and the nucleic acid can create a self-assembled structure via counterion interactions. The purpose of incorporating a nucleic acid into a transfer vehicle, such as a lipid nanoparticle, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some aspects, a suitable delivery vehicle is capable of enhancing the stability of the nucleic acid contained therein and / or facilitate the delivery of nucleic acid to the target cell or tissue.

[0095] In some aspects, the encapsulated nucleic acid can include mRNA. In some aspects, the encapsulated nucleic acid can include DNA. In some aspects, the encapsulated nucleic acid can include one or more modified nucleotides.

[0096] In some aspects, the target antigen does not occur naturally (i.e., nonnatural or not naturally occurring antigen including, but not limited to epitopes comprising one or more D- substituted amino acid or amino acid anlaogs). In some such aspects, the target antigen can include a detectable moiety (such as for example, a radiolabel, fluorophore, a membrane-bound reporter, a member of a binding pair, such as biotin / streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence.

[0097] Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8- ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5- Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6- CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4- I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™;Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA- BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bisBTC; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™-3; Bodipy492 / 515; Bodipy493 / 503; Bodipy500 / 510; Bodipy; 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC- 5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green- 1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP / YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Diehl orodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16- ASP); Diehl orodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC 18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd InducedFluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxy stilbamidine); Fluor-Ruby; FluorX; FM 1- 43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin; Hydroxy stilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; Ly soSensor Blue; Ly soSensor Green; Ly soSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO- 1 PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phy cocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™(super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF 1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO- 1; YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.

[0098] A modifier unit such as a radionuclide can be incorporated into or attached directly to any of the compounds described herein by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine- 124, astatine-210, carbon-11, carbon- 14, nitrogen- 13, fluorine- 18. In another aspect, the radionuclide can be attached to a linking group or bound by a chelating group, which is then attached to the compound directly or by means of a linker. Examples of radionuclides useful in the apset include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling techniques such as these are routinely used in the radiopharmaceutical industry.

[0099] In some aspects, the target antigen can include 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more)to SEQ ID NO: 2. In some aspects, the target antigen can include SEQ ID NO: 2. In some aspects, the target antigen can consist of SEQ ID NO: 2.

[0100] In another aspect, provided is a synthetic antigen including 80% similarity or more (e.g., 81% similarity or more, 82% similarity or more, 83% similarity or more, 84% similarity or more, 85% similarity or more, 86% similarity or more, 87% similarity or more, 88% similarity or more, 89% similarity or more, 90% similarity or more, 91% similarity or more, 92% similarity or more, 93% similarity or more, 94% similarity or more, 95% similarity or more, 96% similarity or more, 97% similarity or more, 98% similarity or more, 99% similarity or more) to SEQ ID NO: 2. In some aspects, the synthetic antigen can include SEQ ID NO: 2. In some aspects, the synthetic antigen can consist of SEQ ID NO: 2.METHODS

[0101] In an aspect, provided is a method of engineering a cell to express a target antigen, the method including exposing the cell to a nanoparticle, the nanoparticle including: one or more lipids including an ionizable lipid, a cholesterol, a phospholipid, a PEGylated lipid, or any combination thereof; and an encapsulated nucleic acid encoding a target antigen.

[0102] In some aspects, the cell can be cancerous or abnormal. In some aspects, the cell can be a hematologic cancer cell, a lymphoma cell, a colorectal cancer cell, a colon cancer cell, a lung cancer cell, a head and neck cancer cell, an ovarian cancer cell, a prostate cancer cell, a testicular cancer cell, a renal cancer cell, a skin cancer cell, a melanoma cell, a cervical cancer cell, a brain cancer cell, a pancreatic cancer cell, or a breast cancer cell. In some aspects, the cell can be derived from a solid tumor. In other aspects, the cell can be derived from acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, or multiple myeloma. In yet other aspects, the cell can be derived from a leukemia, a lymphoma, a sarcoma, or a carcinoma, any of which may originate in the marrow, brain, lung, breast, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, liver, kidney, intraperitoneum, bone, joint, or eye. In other aspects, the cell can be healthy. In some aspects, the cell can be human.

[0103] In some aspects, the nanoparticle can be any of the disclosed nanoparticles described above.

[0104] In some aspects, the method can be carried out in vitro, in vivo, or ex vivo.

[0105] In another aspect, provided is an engineered cell produced by the any of the disclosed methods of engineering a cell.

[0106] In yet another aspect, provided are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer, solid tumor, and or metastasis (including,but not limited to breast cancer, colon cancer, brain cancer, lung cancer, prostate cancer, or melanoma) in a subject, the method comprising a) providing to the subject the nanoparticle of any preceding aspect and b) subsequently providing to the subject an immunotherapy (such as, for example, antibodies that block PD-1, PD-L1, CTLA-4, PD-L2 IDO, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT) B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), TIM3, or LAG-3) and adoptive cell therapies such as, for example chimeric antigen receptor (CAR) T cells, CAR Natural Killer (NK) (CAR NK cells), CAR Macrophage (CARMAC) CAR NK T cells, Tumor Infiltrating Lymphocytes (TILs), and / or Marrow Infiltrating Lymphocytes (MILs)). For example, disclosed herein are methods of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing a cancer, solid tumor, and or metastasis (including, but not limited to breast cancer, colon cancer, brain cancer, lung cancer, prostate cancer, or melanoma) in a subject, the method comprising a) providing to the subject (including, but not limited to a lipid nanoparticle, a liposome, or a polymeric nanoparticle), the nanoparticle comprising one or more lipids comprising an ionizable lipid (such as, for example, cKK-E12, SM102, MC3, Lipid-5, Alc- 0315, LP01, Lipid A9, or any combination thereof), a cholesterol, a phospholipid (such as, for example, DSPC, DOPE, or any combination thereof), a PEGylated lipid (such as, for example, ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof), or any combination thereof; and an encapsulated nucleic acid (such as, for example, DNA, mRNA, cell free DNA (cfDNA), long noncoding DNA (IncDNA), or microRNA (miRNA)) encoding a target antigen (including, but not limited to non-naturally occurring antigens such as, for example a target antigen that comprises 80% similarity or more to SEQ ID NO: 2); and b) subsequently providing to the subject an immunotherapy (including, but not limited to immune checkpoint inhibitors (such as, for example, antibodies that block PD-1, PD-L1, CTLA-4, PD- L2 IDO, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT) B- and T- lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), TIM3, or LAG-3) and adoptive cell therapies such as, for example, tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (NILs), chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) (CAR NK) cells, CAR macrophage (CARMACs), or CAR NK T cells expressing a CAR, T cell receptor, or NK receptor which recognizes the target antigen). In some aspects, at least about 15% of the cancer or tumor cells express the target antigen. In some aspects, step b) is carried out from about 24 hours to about 72 hours after step a).

[0107] In some aspects, the nanoparticle can be any of the disclosed nanoparticles described above.

[0108] In some aspects, the immunotherapy can include an immune checkpoint inhibitor. In some such aspects, the immune checkpoint can include an antibody that blocks PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT- 011, MK-3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvaluniab, MDX- 1105 (BMS-936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA-4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP- 675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7- H3, T cell immunoreceptor with Ig and ITIM domains (TIGIT)(such as, for example BMS- 986207, OMP-313M32, MK-7684, AB-154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, JNJ-61610588, CA-170), TIM3 (such as, for example, TSR- 022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).

[0109] In some aspects, the immunotherapy can include an adoptive cell therapy. In some such aspects, the adoptive cell therapy can include the administration of tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (NILs), chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) (CAR NK) cells, CAR macrophage (CARMACs), or CAR NK T cells expressing a CAR, T cell receptor, or NK receptor which recognizes the target antigen.

[0110] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti-cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA®(Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil— Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clofarabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR-U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil— Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EV ACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA®(Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil— Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil— Topical), Fluorouracil Injection, Fluorouracil— Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDASIL® (Recombinant HPV Quadrivalent Vaccine), GARDASIL 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINEOXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMfDUM® (Ifosfamide), IL- 2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine 1 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado- Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine,LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, MITOZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPP A, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, RecombinantHuman Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonaval ent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R- EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R- ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, SOMATULINE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, T ARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil— Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine I 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASARPFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223Dichloride), XTANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / or ZYTIGA® (Abiraterone Acetate).[OHl] In some aspects, step a) can include administering at least about 0.05 mg / kg (e.g., at least about 0.1 mg / kg, at least about 0.15 mg / kg, at least about 0.2 mg / kg, at least about 0.25 mg / kg, at least about 0.3 mg / kg, at least about 0.35 mg / kg, at least about 0.4 mg / kg, at least about 0.45 mg / kg, at least about 0.5 mg / kg, at least about 0.55 mg / kg, at least about 0.6 mg / kg, at least about 0.65 mg / kg, at least about 0.7 mg / kg, at least about 0.75 mg / kg, at least about 0.8 mg / kg, at least about 0.85 mg / kg, at least about 0.9 mg / kg, at least about 0.95 mg / kg, at least about 1 mg / kg) nanoparticle. In some aspects, step a) can include administering up to about 1 mg / kg (e.g., up to about 0.95 mg / kg, up to about 0.9 mg / kg, up to about 0.85 mg / kg, up to about 0.8 mg / kg, up to about 0.75 mg / kg, up to about 0.7 mg / kg, up to about 0.65 mg / kg, up to about 0.6 mg / kg, up to about 0.55 mg / kg, up to about 0.5 mg / kg, up to about 0.45 mg / kg, up to about 0.4 mg / kg, up to about 0.35 mg / kg, up to about 0.3 mg / kg, up to about 0.25 mg / kg, up to about 0.2 mg / kg, up to about 0.15 mg / kg, up to about 0.1 mg / kg, up to about 0.05 mg / kg) nanoparticle.

[0112] It is considered that step a) can include administering an amount of nanoparticle ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, step a) can include administering from about 0.05 mg / kg to about 1 mg / kg (e.g., from about 0.1 mg / kg to about 0.95 mg / kg, from about 0.15 mg / kg to about 0.9 mg / kg, from about 0.2 mg / kg to about 0.85 mg / kg, from about 0.25 mg / kg to about 0.8 mg / kg, from about 0.3 mg / kg to about 0.75 mg / kg, from about 0.35 mg / kg to about 0.7 mg / kg, from about 0.4 mg / kg to about 0.65 mg / kg, from about 0.45 mg / kg to about 0.6 mg / kg, from about 0.5 mg / kg to about 0.55 mg / kg, from about 0.05 mg / kg to about 0.55 mg / kg, from about 0.1 mg / kg to about 0.5 mg / kg, from about 0.15 mg / kg to about 0.45 mg / kg, from about 0.2 mg / kg to about 0.4 mg / kg, from about 0.25 mg / kg to about 0.35 mg / kg, from about 0.5 mg / kg to about 1 mg / kg, from about 0.55 mg / kg to about 0.95 mg / kg, from about 0.6 mg / kg to about 0.9 mg / kg, from about 0.65 mg / kg to about 0.85 mg / kg, from about 0.7 mg / kg to about 0.8 mg / kg) nanoparticle.

[0113] In some aspects, the nanoparticle can be administered intratum orally.

[0114] In some aspects, after step a), at least about 15% (e.g., at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%) of the cancer or tumor cells may express the target antigen.

[0115] In some aspects, step b) can be carried out at least about 24 hours (e.g., at least about 28 hours, at least about 32 hours, at least about 36 hours, at least about 40 hours, at least about 44 hours, at least about 48 hours, at least about 52 hours, at least about 56 hours, at least about 60 hours, at least about 64 hours, at least about 68 hours, at least about 72 hours) after step a). In some aspects, step b) can be carried out up to about 72 hours (e.g., up to about 68 hours, up to about 64 hours, up to about 60 hours, up to about 56 hours, up to about 52 hours, up to about 48 hours, up to about 44 hours, up to about 40 hours, up to about 36 hours, up to about 32 hours, up to about 28 hours, up to about 24 hours) after step a).

[0116] It is considered that step b) can be carried out any duration after step a) ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, step b) can be carried out from about 24 hours to about 72 hours (e.g., from about 28 hours to about 68 hours, from about 32 hours to about 64 hours, from about 36 hours to about 60 hours, from about 40 hours to about 56 hours, from about 44 hours to about 52 hours, from about 24 hours to about 48 hours, from about 28 hours to about 44 hours, from about 32 hours to about 40 hours, from about 48 hours to about 72 hours, from about 52 hours to about 68 hours, from about 56 hours to about 64 hours) after step a).

[0117] In some aspects, the cancer or solid tumor can be hematologic cancer, lymphoma, colorectal cancer, colon cancer, lung cancer, head and neck cancer, an ovarian cancer, prostate cancer, testicular cancer, renal cancer, skin cancer, melanoma, cervical cancer, brain cancer, pancreatic cancer, or breast cancer. In some aspects, the subject can have a solid tumor. In other aspects, cancer can be acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, or multiple myeloma. In yet other aspects, cancer or solid tumor can be leukemia, lymphoma, sarcoma, or carcinoma, any of which may originate in the marrow, brain, lung, breast, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, liver, kidney, intraperitoneum, bone, joint, or eye.EXAMPLESExample 1: Methods and composition for lipid nanoparticle (LNP) mediated delivery of mRNA encoded synthetic antigens for universal tumor targeting by CAR T cells

[0118] Cytotoxic T cells functionalized with chimeric antigen receptors (CARs) have demonstrated tremendous clinical benefits combatting hematological malignancies, leading to curative responses in patients with leukemia and myeloma. Briefly, CARs are synthetic surface receptors expressed by immune cells that enable targeting of cell surface antigens (including proteins, lipids, and sugars) independent of presentation on the major histocompatibility complex (MHC), thus enabling directed T cell cytotoxicity towards virtually any antigen of interest. However, the scarcity of tumor specific antigen expression on the surface of malignant cells poses a significant hurdle in CAR T cell therapy as low levels of target antigen expression in healthy organs can lead to on-target off-tumor toxicity. Previous studies have examined the methods and composition for designing synthetic antigens and cognate CARs to sensitize tumors to T cell-mediated cytotoxicity.

[0119] Encoding synthetic antigens in mRNA facilitates the transient synthesis and expression of synthetic antigens on the surface of tumor cells without risk of permanent genomic integration. Disclosed herein is a lipid nanoparticle (LNP) encapsulating synthetic antigen encoding mRNA for in situ redirection of universal CAR T cells towards tumor cells. Synthetic antigen expression on tumors using LNPs protects mRNA from degradation in vivo and helps its entry into cells enabling protein production. While viral vectors, such as AAV, induces strong immune responses, the low immunogenicity of LNPs enables repeat dosing. A study was conducted which demonstrates that the delivery of CAR T cells following LNP- mediated VHH expression improves therapeutic outcomes in preclinical models of cancer without measurable off-tumor toxicity. This platform renders tumor cells, including those which are otherwise untargetable, vulnerable to T cell recognition and enables the use of a single CAR construct to treat tumors lacking immunogenic antigens, ultimately expanding the application of CAR T cell therapies across cancer types.Technical Description

[0120] Lipid nanoparticle (LNP) formulations for synthetic antigen encoding mRNA encapsulation'. LNPs are a nanoscale structure which encapsulate and protect their cargo from degradation and enhance uptake. Here, LNPs including an ionizable lipid (such as but not limited to cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9), phospholipid (such as but not limited to DSPC, DOPE), PEGylated lipid (such as but not limited to ALC-0159, DMG-PEG, DSPE-PEG), and cholesterol with various molar ratios (TABLE 1) is prepared toencapsulate synthetic antigen encoding mRNA. Briefly, LNPs are prepared by mixing the lipid mixtures at indicated molar ratios in ethanol, combined with of mRNA in acetate buffer and injected into a microfluidic mixing device at a total flow rate of 12 mL / min (3 : 1 flow rate ratio of mRNA in acetate buffer to lipid mixtures in ethanol). The resultant LNPs were diluted 40* in PBS and concentrated using 10 kDa Amicon spin filters and stored at -80C with 8% sucrose. LNP stability was assessed by particle diameter as measured by dynamic light scattering (DLS) measurements 1 day and 1 year post LNP production (FIG. 1). MC3, LP01, LPA9, ALC0315, and Spark appeared to aggregate as particle diameters increased substantially while SM102, cKK-E12, and Lipid 5 based LNPs maintained similar diameter after long-term storage (~100nm) indicating high stability.TABLE 1. Compositions of various LNP formulations.

[0121] LNP-mediated delivery of synthetic antigens to tumor cells'. In one embodiment, LNPs are delivered to cancer cells to induce synthetic antigen expression on their cell surface. The study screened the transfection efficiency of various LNP formulations (see TABLE 1) on the triple negative human breast cancer (TNBC) cell line MDA-MB-468. To do so, the study encapsulated mRNA encoding for NanoLuc luciferase and measured tumor cell luminescence following in vitro co-incubations of tumor cells and LNPs at a concentration of Ipg / mL with or without 1 pg / mL of apolipoprotein E (ApoE). All LNPs formulations tested led to an increase in luciferase signal from tumor cells, either with or without supplementing with ApoE. The addition of ApoE enhanced transfection efficiency across several formulations, including MC3, SM102, Lipid-5, Lipid A9, Alc-0315, and cKK-E12. cKK-E12 LNPs loaded with NanoLuc mRNA led to the highest transfection efficiency (referred to nLuc-LNP hereafter).Subsequently, surface expression of the VHH synthetic antigen was assessed following tumor co-incubation with the VHH mRNA-loaded cKK-E12 formulation on 5 human and 5 murine tumor cell lines by flow cytometry. Expression of the VHH synthetic antigen was achieved across all tested tumor cell lines, which span brain, TNBCs, colorectal, and lung malignancies of either mouse or human origin. Expression kinetics were measured on the murine TNBC line E0771 and human MDA-MB-468 where VHH was expressed on the cell surface for over 5 days following a single treatment with cKK-E12 LNPs loaded with VHH-mRNA (referred to VHH-LNP hereafter).

[0122] In situ delivery of synthetic antigens by LNP'. To achieve local expression of the VHH synthetic antigen within the tumor, tumors were treated with intratumoral injections of LNPs. Intratumoral injections of 5 pg of nLuc-LNPs led to the detection of luciferase signal in the tumor with minimal to no detection of luciferase signal in other organs. Viral delivery methods (such as AAV), can elicit strong immune responses that generate neutralizing antibodies and eliminate the option to re-dose the same therapy, thus precluding the potential to for multiple synthetic antigen treatments. LNPs, which are primarily lipid-based, are suitable for repeated dosing due to their proven safety profile and tolerability. This study demonstrates the ability to re-dose LNPs in tumors using nLuc-LNP, and demonstrate that this formulation leads to VHH expression following intratumoral injections of VHH-LNP. Importantly, this study demonstrates that, on its own, this LNP formulation does not affect tumor growth.

[0123] LNP-mediated delivery of synthetic antigens enables the redirection of a universal CAR towards tumors'. LNP-mediated delivery of synthetic antigen encoding mRNA enables the redirection of a universal, synthetic-antigen targeting CAR T cell toward transfected tumor cells. Here, the study demonstrates that IFN-y production and tumor cell cytotoxicity increase when human or mouse VHH CAR T cells are co-incubated in vitro with either mouse (E0771) or human (MDA-MB-468) tumor cells treated increased doses of VHH-LNP. Dose dependent increase in cytotoxicity against wildtype tumor cells were observed as LNP dose increased. Furthermore, the in situ delivery of VHH-LNP to E0771 tumors, followed by treatment with murine VHH CAR T cells, led to the significant delay of tumor growth and enhanced survival in a syngeneic mouse model of TNBC. Similarly, compared to untreated tumors, treatment of NSG mice bearing human TNBC tumors (MDA-MB-468s) with VHH-LNPs and VHH CAR T cells led to the significant delay in tumor growth.

[0124] Heterogenous antigen expression within a tumor and interpatient variation in expression limit the broad applicability of a single CAR construct. Human breast cancers, for example, are classified as estrogen receptor (ER)-positive when as few as 1% of tumor cellsexpress ER. Additionally, low antigen density and antigen loss, such as through trogocytosis and acquired mutations, render CAR T cells ineffective over time and raise concerns with antigen escape. To test the ability of synthetic antigen treatment to treat heterogenous tumors, the study inoculated NSG mice with tumors where 14% of the population express the HER2 antigen and compared the therapeutic efficacy of HER2 CAR T cells and synthetic antigen treatment (VHH-LNP + VHH CAR). While HER.2 CAR treatment slowed tumor growth, synthetic antigen treatment led to the complete clearance of tumors. Thirty-five days following the initiation of treatment, remaining tumors were dissociated and HER2 expression within tumor cells was measured, showing that tumors treated with HER2 CAR T cells escaped recognition due to HER2 antigen loss, while control groups maintained HER2 expression. These data demonstrate the ability for LNP -mediated delivery of synthetic antigens in combination with synthetic antigen CARs to treat antigen negative or heterogenous tumors.Example 2: Sensitizing solid tumors to CAR-mediated cytotoxicity by lipid nanoparticle delivery of synthetic antigens

[0125] A study was conducted which developed synthetic antigens (syntAgs) to sensitize solid tumors to CAR T cell therapy and bypass tumor antigen discovery. An ideal synthetic antigen would be orthogonal to the human proteome, small and genetically encodable for delivery by viral and nonviral approaches, stably expressed and targetable by single-chain variable fragments (scFvs) for CAR T cell design. Considering these criteria, the study repurposed a xenogeneic protein, the antigen binding fragment of the heavy-chain-only camelid antibodies (VHH), as a synthetic antigen. Its small genetic footprint (-375 bp), thermal stability, protease resistance, and pH stability have facilitated numerous in vivo applications, including as neutralizing agents, fusion proteins for imaging, payload carriers for targeted vaccine delivery, and chimeric antigen receptors. The study delivered VHH as an mRNA- encoded syntAg using lipid nanoparticles (LNPs) and demonstrated that its expression on the surface of tumor cells results in cytotoxicity by aVHH CAR T cells. The study showed that aVHH CAR T cells exhibit low off-target toxicity in cocultures with peripheral blood mononuclear cells (PBMCs), and are well-tolerated in naive C57BL / 6J mice up to 100 days following adoptive transfer by serum chemistry analysis. In multiple syngeneic and xenograft models of cancer bearing VHH-treated tumors, the study showed that the adoptive transfer of aVHH CAR T cells significantly reduced tumor burden, improved survival, induced epitope spread, protected tumor rechallenge, and mitigated antigen escape. This data shows that sensitizing tumors to CAR-mediated cytotoxicity using synthetic antigens could potentiate antitumor immunity and improve responses against solid tumors.Methods

[0126] Statistics and reproducibility. Power analyses were performed using G*Power 3.1.9.6 (HHU) based on effect sizes determined by previous studies. Differences in animal survival (Kaplan-Meier survival curves) were analyzed by two-sided Mantel-Cox test. For statistical analysis of two groups, an unpaired Student’s t-test was applied. For grouped analyses, the study performed a two-way ANOVA. Statistical analysis was performed using GraphPad Prism (Version 10.3.1). Data are presented as mean and s.d. or s.e.m. (error bars) as defined in the legends. Differences were considered significant when the p values were <0.05. Data distribution was assumed to be normal but this was not formally tested. Mice were inoculated with tumors and then randomized prior to allocation to experimental groups. For the remaining studies, randomization was not relevant, as these experiments were conducted on uniform biological materials, such as commercially sourced cell lines. The investigators were not blinded to group allocation during experiments and outcome analysis. Blinding was not feasible for both the in vitro and in vivo studies, as these experiments were conducted by individual investigators who were aware of the experimental groups. Additionally, blinding was not considered relevant, as the data analysis was performed quantitatively (e.g., flow cytometry, ELISA) and did not involve subjective, qualitative assessments. Mice were randomly assigned to experimental groups, but the experiments were not randomized, and the investigators were not blinded to group allocation or outcome assessment. No data were excluded from the analyses. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

[0127] Statistical analysis'. Appropriate statistical analyses were performed using GraphPad Prism (*p<0.05, **p<0.01, *** p<0.001, ****p<0.0001). Central values represent mean and error bars depict s.d. or s.e.m. as defined. Flow cytometry data were analyzed using Flow Jo X. Power analyses were performed using G*Power 3.1.9.6 (HHU).

[0128] Animals'. Six- to twelve-week-old female C57BL / 6J and NSG mice were used. C57BL / 6J mice were purchased from the Jackson Laboratory (000664). B6-HER2 mice were purchased from Jackson Laboratory (010562), bred in-house, and used at 2-6 months of age. Tumor dimensions were measured with calipers in three dimensions and reported as an ellipsoidal volume. Following approved IACUC protocol, a maximal tumor size of 1.5 cm in any direction was not exceeded in any experiment.

[0129] Cell culture and generation of cell lines'. Cell lines used, including their sources, are provided in TABLE 2. HEK293T and MC38 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin / streptomycin. E0771 and SB28 cells were cultured in RPMI-1640 with 10% FBS, 1% penicillin / streptomycin. SB28 cells were maintained in RPMI-1640 supplemented with 10% FBS and 1% penicillin / streptomycin, 1% HEPES, 1% Glutamax, 1% non-essential amino acids, 1% sodium pyruvate, and 0.5 mM 2-Mercaptoethanol. VHH- positive cell lines were generated by lentiviral transduction of tumor cells with the VHH gene driven by the EFla core promoter in a LeGO-C lentiviral backbone. VHH+ cells were single cell sorted using the BD Fusion in the Georgia Institute of Technology’s Cellular Analysis Core using the BD FACSDIVA v8 software (BD Biosciences). Similarly, E0771-Thyl.l and MC38-Thyl.l were generated by lentiviral transduction of the Thy 1.1 gene, single cell sorted, and stably maintained using Blasticidin. All cells were cultured at 37°C in 5% CO2.TABLE 2. Cell lines used.

[0130] Construction of CAR T cells'. REmAb™ Protein Sequencing with WILD™ analysis (Rapid Novor) was performed on the MonoRab™ Rabbit Anti-Camelid VHH Antibody toobtain the anti-VHH scFv sequence. The human CAR is composed of a human CD8a signal peptide, antigen-specific scFv (anti-GCN4 or anti-VHH scFv), CD8a hinge and transmembrane domains, and the 4-1BB and CD3(^ intracellular domains. The human aCD19 CAR (US9499629B2) was kindly provided by Dr. Krishnendu Roy (Georgia Institute of Technology) and includes a aCD19 scFv, CD8a hinge and transmembrane domain, and the 4- 1BB and CD3(^ signaling domains. The human aHER2 CAR (US20180326032A1) was kindly provided by Dr. Saul Priceman (City of Hope) and includes aHER2 scFv, IgG4 hinge and CD8a transmembrane domain, 4-1BB and CD3(^ signaling sequences. The murine CARs are composed of a mouse CD8 signal peptide, antigen-specific scFv (anti-GCN4, anti-VHH, 2173 [anti-human EGFRviii], or Trastuzumab-derived scFv [anti-human HER2]), CD8a hinge and transmembrane domain, and the CD28 and CD3(^ intracellular domains. The CAR constructs co-express a eGFP reporter using the T2A sequence and were generated using custom DNA fragments (Eurofins Genomics). Human CAR constructs were cloned into a lentiviral vector using the BsiWI and Nhel restriction sites while murine CAR constructs were cloned into the pMKO.l retroviral vector using the EcoRI and Notl.

[0131] In vitro transcription of syntAg mRNA: Plasmid templates were linearized with Notl-HF overnight at 37°C, purified the following day by sodium acetate precipitation and subsequently rehydrated with nuclease-free water. IVT was performed using the HiScribe T7 Kit (NEB) following the manufacturer’ s instructions, using complete substitution of uridine for Nl-methylpseudouridine-5’ -triphosphate. The RNA product was treated with DNase for 30 min and purified using lithium chloride precipitation. RNAs were capped using guanylyl transferase and 2’-O-methyltransferase (Aldevron) to generate a Cap-1 structure, purified by lithium chloride precipitation, treated with alkaline phosphatase (NEB), and re-purified. The concentration of the purified mRNA was measured using a Nanodrop and stored at -80°C at 1-4 mg / mL. Purified RNA product was analyzed by gel electrophoresis to ensure purity.

[0132] Lipid Nanoparticle (LNP) Synthesis'. Lipid components were mixed in ethanol according to the molar ratios described in FIGS. 6A-6B and mRNA was diluted in 100 mM pH 5 sodium acetate buffer to a concentration of 0.1 mg / mL. LNPs were synthesized by mixing lipid components and mRNA solutions using NanoAssemblr™ Ignite™ (Precision Nanosystems) at a lipid-to-mRNA mass ratio of 20:1, volumetric ratio of 1 :3, and total flow rate of 12 mL / min. The formulated LNPs were buffer exchanged into PBS by repeated centrifugation using 10 kDa Amicon™ Ultra- 15 Centrifugal Filter Unit at 2000xg to reach at least 300-fold dilution of the formulation buffer. The size of the LNPs was measured using Malvern Zetasizer. LNPs were stored in sucrose (8% w / v) and kept at -80°C until use.

[0133] To quantify the mRNA concentrations and encapsulation efficiency, Invitrogen™ Quant-it™ RiboGreen RNA Assay Kit was used following the manufacturer’s protocol. Samples were analyzed using a fluorescence microplate reader (Ex / Em 485 / 530nm). The mRNA encapsulation efficiency was calculated using this equation:

[0134] Evaluation of syntAg expression kinetics following in vitro transfection'. Expression of mRNA-encoded syntAg constructs were first evaluated by in vitro transfection into target cell lines using the lipoplex jetMESSENGER™ mRNA transfection reagent following manufacturer’s protocol. Expression kinetics were analyzed via flow cytometry by staining for SunTag expression using an anti-GCN4 antibody or for VHH expression using an anti-Camelid VHH antibody. Samples were collected using the Accuri™ C6 Plus flow cytometer and its software (vl.0.27.1) at indicated time points. Subsequent assessment of VHH expression kinetics by LNP-mediated delivery was evaluated by loading VHH mRNA into various LNP formulations and coincubating with tumor cells at indicated doses in 96-well plates with or without 1 pg / mL ApoE in complete media. Expression levels and kinetics were analyzed via flow cytometry using the Northern Lights™ (Cytek) following staining, and analyzed using SpectroFlo (Version 3.2.1).

[0135] In vivo kinetics of expression '. Evaluation of in vivo LNP kinetics was conducted using nLuc or Flue loaded LNP. C57BL6 / J mice were inoculated with 1 x lO6MC38 or 5* 105E0771 tumor cells. When tumors reached -100 mm3, 5 pg of nLuc-LNPs or Fluc-LNPs were injected intratum orally. Luciferase expression was quantified by IVIS imaging at indicated timepoints using appropriate substrates (nLuc using Nano-Glo(R) in vivo substrate; Flue using D-Luciferin, Potassium Salt prepared following manufacturer’s instructions). Four days after injection, indicated cohorts received a second nLuc-LNP or Fluc-LNP (5 pg) injection to evaluate luminescence by IVIS.

[0136] Lenti- and retroviral production'. Retrovirus was made by co-transfection with pCL-Eco and pMKO. l retroviral vectors encoding for murine CARs in HEK293T cells using TransIT-293. Supernatant was collected 48 hrs later, filtered through a 0.45 pm syringe filter, and concentrated using Retro-Concentin Virus Precipitation Solution following the manufacturer’s protocol and immediately used for transduction of primary murine T cells. Lentivirus was produced by co-transfection of lentiviral expression plasmids with psPAX2 andpMD2.G using TransIT-LTl into HEK293T cells. Supernatant was collected after 48 hrs, filtered through a 0.45 pm syringe filter, concentrated using PEG-itTM Virus Precipitation Solution following the manufacturer’s protocol and stored at -80°C until use.

[0137] Primary murine CAR T cell production'. Cells from Pmel-1, P14 and C57BL / 6 mouse spleens were harvested by gently dissociating the tissues using frosted glass slides, then centrifuged at lOOOxg for 5 min and resuspended in lx RBC lysis buffer for 5 min at 4°C. lx PBS was added to quench the lysis reaction, and cells were centrifuged and resuspended in murine T cell media (mTCM; RPMI + 10% FBS + 1% Pen / Strep + lx NEAA + lx Sodium Pyruvate + 50 pM Beta-mercaptoethanol + 100 lU / mL rhIL-2) and passed through a 40 pm cell strainer. Pmel-1 were cultured in the presence of 1 pM human gplOO (Pmel-1). CD3+ cells from C57BL / 6 mice isolated using the StemCell EasySep Mouse T Cell Isolation kit following the manufacturer’s protocol. After isolation, C57BL / 6 CD3+ T cells were resuspended at a concentration of 1 x 106cells / mL in mTCM supplemented with 5 pg / mL aCD28 antibody (BD) and cultured on an aCD3 antibody-coated plate (3 pg / well). Two days after activation, cells were collected, washed, and resuspended at l +I O6cells / mL in concentrated retroviral supernatant supplemented with 100 lU / mL hIL2 and 8 pg / mL polybrene. Spinfection was performed in a U-bottom 96-well plate at 2,000xg for 90 min at 32°C. Transduced cells were maintained at I +I 06cells / mL in complete murine T cell media supplemented with 100 lU / mL hIL-2 and passaged daily until use on Day 5-6. Murine CD 19 CAR expression was evaluated by staining with Alexa Fluor™ 647-F(ab')2 Goat Anti-Mouse IgG at a 1 :800 dilution. For the murine VHH, SunTag, and hHER2 CARs, expression was evaluated by surface staining with biotinylated antigen (biotinylated VHH,; biotinylated GCN4, synthesized in-house 100 pg / mL; biotinylated hHER2 10 pg / mL) followed by a secondary stain with streptavidin-APC at a 1 :500 dilution. For staining, biotinylated SunTag was synthesized on Rink Amide ProTide (LL) resin using CEM Liberty Blue, including Fmoc deprotection in piperidine, amino acid coupling in N,N'-diisopropylcarbodiimide and Oxyma Pure, N-terminal biotinylation by biotin p- nitrophenyl ester in presence of Oxyma Pure. The peptide was cleaved off resin in trifluoroacetic acid, precipitated in diethyl ether, dried overnight, and resuspended in 10 mg / mL in water for storage at -20°C. Additionally, surface expression of CD3, CD4 and CD8 was quantified by flow cytometry after staining T cells with aCD4 and aCD8 antibodies as described above.

[0138] Primary human CAR T cell production '. Peripheral blood was drawn from healthy human donors. PBMCs were isolated using Lymphoprep density gradient medium and SepMate-15mL tube, according to manufacturer’s instructions. CD3+ cells were then isolatedusing the EasySep Human CD3 Positive Selection Kit II and activated using Dynabeads at a 3: 1 bead-to-cell ratio. Activated cells were cultured in complete human T cell media (hTCM; X-vivo 10 , 5% Human AB serum, 10 mM N-acetyl L-Cysteine, 55 uM 2-mercaptoethanol supplemented with 50 U / mL rhIL-2 for 24 hours at 37°C in 5% CO2. Concentrated lentivirus was added to a 24-well suspension culture plate coated with Retronectin according to the manufacturer’s instructions and centrifuged at l,200xg for 90 minutes at 37°C. Following centrifugation, activated human T cells in human T cell media supplemented with 100 units / mL of hIL-2 were added to each well and the plate was spun at l,200xg for 60 minutes at 37°C. Cells were incubated on the virus-coated plate for 24 hours before expansion. 7 days after activation, T cells were supplemented with Dynabeads at a 1 : 1 bead to cell ratio. The beads were removed on Day 9. Cells were maintained at a concentration of 0.7-2x 106cells / mL until use on Day 10-14.

[0139] Biodistribution Study. Organs were harvested 24 hrs following intratumoral injection of nLuc-LNPs and immediately placed on ice cold lx PBS- / - until imaging. Organs were treated with Nano-Gio Live Cell Assay System initially diluted 20x in provided buffer and then further diluted 50x in IxPBS- / - before use. Prior to IVIS imaging, organs were dipped in the diluted substrate for 5 min at 37°C. Excess liquid was removed by tapping the tissue on a KimWipe prior to placing on black construction paper for imaging.

[0140] Liver function analysis'. Naive C57BL6 / J mice received either PBS, untransduced T cells, or 5x 106CAR T cells. 7 days following administration, blood was drawn via the jugular vein and collected in serum separator tubes and sent out for blood chemistry testing at Antech Diagnostics. To assess longitudinal toxicity CAR T cells, peripheral blood samples were collected at each timepoint into BD Microtainer tubes and left undisturbed for 30 minutes before centrifugation at 2500xg for 10 minutes. Serum was stored at -80°C until analysis. Collected samples were thawed and evaluated for AST and ALT levels using AST and ALT ELISA kits following the manufacturer’s instructions.

[0141] PBMC Toxicity Assay: PBMCs were isolated from whole blood using Lymphoprep™ and SepMate™ PBMC Isolation Tubes following manufacturer’s protocol. Isolated PBMCs were cocultured with human aVHH CAR T or aCD19 CAR T cells of the matching donor at 1 : 1 ratio for 48 hours. Supernatant was collected after incubation and assayed for human interferon gamma (IFN-y), tumor necrosis factor-alpha (TNF-a), and interleukin-6 (IL-6) with ELISA kits according to the manufacturer’s protocols.

[0142] In vitro cytotoxicity assays: Tumor cells were transfected in a 96-well plate with 100 ng of either VHH or SunTag mRNA using jetMESSENGER™ transfection reagent kitfollowing manufacturer instructions. For LNP-mediated transfections, tumor cells were treated with 1-100 ng of VHH-LNP. Transfected target cells were then cocultured with either untransduced, aVHH CAR or aSunTag CAR T cells at the indicated effector to target cell ratio. Supernatant was collected after incubation and assayed for IFN-y and cytotoxicity with species- matched IFN-y ELISA Kit or using a lactase dehydrogenase (LDH) release assay, respectively, according to the manufacturer’s instructions.

[0143] Comparison of murine CAR T cells sourced from either Pmel-1 or C57BL6 / J splenocytes was conducted by using MC38-Rluc or MC38-VHH-Rluc. Tumor cells were seeded in 96 well plate at 2* 104cells / well. Untransduced or CAR T cells were added at a 1 : 1 E:T ratio, and cytotoxicity was assessed by a luminescence. To do so, ViviRen™ Live Cell Substrate was added to wells 24 hrs after coculture and luminescence was quantified by IVIS Spectrum CT. The supernatant from the coculture was collected and IFN-y levels were quantified using an IFN-y Mouse ELISA Kit according to the manufacturer’s protocol.

[0144] Validating VHH CAR functionality in vivo against tumors constitutively expressing VHH: C57BL6 / J mice inoculated with either U I O6MC38-VHH, 5x l05E0771-VHH, or 5x l05E0771-wt tumor cells. For E0771 experiments, cells were implanted i.d. in the left mammary fat pad (fourth). For MC38 experiments, cells were implanted s.c. into the left flank. Tumor burden, quantified as 0.52 x length x width x depth, was monitored until average tumor volume was approximately 100 mm3before initiating treatment. Mice were sublethally irradiated with 500 cGy and 5x l06CAR transduced pmel-1 splenocytes were adoptively transferred via tail vein. Recombinant human IL-2 (rhIL-2) was administered intraperitoneally twice daily for 3 days. Mice were classified as complete (CR) or partial responders (PR), or as having progressive disease (PD) or stable disease (SD) based on the RECIST criteria. On Day 18 post ACT, MC38-VHH tumors and lymph nodes were isolated for flow cytometry analysis. For E0771-VHH experiments, CRs were rechallenged 45 days after initial treatment with 5x l05syntAg negative (E0771-wt) tumor cells in the right mammary fat pat (fourth).

[0145] Flow cytometry analysis of tumor infiltrating lymphocytes'. Antibodies used, with vendor information and dilutions, are provided in TABLE 3. Unless otherwise noted, all antibodies were used at a 1 : 100 dilution. Single cell suspensions from spleens and lymph nodes were prepared by homogenizing tissue between the frosted glass slides. Homogenized cells were passed through a 40 pm cell strainer and depleted of red blood cells using lx RBC lysis buffer. Less than 1g of MC38-VHH tumors were dissociated using the Mouse Tumor Dissociation Kit and gentleMACS Dissociator. TILs were isolated from the single cell suspension using a density gradient with Percoll Centrifugation Media and DMEM Media(10% FBS, 1% Pen-strep) at a 44:56 volume ratio. The panel used for staining is as follows: anti-CD8-BV786 (53-6.7), anti-Thyl.l-BV711 (OX-7). Cell viability was assessed by staining with LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit) following manufacturer’s instructions. To detect VHH CAR expression, biotinylated VHH and streptavidin-Alexa Fluor™ 488 were used. Repsl reactive T cells were detected by Repsl pMHC tetramer staining. To ensure signal specificity, samples were stained with Repsl pMHC tetramers separately conjugated to streptavidin-APC and streptavidin-BV421. For surface staining, cells were blocked with anti- Fc receptor anti-CD16 / CD32, and then stained in FACS Buffer (lx DPBS, 2% FBS, 1 mM EDTA, 25 mM HEPES). Fixation was performed using eBioscience Intracellular Fixation & Permeabilization Buffer Set following the manufacturer’s instructions. Counting beads were added to each sample of stained cells prior to analysis by the LSR Fortessa Flow Cytometer (BD).Attorney Docket No. 10034-319W01TABLE 3. Antibodies used.Atorney Docket No. 10034-319W01

[0146] Tetramer production '. For tetramer staining, tetramers were generated from pMHC monomers and streptavidin. Biotinylated Repsl pMHC monomers were diluted in exchange buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.0) at 0.15 mg / mL. APC- or BV421 -conjugated streptavidin was added to the diluted pMHC monomers in 4 small aliquots to achieve a final 4: 1 pMHC to streptavidin molar ratio. Each aliquot was added to the diluted pMHC monomers efvery 5 min for a total of 4 times. After each addition, the pMHC-streptavidin mixture was mixed thoroughly and incubated on ice for 5 min. 15 min after the last addition, the pMHC tetramer was centrifuged at 16,000xg for 1 min, and the supernatant was collected for cell staining at 1 :50 dilution (0.2 pg / sample).

[0147] SyntAg treatment by LNP-mediated delivery of VHH'. C57BL6 / J mice were inoculated with 5xl05E0771-wt tumor cells i.d. in the left mammary fat pad (fourth). When tumors reached -75-150 mm3, mice were sublethally irradiated with 500 cGy and injected intratum orally with 5 pg of Fluc-LNP or VHH-LNP. The same day, 5xl06aVHH CAR T cells were administered intravenously. rhIL-2 was administered intraperitoneally twice daily for 3 days. Alternatively, NSG mice were inoculated with 5xl06MDA-MB-468-wt tumor cells in the left flank. When tumors reached -50-100 mm3, mice were injected intratumorally with 5 pg of either VHH-LNP with 3 mg / mL ApoE4 (Peprotech #350-04) or PBS. The same day, 5xl06aVHH CAR T cells were administered intravenously.

[0148] To evaluate SyntAg treatment of heterogenous tumors, C57BL6 / J mice inoculated with 5xl05SB28 (10% EGFRviii+) tumor cells subcutaneously in the left flank. Prior to the first treatment, mice were sublethally irradiated with 500 cGy and injected intratumorally with a 5 pg of either Fluc-LNP or VHH-LNP. The same day, 5xl06aVHH CAR T cells were administered intravenously. Recombinant human IL-2 (rhIL-2) was administered intraperitoneally twice daily for 3 days. To evaluate tumor expression of VHH, tumors from a subset of mice were harvested 24 hrs post LNP injection, dissociated using a mouse tumor dissociation kit, and stained using anti-VHH-iFluor 647 and LIVE / DEAD Fixable near-IR Dead Cell Stain Kit. Analysis was performed using the Northern Lights Flow Cytometer (Cytek).

[0149] Similarly, to evaluate SyntAg treatment of human tumors, NSG mice were inoculated with 5xl06MDA-MB-468 (15% HER2+) tumor cells resuspended in 100 pL PBS -I- and injected subcutaneously in the left flank. When tumors reached volume -100 mm3, mice were injected intratumorally with a 5 pg of either VHH-LNP with 3 mg / mL ApoE4. The same day, 5xl06aVHH CAR T cells were administered intravenously, repeated as indicated. To evaluate tumor composition post-treatment, tumors were harvested at endpoint (Day 34),dissociated using a human tumor dissociation kit and stained using the following panel: antihuman p2-microglobulin PE, anti-VHH-iFluor 647, and LIVE / DEAD Fixable near-IR Dead Cell Stain Kit. Analysis was performed by the Northern Lights Flow Cytometer (Cytek).Results

[0150] Expression of mRNA-encoded SyntAgs by multiple tumor types'. The study tested two xenogeneic molecules, SunTag and VHH, as synthetic antigens for CAR targeting. SunTag is a repeat of the small 19-aa epitope EELLSKNYHLENEVARLKK of the yeast transcriptional factor GCN4, is orthogonal to the human proteome, and binds to its cognate scFv with high affinity and specificity. VHHs are small (~15 kDa) antigen binding fragments derived from camelid antibodies and are highly stable in vivo. The study designed synthetic antigen constructs with a membrane anchor for surface expression and retention, a linker to provide flexibility, and either a SunTag or VHH antigen domain for CAR T cell recognition (FIG. 2A). The study first compared the CD4 transmembrane (CD4TM) domain to the glycosylphosphatidylinositol (GPI) anchor from the decay accelerating factor (DAF) as membrane anchors for VHH in wildtype cancer cells by surface staining and flow cytometry following mRNA transfection using jetMESSENGER, a commercial lipoplex formulation (FIG. 2B, FIG. 3) Maximum surface expression, as measured by median fluorescence intensity (MFI), was ~ 10-fold higher for GPI-anchored VHH compared to CD4TM-anchored VHH across the three tumor cell lines (FIG. 2C). Moreover, VHH expression was maintained for 5-12 days for the GPI-anchored construct compared to 2-4 days for CD4TM-VHH (FIG. 2C). Due to higher and sustained expression, GPI was selected as the membrane anchor.

[0151] To evaluate SunTag and VHH as antigen domains, the study designed two SunTag constructs, each linked to the GPI anchor by either a lx repeat of the flexible G4S peptide or a 3x repeat of G4S. As peptides are susceptible to enzymatic or chemical degradation, it was hypothesized that fusing SunTag to a protein could enhance its surface expression. Therefore, the study designed a third construct where SunTag was fused to the C-terminus of VHH. All three GPI-anchored SunTag constructs were indistinguishable from background fluorescence by day 3 post transfection in A549 cells (FIG. 2D), demonstrating that neither linker length nor fusion to VHH affected surface expression of SunTag. Moreover, while VHH-linked SunTag was undetectable by day 3 (FIG. 2D, triangles), the VHH linker remained detectable on 96.7% of cells by flow cytometry 5 days post transfection (FIG. 4), showing that VHH surface expression was longer than SunTag. To confirm this, the study further tested VHH expression by E0771, MC38, and A549 tumor lines and found a half-maximal expression of 4.8 d compared to 1.8 days for lxG4S-linked SunTag (FIG. 2D). Altogether, the expression ofGPI-anchored VHH by murine and human cell lines was sustained for at least 5 days (FIG. 2E, FIG. 4). For comparison, the Kb-SIINFEKL pMHC complex has a half-life of ~8 hrs on the surface of MC38 cells (FIG. 5), while the half-life of the HLA-A*02:01-gpl00i54-i62 complex on human immune cells such as dendritic cells, B cells, and monocytes is 1.5-22.5 hrs. Based on the sustained expression of VHH (FIGS. 2A-2E), the VHH synthetic antigen was selected for subsequent validation.

[0152] LNP -mediated mRNA delivery of syntAgs to broad tumor types'. The study next developed a lipid nanoparticle (LNP) formulation for syntAg delivery to tumors. The study evaluated 8 LNP formulations of distinct ionizable lipids, cholesterol, phospholipids, and PEGylated lipids (FIGS. 6A-6B) loaded with GPI-nLuc encoding mRNA (FIG. 7A). The tudy screened each formulation on MDA-MB-468 tumor cells and evaluated luminescent signal by IVIS with and without the addition of ApoE, which adsorb to LNPs to increase their cellular uptake via the ApoE-LDLR pathway (FIG. 7B). Among the LNP formulations tested, transfection with cKK-E12 LNPs resulted in the highest luminescent signal of ~4xl08photons / sec (FIG. 7B). The study expanded this evaluation of cKK-E12 LNPs to deliver VHH mRNA (VHH-LNPs) by testing additional murine (SB28, E0771, MC38 and GL261) and human cancer cells (MDA-MB-468, A549, DU145, and IMR-32) (FIGS. 7C-7D). VHH surface expression was detected on all cell types at the lowest concentration tested of 0.1 ng of encapsulated mRNA per 2>< 104cells (FIG. 7D). The addition of ApoE improved transfection efficiency at doses of 0.1-1 ng in 7 out of 8 cell lines, and at 5-50 ng for DU145 cells. Increasing the VHH-LNP dose up to 100 ng resulted in an expression frequency greater than 84.6% across all cell lines, independent of ApoE, and extended expression for up to 9 days (FIG. 7D, FIG. 8). Taken together, these data demonstrate that cKK-E12 LNPs can effectively deliver syntAg-encoding mRNA for its sustained surface expression on a broad range of tumor types.

[0153] aVHH CAR T cells recognize & kill VHH-expressing tumor cells'. To design a murine CAR to target the VHH syntAg, an anti-VHH scFv sequence was fused to the murine CD8a hinge and transmembrane, CD28 costimulatory, and the CD3(^ signaling domains. Similarly, human aVHH CARs included the anti-VHH scFv, but with human sequences for the CD8a hinge and transmembrane, 4- IBB costimulatory, and CD3(^ signaling domains (FIGS. 9A-9B) The study also designed aSunTag CARs by replacing the VHH-targeting scFv with an anti-GCN4 scFv as a negative control. After viral transduction, both aVHH and aSunTag CAR constructs were found to be stably expressed on the surface of primary monoclonal (Pmel-1) or polyclonal (C57BL6 / J splenocytes) murine T cells (FIGS. 10A-10C) and humanT cells (FIG. 9C). To confirm antigen-dependent activation, murine aVHH CAR T cells were co-incubated with E0771 breast cancer cells transfected with either VHH or SunTag, and significant upregulation of activation markers CD25 and CD69 in greater than 94% of CAR T cells expressing the cognate synthetic antigen was observed (FIG. 11, FIGS. 12A-12C). To assess CAR T cell cytotoxicity, aVHH CAR T cells or control aSunTag CAR T cells produced interferon gamma (IFN-y) at significantly higher levels over wildtype T cells when cocultured with VHH- or SunTag-expressing tumor cells (****p < 0.001), respectively (FIG. 9D). By contrast, no significant elevation was detected when cultured alone or with tumor cells expressing a mismatched synthetic antigen (FIG. 9D). IFN-y levels were statistically identical among engineered murine CAR T cells sourced from either pmel-1 or C57BL6 / J mice (FIG. 12B)

[0154] Next, the study assessed aVHH CAR T cell killing of VHH-expressing tumor cells. At a 2: 1 E:T ratio, no cytolytic activity was observed by untransduced (UTD) murine T cells on either SunTag- or VHH-transfected E0771 tumor cells or by CAR T cells cultured on tumor cells expressing a mismatched synthetic antigen. To determine potential for non-specific toxicity, aVHH and aSunTag CAR T cells were cultured with untransfected or transfected A549 tumor cells at six E:T ratios ranging from 1 : 10 to 20: 1. No cytolytic activity on untransfected, VHH-transfected, SunTag-transfected tumor cells across all E:T ratios tested was observed (FIG. 9F). When aVHH CAR T cells were co-incubated with their cognate synthetic antigen, however, significant increases in cytotoxicity for both murine (****p<0.001, 63.2%) (FIG. 9E, FIG. 12C) and human CAR T cells (****p<0.001) were observed (FIG. 9F). Both murine and human VHH CAR T cells killed target cells more potently than their aSunTag CAR T cell counterparts (FIGS. 9E-9F). Murine VHH CAR T cells killed 16.6% more tumor cells at a 2: 1 E:T ratio (VHH: 63.2% vs. ST : 46.6%) and human VHH CAR T cells showed a 1.7-fold lower E:T ratio required to generate a 50% killing response (EC50: VHH=0.9 vs. STM.5), consistent with earlier data which showed higher secretion of IFN-y by aVHH CAR T cells (FIG. 9D). Collectively, this data shows that tumor cells expressing the VHH synthetic antigen can be recognized and killed by both murine and human CAR T cells.

[0155] aVHH CAR T cells are well tolerated systemically. It was postulated that aVHH CAR T cells would exhibit minimal off-tumor toxicity since its target antigen is not natively found in the mouse or human proteome. To test this, primary murine aVHH CAR T cells were administered intravenously into immunocompetent and naive C57BL / 6J mice (FIG. 13A). Compared to saline controls or mice receiving an equal number of UTD T cells, the study found no significant elevations in serum levels of alanine aminotransferase (ALT) or aspartatetransaminase (AST) (FIG. 13B) up to 100 days post transfer nor did the study find elevations in other serum chemistry values, including total protein, urea nitrogen (BUN), creatinine, phosphorous, and calcium, as measured on Day 7 (FIG. 14A). Moreover, no significant difference was observed in body weight between naive mice receiving saline, UTD T cells, or aVHH CAR T cells (FIG. 14B). By contrast, B cell depletion and upregulation of AST and ALT by murine CAR T cells targeting the endogenous antigens CD 19 and HER2, respectively, was observed, indicative of systemic toxicity (FIGS. 14C-14D). These data demonstrate that murine aVHH CAR T cells were well tolerated systemically, with no appreciable abnormalities in renal and liver function, in body weight, or in B cell counts.

[0156] The study next determined off-tumor toxicity of human aVHH CAR T cells by quantifying in vitro immunogenicity against donor-matched PBMCs, benchmarking their activity against CAR T cells targeting the CD 19 antigen. PBMCs from healthy donors were co-incubated with UTD, aVHH CAR or aCD19 CAR T cells at a 1 : 1 E:T ratio, and the release of inflammatory cytokines IFN-y, TNF-a, and IL-6 after 48 hours was measured (FIGS. 13C- 13D) The co-incubation of PBMCs with aCD19 CAR T cells led to a significant increase in all the measured inflammatory cytokines compared to UTD T cells (*p<0.05, **p<0.01) whereas co-incubation with aVHH CAR T cells did not lead to detectable cytokine secretion, underscoring improved safety of human CAR T cells targeting VHH rather than CD 19.

[0157] aVHH CAR T cells enhance antitumor immunity in solid tumors'. To evaluate the antitumor efficacy of aVHH CAR T cells, the study first compared tumor growth kinetics of E0771 and MC38 tumor cells stably transduced to express VHH and found that VHH expression did not alter tumor growth compared to their wildtype counterparts (FIGS. 15A- 15C). Similarly, since aVHH CAR T cells target a xenogeneic protein not found in murine tissue, it was unclear if the adoptive transfer of aVHH CAR T cells alone would alter tumor growth in the absence of target antigen. To test this, the study compared tumor growth kinetics of wildtype E0771 tumors treated with UTD or aVHH CAR T and found no changes to tumor growth compared to saline-treated mice (FIG. 16). These data show that neither VHH expression nor aVHH CAR T cell treatment alone is sufficient to reduce tumor burden.

[0158] The study compared the anti-tumor efficacy of aVHH CAR T cells against aHER2 CAR T cells as a benchmark (FIG. 17A). MDA-MB-468 breast cancer cells transduced with both the VHH and HER2 antigen (468-HV) were co-incubated with UTD, aVHH CAR, or aHER2 CAR T cells at a 2: 1 E:T ratio. No cytolytic activity by UTD T cells and a significant increase in cytotoxicity by both aVHH (****p<0.001, 99.3%) and aHER2 (****p<0.001, 98.3%) CAR T cells were observed (FIG. 17B). Notably, the potency of aVHH CAR T cellswas indistinguishable from aHER2 CARs. Following adoptive cell transfer in cohorts of mice bearing 468-HV tumors (FIG. 17C), tumor clearance was observed in mice treated with either aHER2 or aVHH CAR T cells (UTD vs. aVHH CAR: ***p<0.001, UTD vs. aHER2 CAR: f f fpO.OOl) but not in control mice treated with UTD T cells, demonstrating that the efficacy of VHH-targeting CARs is comparable to a conventional CAR targeting an endogenous antigen.

[0159] To evaluate the potential for antigen spread, murine aVHH CAR T cells were adoptively transferred into immunocompetent mice bearing syngeneic MC38 colon adenocarcinoma tumors that were stably transduced to express VHH. Compared with control aSunTag CAR T cells, transfer of aVHH CAR T cells significantly reduced tumor burden (FIGS. 18A-18B); within 18 days of adoptive cell transfer, tumor volumes within the control group grew on average 9-fold, whereas those treated with aVHH CAR T cells grew ~2.5-fold. Flow cytometric analysis of the tumor, spleen, and the ipsilateral and contralateral lymph nodes revealed that aVHH CAR T cell treatment increased the number of CD8+ tumor infiltrating lymphocytes (TILs) compared to treatment with control CAR T cells (**p<0.01, FIG. 18C). Additionally, CAR+ CD8 T cells included a higher frequency of the CD8+ TILs within mice receiving aVHH CAR T cells (55% vs. 22%, **p<0.01, FIG. 18C). To determine whether CAR targeting of VHH could prime endogenous immunity against a different tumor antigen, the study quantified the percentage of endogenous T cells reactive against Repsl, an H-2Db- restricted mutant neoepitope in MC38 cells. A significant increase in CAR-negative, Repsl- reactive CD8+ T cells (*p<0.05, FIG. 18C, FIG. 19) was observed in the tumor as well as in the ipsilateral lymph nodes (*p<0.05, FIG. 18D), which was not observed in animals given control CAR T cells (FIG. 18D). These data support that CAR-mediated killing of VHH+ tumors primes endogenous T cells against tumor antigens and promotes epitope spread.

[0160] The study further sought to evaluate the antitumor efficacy of aVHH CAR T cells against triple negative breast cancer (TNBC), which lack estrogen and progesterone receptors (ER / PR) and HER2 to make them unresponsive to endocrine and anti-HER2 therapies. The study tested aVHH CAR T cells in mice bearing E0771 tumors, a TNBC originally isolated from a spontaneous mammary tumor in a female C57BL / 6 mouse. Following adoptive transfer of aVHH CAR T cells (FIG. 18E), 3 of 4 mice treated with aVHH CAR T cells were complete responders for at least 100 days after ACT (CR = 3 / 4, blue traces, FIG. 18F), whereas mice that received control CARs reached endpoint criteria (tumor volume > 1,000 mm3) within 30 days of treatment (CR = 0 / 4, black traces, FIG. 18F). To determine whether treatment could initiate epitope spread and provide protection against the wildtype tumor, either naive mice orcomplete responders were rechallenged with wildtype VHH-negative E0771 tumor cells in the contralateral mammary fat pad 45 days after initial therapy (FIG. 18E). While naive mice grew tumors that reached endpoint in 28-32 days, complete responders rechallenged with VHH- negative E0771 tumors were resistant to tumor growth (****p<0.0001, FIG. 18G). Additionally, survival of rechallenged mice was significantly extended by at least 70 days over mice treated with control CAR T cells (**p<0.01, FIG. 18H).

[0161] SyntAg treatment triggers potent antitumor responses'. The study next sought to explore whether in situ delivery of syntAgs by LNPs could sensitize wildtype tumors to CAR T cell cytotoxicity. The study evaluated the ability of aVHH CAR T cells to kill TNBC tumor cells transfected with VHH-LNPs in vitro (FIG. 20A). At doses up to 100 ng of VHH-LNP, no cytolytic activity by UTD murine or human T cells on E0771 and MDA-MB-468 tumor cells, respectively, was observed (FIG. 20B). However, when aVHH CAR T cells were coincubated with VHH-LNP -transfected target cells, a significant increase in cytotoxicity was observed against both E0771 and MDA-MB-468 cells at doses above 1 ng (E0771 : ****p<0.001, MDA-MB-468: **p<0.01 to ****p<0.001) (FIG. 20B). To validate cKK-E12 LNPs for tumor transfection, LNPs were loaded with mRNA encoding a GPI-anchored nLuc reporter to enable analysis of tissues by luminescent imaging. Twenty-four hours after intratumoral injection, tumors treated with nLuc-LNPs showed an 83 -fold increase in luminescence compared to tumors treated with LNPs loaded with control mRNA (****p<0.001, FIG. 21A). Low levels of luminescent signal was detected in the spleen and tumor draining lymph node (tdLN) whereas negligible signals were detected from the other major organs. The study also measured tumor luminescence following multiple i.t. injections of nLuc-LNPs and found that a second dose 4 days following the initial injection increased the luminescent signal, demonstrating the ability to boost mRNA delivery with repeated injections (**p<0.01, FIG. 21B).

[0162] To validate the expression of VHH by tumors following i.t. injections of VHH- LNPs, injected wildtype tumors were injected intratum orally with 5 pg VHH-LNPs, and the median fluorescence intensity (MFI) of dissociated tumor cells was quantified. The MFI of VHH-LNP-treated tumor cells was 33 -fold over cells obtained from saline-injected tumors (*p<0.05), corresponding to a mean frequency of 26.1% VHH+ tumor cells (FIGS. 20C-20D). Notably, VHH-LNP injection at this dose did not alter tumor growth (FIG. 22A), nor did the administration of aVHH CAR T cells to Fluc-LNP treated mice (FIG. 20E). By contrast, 2 treatment doses of both systemically administered aVHH CAR T cells and an intratumoral injection of VHH-LNPs led to a significant delay in tumor growth in a murine model of TNBC(****p<0.001, FIG. 20E) as well as improvement in median survival from 21-24 days in control cohorts to 35 days in syntAg-treated mice. By increasing the number of syntAg treatments from 2 to 5 doses, a further improvement in median survival to 61 days was observed (FIG. 20F, FIG. 22B). Next, to evaluate LNP -mediated syntAg treatment of a murine model of human TNBC, VHH-LNP was administered, followed by adoptive transfer of human aVHH CAR T cells into MDA-MB-468 tumor bearing NSG mice (FIG. 20G). Control cohorts treated with saline or VHH-LNPs alone grew from ~80 mm3to an average tumor volume of -600 mm358 days after initial treatment, while the tumor burden in mice that received VHH-LNPs and aVHH CAR T cells decreased to 40 mm3 (****p<0.001, FIG. 20H). Taken together, these data demonstrate that LNP-mediated delivery of mRNA-encoded VHH sensitizes wildtype tumors to immune recognition, leading to improved antitumor responses in vivo.

[0163] SyntAg treatment enhances control of heterogeneous tumors'. Unlike certain hematologic malignancies where endogenous antigens like CD 19 and B cell maturation antigen (BCMA) are expressed on most tumor cells, the expression of targetable antigens in solid tumors is highly heterogenous. Targeting endogenous antigens can exert selective pressure on tumors, promoting antigen-negative relapse that is refractory to initial therapy. The study therefore sought to evaluate whether LNP-mediated delivery of the VHH syntAg combined with aVHH CAR T cell treatment could improve therapeutic outcomes over conventional CARs in preclinical models of heterogenous tumors. Prior to in vivo assessment, the study compared the ability of VHH-LNP to transfect both epidermal growth factor receptor variant III (EGFRvIII)+ and EGFRvIII- SB28 tumor cells, a murine glioblastoma cell line characterized by an immunogenicity profile similar to human GBM. Following in vitro transfection with 100 ng of VHH-LNP, high surface expression of VHH was observed as quantified by flow cytometry 18 hours after transfection in both EGFRvIIU- and EGFRvIII- tumor cells (EGFRvIIU- 99.9% VHH+, EGFRvIII- 99.3% VHH+). VHH expression among EGFRvIII-positive and -negative tumors were indistinguishable from one another (FIG. 23A).

[0164] Next, immunocompetent mice were inoculated with a tumor including 10% EGFRvIIU- and 90% EGFRvIII- SB28 cells (FIG. 23B), a heterogenous antigen composition comparable to that of glioblastoma patients eligible for EGFRvIII CAR T cell therapy in a recent clinical study. Once SB28 tumors with heterogenous EGFRvIII composition reached -100 mm3, a 5 pg dose of VHH-LNP was administered intratum orally. Consistent with in vitro transfection, VHH-LNP treatment led to a significant increase in VHH expression on SB28 tumors in vivo (52.0% VHH+, ***p<0.001) (FIGS. 23C-23D), while the same treatment did not alter EGFRvIII expression which remained at 7.8% for both treated and untreated groups.Using murine anti-human EGFRvIII CAR T cells as a benchmark, tumor-bearing mice were treated intratum orally with either saline or 5 pg of VHH-LNP followed by adoptive transfer of either VHH-targeting or EGFRvIII targeting murine CAR T cells once a week for a total of three treatments (FIG. 23E). Compared to untreated mice, a significant increase in survival in groups receiving VHH-LNPs alone or EGFRvIII CAR T cells was not observed (FIG. 23F). All mice treated with EGFRvIII CAR T cells reached endpoint criteria (tumor volume > 1,000 mm3) within 29 days post treatment with a median survival of ~27 days, whereas mice that received both VHH-LNPs and aVHH CAR T cells exhibited significant improvement in survival (**p<0.01) with median survival improved by at least 48 days.

[0165] The study next evaluated syntAg treatment on a heterogenous model of breast cancer. A heterogenous mixture of MDA-MB-468 tumor cells where 15% of the tumor expressed human HER2 was inoculated (FIG. 24A). Following tumor engraftment, mice were treated intratum orally with either saline or 5 pg of VHH-LNP followed by adoptive transfer of either VHH-targeting or HER2 targeting human CAR T cells once a week for a total of three treatments (FIGS. 24B-24C). VHH-LNP treatment alone did not alter tumor growth compared to untreated mice. On the contrary, treatment with VHH-LNPs and aVHH CAR T cells led to a significant decrease in tumor burden 16 days following the initiation of treatment, which continued to decrease until no palpable tumors were detected by day 34. Treatment of the heterogenous tumors with CAR T cells targeting the endogenous HER2 antigen, however, did not clear the tumor. To determine whether antigen escape contributed to the lack of tumor clearance, the study assessed HER2 expression within remaining tumors. Compared to control cohorts which maintained expression of HER2 on the surface of tumor cells (Untreated: 23.8%; VHH-LNP: 22.1%), mice treated with aHER2 CAR T had a significant decrease in the frequency of HER2+ tumor cells to 1.1% (*p<0.05, FIGS. 24D-24E). Collectively, this data indicates that syntAg treatment has the potential to mitigate antigen escape in tumors with heterogenous expression of endogenous antigens.Discussion

[0166] The scarcity of tumor-specific antigens limits the safety and efficacy of CAR T cell therapies for solid tumors. This study developed synthetic antigens to sensitize solid tumors for recognition and elimination by CAR T cells. Unlike tumor-associated antigens, the study designed a synthetic antigen VHH that is orthogonal to the human proteome and has a small genetic footprint for efficient delivery to tumors by LNPs. It was found that a GPI membrane anchor linked to a CAR recognition domain led to stable VHH surface expression by tumor cells following mRNA transfection. To deliver VHH to tumors in vivo, the study tested LNPformulations including distinct ionizable lipids and found that particles formulated with cKK- E12 successfully transfected all tumor cells tested of both mouse and human origin. The study also designed and validated both human and murine aVHH CAR T cells, which showed potent antigen-dependent activation, cytokine production, and cytotoxicity upon recognition of surface-expressed VHH on tumor cells in vitro. Importantly, aVHH CAR T cells exhibited potent antitumor activity in vivo against VHH+ tumors in syngeneic models of colorectal and TNBC, which led to recruitment of endogenous tumor-reactive cells, resistance to tumor rechallenge, and extended survival. The study further demonstrated that LNP-mediated delivery of VHH combined with aVHH CAR T cells led to the recognition and killing of tumors in both syngeneic and xenograft tumor models of glioblastoma and TNBC, which reduced tumor burden, improved survival, and mitigated antigen escape in heterogenous tumors.

[0167] The majority of tumor antigens under evaluation as CAR targets are also expressed at low levels in healthy tissue. Consequently, on-target off-tumor toxicity with CAR T cells remains a primary safety concern including severe respiratory distress, hearing loss, vestibular dysfunction, liver toxicity, and neurotoxicity. As a synthetic antigen derived from camelids, VHH is not expressed by healthy tissue. This data demonstrates that murine aVHH CAR T cells are well tolerated in vivo, as measured by serum chemistry and body weights of non-tumor bearing mice. Moreover, this study shows that the co-incubation of human aVHH CAR T cells with donor-matched PBMCs does not elicit expression of CRS-related cytokines, unlike CAR T cells targeting CD 19. Nonetheless, due to differences in the mouse and human proteome and the limitations of preclinical models, murine studies and in vitro toxicity assays may not fully recapitulate CAR T cell cytotoxicity in humans and thus, as with any new CAR T cell construct under consideration, future studies may be warranted to assess the potential off-tumor activity of aVHH CAR T against healthy human tissue. Notably, however, the compact size, low immunogenicity, and stability of VHHs shown in numerous preclinical studies as well as its FDA-approval as a neutralizing agents against the von Willebrand Factor further support that this class of molecules has the potential for clinical translation.

[0168] In addition to antigen identification, each CAR construct requires independent optimization for parameters such as affinity, linker lengths, and intracellular domains. Streamlining CAR design with a single optimized CAR construct that could be used for multiple types of tumors could accelerate clinical testing. Other strategies to deliver T cell targets to tumor cells including antibody-mediated delivery of immunodominant viral epitopes, intratumoral or systemic delivery of sfGFP using tumor colonizing bacteria (E. coli Nissle1917), intratumoral injection of membrane anchoring FITC, and the local delivery of CD 19 using oncolytic virus (OV), for subsequent treatment with their cognate CAR T cells. In this study, it was observed that CAR T cell clearance of tumors expressing the VHH syntAg primed endogenous T cells against neoantigens, as indicated by the increased number of Repsl tetramer positive T cells in the tumor and draining lymph nodes in the syngeneic MC38 murine model of colon adenocarcinoma. Indication of epitope spread in mice bearing TNBC tumors where complete responders to aVHH CAR T cell therapy were resistant to rechallenge to wildtype tumor cells was also observed, indicating the establishment of anti-tumor immunity against tumor antigens that were not targeted by CAR T cells. It is envisioned that intratumoral delivery of VHH could be used to kickstart antitumor immunity, analogous to in situ vaccination strategies such as intratumoral delivery of TLR agonists, radiation, and cytokines which use the tumor to prime or enhance immune responses. Outside of LNPs, an increasing number of FDA-approved viral (e.g., adeno-associated viruses or the oncolytic virus T-VEC73) and non-viral (e.g., polymeric nanoparticles) gene delivery vehicles are amenable for intratumoral or locoregional delivery. Whereas viral vectors are limited by preexisting or treatment-induced immunity, LNPs can be re-administered to increase the expression of the syntAg where a single dose may not be sufficient to generate durable antitumor responses. Treatment efficacy may also be boosted by using circular RNA or self-amplifying mRNA to extend VHH expression, or by co-delivery of VHH mRNA with adjuvants, cytokines, or costimulatory molecules that promote a proinflammatory tumor microenvironment. While non- viral vectors lack cell-type specificity, non-specific delivery to cells within the tumor microenvironment may prove advantageous, such as by the depletion of immunosuppressive stromal cells. With emerging high throughput approaches, it is also possible to further optimize LNP formulations for improved delivery and tumor-tropism in vivo. Altogether, this study provide support for the design and use of syntAgs to promote CAR-mediated tumor cytotoxicity for solid tumors with otherwise limited antigen targets.

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Nature Protocols 1, 1120-1132 (2006).90 US9499629B291 US20180326032A1SEQUENCESSEQ ID NO: 1 atgaaatgggtcacatttatatctctgctcttccttttctcttcagcctacagccaggtacagttgcaggagtccggaggtggtctggtaca accaggtgggtccctcagattgtcttgtgcagctagtggctttacgctcgactactattatatcgggtggtttcggcaagcaccgggtaaa gagagggaggctgttagctgtatcagcggctcttcagggtccacgtattaccctgacagtgttaaagggagatttaccatatcccgcgat aacgcaaagaacactgtgtacttgcagatgaatagcctgaagcccgaggacacagccgtttactactgtgccacgattcgctcctcttc atggggaggatgcgttcattacgggatggattactggggcaaaggcactcaggtgacggttagctctggaggcgggggcagccacg agaccacccccaacaaggggagcgggaccacgtccggcacaactagactgctttccggccatacatgctttacacttactgggctgct ggggactcttgtaactatggggctcctcacatgaSEQ ID NO: 2MKWVTFISLLFLFSSAYSQVQLQESGGGLVQPGGSLRLSCAASGFTLDYYYIGWFRQ APGKEREAVSCISGSSGSTYYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC ATIRSSSWGGCVHYGMDYWGKGTQVTVSSGGGGSHETTPNKGSGTTSGTTRLLSGH TCFTLTGLLGTLVTMGLLT

Claims

What is claimed is:

1. A nanoparticle comprising: one or more lipids comprising an ionizable lipid, a cholesterol, a phospholipid, a PEGylated lipid, or any combination thereof; and an encapsulated nucleic acid encoding a target antigen.

2. The nanoparticle of claim 1, wherein the ionizable lipid comprises cKK-E12, SM102, MC3, Lipid-5, Alc-0315, LP01, Lipid A9, or any combination thereof.

3. The nanoparticle of any one of claims 1-2, wherein the nanoparticle comprises from about 30 mol% to about 60 mol% of the ionizable lipid.

4. The nanoparticle of any one of claims 1-3, wherein the nanoparticle comprises from about 35 mol% to about 50 mol% of the cholesterol.

5. The nanoparticle of any one of claims 1-4, wherein the phospholipid comprises DSPC, DOPE, or any combination thereof.

6. The nanoparticle of any one of claims 1-5, wherein the nanoparticle comprises from about 5 mol% to about 20 mol% of the phospholipid.

7. The nanoparticle of any one of claims 1-6, wherein the PEGylated lipid comprises ALC-0159, DMG-PEG, DSPE-PEG, PEG14-2000, or any combination thereof.

8. The nanoparticle of any one of claims 1-7, wherein the nanoparticle comprises from about 0.5 mol% to about 5 mol% of the PEGylated lipid.

9. The nanoparticle of any one of claims 1-8, wherein a ratio of PEGylated lipid to total lipids is from about 0.5:1 to about 2:1.

10. The nanoparticle of any one of claims 1-9, wherein the nanoparticle is a lipid nanoparticle, a liposome, or a polymeric nanoparticle.

11. The nanoparticle of any one of claims 1-10, wherein the encapsulated nucleic acid comprises mRNA.

12. The nanoparticle of any one of claims 1-11, wherein the target antigen is not naturally occurring.

13. The nanoparticle of claim 12, wherein the target antigen comprises a detectable moiety.

14. The nanoparticle of any one of claims 12-13, wherein the target antigen comprises 80% similarity or more to SEQ ID NO: 2.

15. A method of engineering a cell to express a target antigen, the method comprising exposing the cell to a nanoparticle, the nanoparticle comprising: one or more lipids comprising an ionizable lipid, a cholesterol, a phospholipid, a PEGylated lipid, or any combination thereof; and an encapsulated nucleic acid encoding a target antigen.

16. The method of claim 15, wherein the cell is cancerous or abnormal.

17. The method of claim 16, wherein the cell is a breast cancer cell, a colon cancer cell, a brain cancer cell, a lung cancer cell, a prostate cancer cell, or a melanoma cell.

18. The method of any one of claims 15-17, wherein the nanoparticle is the nanoparticle of any one of claims 1-14.

19. The method of any one of claims 15-18, wherein the cell is human.

20. The method of any one of claims 15-19, wherein the method is carried out in vitro, in vivo, or ex vivo.

21. An engineered cell produced by the method of any one of claims 15-20.

22. A method of treating and / or preventing a cancer or solid tumor in a subject, the method comprising: a) providing to the subject a nanoparticle comprising: one or more lipids comprising an ionizable lipid, a cholesterol, a phospholipid, a PEGylated lipid, or any combination thereof; and an encapsulated nucleic acid encoding a target antigen; and b) subsequently providing to the subject an immunotherapy.

23. The method of claim 22, wherein the nanoparticle is the nanoparticle of any one of claims 1-14.

24. The method of any one of claims 22-23, wherein the immunotherapy comprises an immune checkpoint inhibitor.

25. The method of claim 24, wherein the immune checkpoint inhibitor comprises an antibody that blocks PD-1, PD-L1, CTLA-4, PD-L2 IDO, B7-H3, B7-H4, T cell immunoreceptor with Ig and ITIM domains (TIGIT) B- and T-lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), TIM3, or LAG-3.

26. The method of any one of claims 22-25, wherein the immunotherapy comprises an adoptive cell therapy.

27. The method of claim 26, wherein the adoptive cell therapy comprises the administration of tumor infiltrating lymphocytes (TILs), marrow infiltrating lymphocytes (NILs), chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) (CAR NK) cells, CAR macrophage (CARMACs), or CAR NK T cells expressing a CAR, T cell receptor, or NK receptor which recognizes the target antigen.

28. The method of any one of claims 22-27, wherein step a) comprises administering from about 0.05 mg / kg to about 1 mg / kg nanoparticle.

29. The method of any one of claims 22-28, wherein the nanoparticle is administered intratum orally.

30. The method of any one of claims 22-29, wherein, after step a), at least about 15% of the cancer or tumor cells express the target antigen.

31. The method of any one of claims 22-30, wherein step b) is carried out from about 24 hours to about 72 hours after step a).

32. The method of any one of claims 22-31, wherein the cancer or solid tumor is breast cancer, colon cancer, brain cancer, lung cancer, prostate cancer, or melanoma.

33. A synthetic antigen comprising 80% similarity or more to SEQ ID NO: 2.

34. The synthetic antigen of claim 33, wherein the synthetic antigen comprises 90% similarity or more to SEQ ID NO: 2.

35. The synthetic antigen of any one of claims 33-34, wherein the synthetic antigen comprises SEQ ID NO: 2.

36. The synthetic antigen of any one of claims 33-35, wherein the synthetic antigen consists of SEQ ID NO: 2.

Images