WO2025087266A1

WO2025087266A1 - Immunomodulatory mrna cassettes, and uses thereof

Info

Publication number: WO2025087266A1
Application number: PCT/CN2024/126641
Authority: WO
Inventors: Hongya HAN; Bo YING; Jinjuan MAO; Jingshu MA; Jijun Yuan
Original assignee: Abogen Biosciences (Shanghai) Co., Ltd.
Priority date: 2023-10-23
Filing date: 2024-10-23
Publication date: 2025-05-01

Abstract

The present invention relates to nucleic acid vaccines that specifically express immunomodulatory polypeptides to increase efficacy of target antigen presentation by antigen presenting cells (APCs) to lymphocytes. The present invention further relates to uses of the vaccines for the preparation of compositions, methods of stimulating an immune response, methods of treating or preventing disease (e.g., cancer, viral infections), and kits comprising the vaccines.

Description

IMMUNOMODULATORY MRNA CASSETTES, AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to International Patent Application No.: PCT/CN2023/125895 filed on October 23, 2023, the content of which is incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a computer readable Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “FPCH24160376P. 241022. SEQUENCE LISTING. xml” , was created on October 22, 2024, and is 345 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to nucleic acid vaccine, in particular mRNA-based vaccines against, e.g., cancers or infectious diseases. Further provided herein are pharmaceutical compositions comprising the vaccines, methods of preventing, treating or managing diseases, e.g., viral infection or cancer burden, using the vaccines, and kits comprising the vaccines.

BACKGROUND OF THE INVENTION

RNA molecules produced by in vitro transcription offer the potential for valuable and much needed pharmaceutical compositions by allowing the delivery of genetic material to patient cells capable of being translated into proteins of interest. Most mRNA cancer vaccines are therapeutic vaccines, and therefore are designed to boost cancer-specific immune cell activity. Subsequently, these therapeutic mRNA vaccines are designed to target tumor-associated antigen (TAA) expressing or tumor-specific antigen (TSA) harboring cancer cells theoretically to clear cancer cells or reduce tumor burden.

These mRNA vaccines are selectively taken up into the cytoplasm of dendritic cells (DCs) , where the mRNA encoding the antigens is translated. The resulting antigen peptide then is degraded in proteasomes to be processed into MHC-restricted antigen epitopes and then transported to the endoplasmic reticulum (ER) to loaded onto MHC class I molecules. This process results in the formation of stable antigen peptide-MHC complexes on the surface of DCs for presentation of antigens to T cells and subsequent antigen-specific T cell activation and expansion. Moreover, a fraction of the expressed protein could be sorted by antigen-presenting cells (APC) and then localized into endosome, where could be loaded on MHC class II molecules and later engaged by CD4⁺ T cell.

Optimal antigen specific CD4⁺ and CD8⁺ T cell response requires sufficient antigen presentation. However, the current therapeutic mRNA vaccines display limited clinical efficacy, in line with low levels of DC-presented antigen to T cells that result relatively weak CD8+ T cell response and further differentiate CD4+ T cell into T helper 2 cells. In this regards, existing methods are limited by the immunogenicity of in vitro transcribed RNA molecules, resulting in inefficient expression of the protein of interest within transfected cells. Provided herein are methods and compositions that address such and other needs.

SUMMARY OF THE INVENTION

The present invention provides a novel mRNA-based vaccine that elicits immunity against diseases expressing target antigens, for example HPV+ cancers.

In one aspect, provided herein is an immunomodulating nucleic acid system encoding a target antigen, wherein the system comprises a first coding sequence encoding a helper T cell epitope and a second coding sequence encoding an immunomodulator, wherein the helper T cell epitope and the immunomodulator are configured to modulate immunogenicity of the target antigen upon co-expression with the target antigen. In some embodiments, the helper T cell epitope and the immunomodulator are configured to enhance immunogenicity of the target antigen upon co-expression with the target antigen.

In some embodiments, the system further comprises a third coding sequence encoding a trafficking peptide configured for intracellularly trafficking the target antigen towards proteosome upon expression.

In some embodiments, the system further comprises a fourth coding sequence encoding a signal peptide.

In some embodiments, the system further comprises a fifth coding sequence encoding a ubiquitin peptide.

In some embodiments, the first coding sequence encodes a fusion protein comprising the helper T cell epitope and the target antigen.

In some embodiments, the second coding sequence encodes a fusion protein comprising the immunomodulator and the target antigen; optionally wherein the immunomodulator is positioned N-terminal to the target antigen or is positioned C-terminal to the target antigen in the fusion protein.

In some embodiments, the third coding sequence encodes a fusion protein comprising the trafficking peptide and the target antigen; optionally wherein the trafficking peptide is positioned C-terminal to the target antigen in the fusion protein.

In some embodiments, the fourth coding sequence encodes a fusion protein comprising the signal peptide and the target antigen; optionally wherein the signal peptide is positioned N-terminal to the target antigen in the fusion protein.

In some embodiments, the fifth coding sequence encodes a fusion protein comprising the ubiquitin peptide and the target antigen; optionally wherein the ubiquitin peptide is positioned N-terminal to the target antigen in the fusion protein; optionally wherein fusion protein further comprises a spacer peptide positioned in between the ubiquitin peptide and the target antigen in the fusion protein; optionally the spacer peptide is at least about 25 amino acids in length.

In some embodiments, at least one of the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences are in a first nucleic acid molecule.

In some embodiments of the immunomodulating nucleic acid system, (a) the second coding sequence is in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulatory; (b) the first coding sequence and the second coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator and the target antigen; (c) the first coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the trafficking peptide and the target antigen; (d) the first coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the signal peptide and the target antigen; (e) the first coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the ubiquitin peptide and the target antigen; (f) the second coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the trafficking peptide and the target antigen; (g) the second coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the signal peptide, and the target antigen; (h) the second coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the ubiquitin peptide and the target antigen; (i) the third coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the trafficking peptide, the signal peptide and the target antigen; (j) the third coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the trafficking peptide, the ubiquitin peptide and the target antigen; (k) the fourth coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the signal peptide, the ubiquitin peptide and the target antigen; (l) the first coding sequence, the second coding sequence, and the third coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, and the target antigen; (m) the first coding sequence, the second coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the signal peptide, and the target antigen; (n) the first coding sequence, the second coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the ubiquitin peptide, and the target antigen; (o) the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the trafficking peptide, the signal peptide, and the target antigen; (p) the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the target antigen; (q) the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the trafficking peptide, the signal peptide, the ubiquitin peptide, and the target antigen; (r) the first coding sequence, the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the target antigen; (s) the first coding sequence, the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the target antigen; or (t) the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the ubiquitin peptide, and the target antigen.

In some embodiments, at least one of the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences is in a second nucleic acid molecule, and wherein the first nucleic acid molecule and the second nucleic acid molecule are different molecules.

In some embodiments, the second nucleic acid molecule does not encode the target antigen.

In some embodiments, the first nucleic acid molecule encodes the fusion protein comprising the target antigen; wherein the fusion protein does not comprise the immunomodulator; and wherein the second nucleic acid molecule comprises the second coding sequence encoding the immunomodulator.

In some embodiments, at least the second coding sequence is in the first nucleic acid molecule encoding the fusion protein comprising at least the immunomodulator and the target antigen; and wherein the first nucleic acid molecule further comprises a means for producing the immunomodulator and the target antigen as separate proteins or peptides.

In some embodiments, the means for producing the immunomodulator and the target antigen as separate proteins or peptides comprises a sixth coding sequence encoding a cleavable linker in between the second coding sequence and a coding sequence for the target antigen in the first nucleic acid.

In some embodiments, the cleavable linker is a 2A peptide, selected from the group consisting of P2A, F2A, T2A, and E2A. In some embodiments, the P2A peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 129.

In some embodiments, the helper T cell epitope is a universal CD4 epitope. In some embodiments, the helper T cell epitope is an epitope of tetanus and diphtheria toxoids. In some embodiments, the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE. In some embodiments, the first coding sequence encoding the helper T cell epitope comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72-75 and 171. In some embodiments, the first coding sequence encodes the helper T cell epitope that is a pan DR-binding epitope (PADRE) , and wherein the first coding sequence comprises the nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence of SEQ ID NO: 75 or 171. In some embodiments, the PADRE comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 128.

In some embodiments, the immunomodulator is selected from the group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, and any combination thereof. In some embodiments, wherein the immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING.

In some embodiments, the immunomodulating nucleic acid system comprises (a) the first coding sequence encodes a fusion protein comprising the target antigen and a helper T cell epitope, and the second coding sequence encodes an immunomodulator, wherein the immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING.

In some embodiments, the GM-CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) . In some embodiments, the GM-CSF is a full-length GM-CSF polypeptide. In some embodiments, the full-length GM-CSF polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 108 or 181. In some embodiments, the GM-CSF comprises a truncated GM-CSF or a mutant GM-CSF comprising an amino acid sequence comprising one or more variations compared to the amino acid sequence set forth in SEQ ID NO: 108 or 181, and wherein the truncated GM-CSF or mutant GM-CSF is capable of stimulating macrophage differentiation and proliferation, and/or activating antigen presenting cells (APCs) . In some embodiments, the STING is human STING (hSTING) (V155M) . In some embodiments, the STING comprises a polypeptide comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence of SEQ ID NO: 107.

In some embodiments, the trafficking peptide is derived from one or more polypeptides selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.

In some embodiments, the trafficking peptide is MHC class I trafficking domain. In some embodiments, the trafficking peptide is MHC class I trafficking domain or LAMP3 transmembrane domain (LAMP3 TM). In some embodiments, the third coding sequence encoding the trafficking peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 76-80, 83-86, 172, and 201. In some embodiments, the MHC class I trafficking domain comprises the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 119.

In some embodiments, the fourth coding sequence encoding the signal peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 64-71, 163-164, and 199. In some embodiments, the signal peptide comprises the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequences set forth in SEQ ID NO: 113.

In some embodiments, the ubiquitin peptide comprises a naturally-existing ubiquitin peptide or a functional derivative thereof. In some embodiments, the fifth coding sequence encoding the ubiquitin peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence set forth in SEQ ID NO: 81. In some embodiments, the ubiquitin peptide comprises the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequences set forth in SEQ ID NO: 120.

In some embodiments, the target antigen is selected from the group consisting of an HPV antigen, EGFR, KRAS, an HCC antigen, a portion thereof, and any combination thereof.

In some embodiments, the first nucleic acid molecule is DNA or RNA. In some embodiments, the second nucleic acid molecule is DNA or RNA. In some embodiments, the RNA is mRNA, self-amplifying RNA, or circular RNA. In some embodiments, the one or more coding sequences are codon optimized. In some embodiments, the one or more coding sequences are in one or more mRNA molecule.

In some embodiments, the mRNA molecule further comprises a 5’ untranslated region (UTR) , a 3’ UTR, or both a 5’ UTR and a 3’ UTR. In some embodiments, the 5’ UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 198. In some embodiments, the 3’ UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the mRNA molecule further comprises a 5’ Cap. In some embodiments, the mRNA molecule further comprises a poly (A) sequence. In some embodiments, the poly (A) sequence has a length of about 50 nucleotides or longer.

In some embodiments, the first nucleic acid molecule comprises a chemical modification. In some embodiments, the second nucleic acid molecule comprises a chemical modification. In some embodiments, the chemical modification comprises pseudouridine. In some embodiments, the pseudouridine is 1-methylpseudouridine.

In some embodiments, the coding sequence encoding the fusion protein comprises a nucleic acid sequence having a least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to:

nucleotides 72-1, 436 of SEQ ID NO: 2;

nucleotides 72-1, 523 of SEQ ID NO: 3;

nucleotides 72-2, 423 of SEQ ID NO: 4;

nucleotides 72-1, 709 of SEQ ID NO: 5;

nucleotides 72-1, 304 of SEQ ID NO: 6;

nucleotides 72-1, 295 of SEQ ID NO: 7;

nucleotides 72-2, 396 of SEQ ID NO: 8;

nucleotides 72-1, 220 of SEQ ID NO: 10;

nucleotides 72-1, 208 of SEQ ID NO: 11;

nucleotides 72-1, 178 of SEQ ID NO: 12;

nucleotides 72-1, 175 of SEQ ID NO: 13;

nucleotides 72-1, 178 of SEQ ID NO: 14;

nucleotides 72-1, 781 of SEQ ID NO: 156;

nucleotides 72-1, 781 of SEQ ID NO: 157;

nucleotides 72-1, 781 of SEQ ID NO: 158;

nucleotides 72-1, 790 of SEQ ID NO: 161; or

nucleotides 72-1, 790 of SEQ ID NO: 162.

In some embodiments, the first nucleic acid molecule encoding the fusion protein comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8, 10 to 14, 156 to 158, 161, and 162.

In some embodiments, the first nucleic acid molecule encoding the fusion protein comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8, 10 to14, 156 to 158, 161, and 162.

In some embodiments, the first nucleic acid molecule is in a first vector. In some embodiments, the second nucleic acid molecule is a second vector.

In a related aspect, provided herein is also a vector system comprising the nucleic acid molecules described herein. In some embodiments, the vector system comprises a vector comprising the nucleic acid molecule according to the present disclosure. In some embodiments, the vector system comprises two or more vectors comprising two or more nucleic acid molecules of the present disclosure.

In another related aspect, provided herein is also a polypeptide encoded by the nucleic acid molecule of the immunomodulating nucleic acid system according to the present disclosure.

In another related aspect, provided herein is also composition comprising the immunomodulating nucleic acid system or the vector system or the polypeptide of the present disclosure, and a pharmaceutically acceptable carrier.

In some embodiments, the immunomodulating nucleic acid system or the vector system of the present disclosure is formulated in a lipid nanoparticle (LNP) . In some embodiments, the LNP comprises a cationic lipid. In some embodiments, the LNP comprises a phospholipid. In some embodiments, the LNP comprises a sterol. In some embodiments, the LNP comprises a polymer conjugated lipid.

In some embodiments, the LNP comprises: (a) about 30%to about 55%cationic lipid, (b) about 5%to about 40%phospholipid, (c) about 20%to about 50%sterol, and (d) a polymer conjugated lipid.

In a related aspect of the present disclosure, provided herein is also a method of stimulating an immune response against a heterologous antigen in a subject, comprising administering to the subject an effective amount of the composition according to the present disclosure. In some embodiments, the amount of composition is effective to induce cytotoxic and/or helper T lymphocyte activity in the individual. In some embodiments, the amount of the composition is effective to induce production of antibodies in the individual.

In some embodiments according to any of the methods described above, at least two doses of the composition are administered to the individual. In some embodiments, the at least two doses are administered at least one week apart. In some embodiments, the subject is human. In some embodiments, at least two doses of the composition are administered to the individual. In some embodiments, the at least two doses are administered at least one week apart. In some embodiments, the individual is human.

The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1B provide schematic depictions of exemplary mRNA vaccine constructs. FIG. 1A provides a generalized schematic of an exemplary mRNA expression cassette, wherein the mRNA cassette includes a target antigen domain, immunomodulator domain, signal peptide (SP) and MHC class I trafficking domains, and pan-CD4 epitope domains. The mRNA cassettes may lack SP, MHC class I trafficking domains, and/or immunomodulator domains, or include alternate trafficking peptides. FIG. 1B depicts a schematic overview of exemplary mRNA cassettes ABO-01 through ABO-14. Mut, mutant; UTR, untranslated region; GS linker, glycine and serine linker sequence; SP, signal peptide; P2, P16, P2 and P16 domains of tetanus toxoid; P2A, P2A self-cleavage linker domain; PADRE, pan DR-binding epitope; mGM-CSF, murine granulocyte-macrophage colony-stimulating factor; LAMP1, lysosome-associated membrane protein 1; LAMP3, lysosome-associated membrane protein 3; LAMP3-TM, lysosome-associated membrane protein 3 transmembrane domain; pp65, cytomegalovirus peptide sequence 65; HLA-E, human leukocyte antigen E; hSTING, human stimulator of interferon genes; HLA-DMB, human leukocyte antigen DM beta chain.

FIGs. 2A-2B demonstrate splenic T cell response in mice immunized with mRNA constructs ABO-04 and ABO-05, which both include an immunomodulator domain, compared to the control construct ABO-02, which lacks the immunomodulator domain. FIG. 2A shows light microscope images of murine splenic IFN-γ+ activated T cells assayed using ELISpot three days after final inoculation. FIG. 2B shows quantification of IFN-γ+ spots produced in response to vaccination with mRNA constructs ABO-01 through ABO-08 and PBS sham vaccination. **, p<0.01; ***p<0.001; PBS, phosphate buffered saline.

FIGs. 3A-3B show the impact of including immunomodulator domains in exemplary mRNA expression cassettes in vivo (e.g., ABO-04 and ABO-05 versus control construct ABO-01) . FIG. 3A provides a schematic overview of the tumor vaccination experimental design. TC-1 tumor cells were implanted into C57BL/6 mice, and once tumors reached approximately 6.0 mm³, mice were vaccinated with exemplary mRNA constructs or PBS sham-vaccination on days 0, 7, and 14, and tumor growth was measured over time. FIGs. 3B-3E show tumor growth of PBS sham vaccine recipients (FIG. 3B) , 10 μg ABO-01 (FIG. 3C), 10 μg ABO-04 (FIG. 3D) , and 10 μg ABO-05 (FIG. 3E) . CR, complete response; TGI, tumor growth inhibition; i.m., intramuscular injection.

FIG. 4 shows the immunogenicity of exemplary pan CD4+ T-helper (T_H) epitopes on murine T cells isolated from peripheral blood (e.g., TT_827-841pDT_331–350, TT_632–651, P16, and pan DR-binding epitope (PADRE) ) relative to P2kP16) . Donor-derived peripheral blood mononuclear cells (PBMCs) were plated and stimulated with 15 μM of pan CD4+ T_H epitope peptides or 10 μg/mL conA as positive control. The cells then were tested for the number of IFN-γ+ T cells using an ELISpot assay. IFN-γ+ spots were normalized to antigen control to quantify fold-change in immunogenicity from peptide exposure. conA, concanavalin A.

FIGs. 5A-5B demonstrate splenic T cell response in mice immunized with mRNA constructs ABO-09 through ABO-14, wherein the MHC class I trafficking domain has been replaced with alternative trafficking domains, compared to the control construct ABO-10 that includes the MHC class I trafficking domain. FIG. 5A shows light microscope images of murine splenic IFN-γ+ activated T cells assayed using ELISpot three days after final inoculation. FIG. 5B shows quantification of IFN-γ+ spots produced in response to vaccination with mRNA constructs ABO-09 through ABO-14 and PBS sham vaccination. *, p<0.05.

FIGs. 6A-6B provide schematic depictions of exemplary mRNA vaccine constructs. FIG. 6A provides a generalized schematic of an exemplary mRNA expression cassette, wherein the mRNA cassette includes an HPV target antigen domain, an immunomodulator domain, a signal peptide (SP) domain, an MHC class I trafficking domain, and a pan-CD4 epitope domain. FIG. 6B depicts a schematic overview of exemplary mRNA cassettes ABO-15, ABO-16, ABO-17, ABO-20, and ABO-21. UTR, untranslated region; SP, signal peptide; PADRE, pan DR-binding epitope; mGM-CSF, murine granulocyte-macrophage colony-stimulating factor; hGM-CSF, human granulocyte-macrophage colony-stimulating factor; Cap, 5’ cap; Poly (A) , polyadenylated tail.

FIGs. 7A-7B demonstrate protein expression of the HPV16 E7 peptide in cells transfected with the exemplary mRNA vaccine constructs ABO-15 through ABO-17, wherein the constructs may contain pseudouridine (Ψ) modification. Human expi293F cells were transfected with either 4μg (FIG. 7A) or 1μg (FIG. 7B) of one of the mRNA vaccine constructs ABO-15 through ABO-17 using lipofectamine 2000 and cultured for 24 hours. After incubation, protein was extracted from total cell lysates and used for Western blotting to detect the E7 peptide and normalize to the housekeeping control α-tubulin protein levels.

FIG. 8 demonstrates secretion of murine GM-CSF (mGM-CSF) from cells transfected with the exemplary mRNA vaccine constructs ABO-15 through ABO-17 containing pseudouridinylation (Ψ) modification. Human expi293F cells were transfected with either 4μg or 1μg mRNA vaccine constructs ABO-15 through ABO-17 using lipofectamine 2000 and cultured for 24 hours. After incubation, supernatant was collected and measured for mGM-CSF levels using ELISA.

FIG. 9 demonstrates secretion of human GM-CSF (hGM-CSF) from cells transfected with the exemplary mRNA vaccine constructs ABO-20 and ABO-21 containing pseudouridinylation (Ψ) modification. Human expi293F cells were transfected with either 4μg or 1μg mRNA vaccine constructs ABO-20 and ABO-21 using lipofectamine 2000 and cultured for 24 hours. After incubation, supernatant was collected and measured for hGM-CSF levels using ELISA.

FIGs. 10A and 10B show the impact of including various elements, including the mouse GM-CSF (mGM-CSF) immunomodulator domain, the PADRE helper epitope, the MHC class I trafficking domain (MITD) in exemplary mRNA expression cassettes in vivo (e.g., ABO-05 and ABO-15 versus control construct ABO-01) . FIG. 10A provides a schematic overview of the tumor vaccination experimental design. TC-1 tumor cells were implanted into C57BL/6 mice, and once tumors reached approximately 6.0 mm³, mice were treated with intramuscular injection (i.m. ) of exemplary mRNA constructs or PBS, or sham-vaccination on days 0, 7, and 14, respectively, and tumor growth was measured over time. FIG. 10B shows tumor volume in mice received PBS, sham vaccine (NST) , 5 μg ABO-05, or 5 μg ABO-15 up to 35 days after the first injection.

FIG. 11 provides schematic depictions of exemplary mRNA vaccine constructs. As shown are generalized schematic illustrations of four exemplary mRNA expression cassettes, ABO-24, ABO-25, and ABO-26. UTR, untranslated region; SP, signal peptide; PADRE, pan DR-binding epitope; mGM-CSF, murine granulocyte-macrophage colony-stimulating factor; Cap, 5’ cap; Poly (A) , polyadenylated tail, p2A, cleavable peptide; MITD, MHC class I trafficking domain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel isolated nucleic acids (e.g., an mRNA) comprising a coding sequence encoding a target antigen, a trafficking peptide, a helper T cell epitope, and/or an immunomodulator. Compositions comprising such isolated nucleic acids or polypeptides encoded thereby act to treat, reduce severity, ameliorate symptoms, vaccinate, prophylactically immunize, prevent onset, or elicit an immune response in an individual having or being at risk of having a disease such as a cancer (e.g., an HPV+ cancer) .

After extensive investigation, inventors of the present application discovered that the isolated nucleic acids described herein have several unexpected advantages compared to other vaccine constructs. Vaccination with the composition comprising the isolated nucleic acid described herein reduced tumor burden. When provided after the initiation of tumor growth, vaccination reduced or eliminated tumors. Compared to therapeutic cancer vaccines lacking the trafficking peptide and/or immunomodulator, and optionally the helper T cell epitope, the composition comprising the isolated nucleic acid described herein contributed to: i) improved immunogenicity of antigen-specific cytotoxic T cell response; and ii) improved nucleotide-encoded antigen processing and presentation internally in antigen presenting cells. Therefore, this novel nucleic acid vaccine elicits target antigenic memory to prevent relapse or re-introduction of target antigen-expressing cancers.

Accordingly, in one aspect, the present invention provides an isolated nucleic acid (e.g., an mRNA) comprising a coding sequence encoding a fusion protein comprising a target antigen and a trafficking peptide, wherein the trafficking peptide is derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor. In another aspect, the present invention provides an isolated nucleic acid (e.g., an mRNA) comprising: 1) a coding sequence encoding a fusion protein comprising a target antigen and a helper T cell epitope, and 2) a coding sequence encoding an immunomodulator, wherein the immunomodulator comprises GM-CSF and/or STING. In some embodiments, the isolated nucleic acid is DNA or RNA (e.g., mRNA) . In some embodiments, the isolated nucleic acid is an mRNA, and the mRNA comprises a nucleic acid sequence encoding a signal peptide, a 5’ untranslated region (UTR) , and/or a 3’ UTR. In some embodiments, the isolated nucleic acid further comprises an immunomodulator, e.g., mGM-CSF or hSTING, and/or a helper T cell epitope, e.g., PADRE. In some embodiments, the isolated nucleic acid further comprises an enhancement component, e.g., a ubiquitin peptide that optionally further comprises a C-terminal extension peptide.

In another aspect, there is provided a composition (e.g., a pharmaceutical composition) comprising the isolated nucleic acid described herein, wherein the isolated nucleic acid is optionally formulated in a lipid nanoparticle (LNP) . The compositions (e.g., pharmaceutical compositions) comprising an mRNA, optionally formulated in an LNP, may be administered to an individual as a vaccine. The fusion protein encoded by the isolated nucleic acid (e.g., mRNA) is expressed in vitro and in vivo and induces tumor-specific killing. Therefore, the composition (e.g., pharmaceutical composition) may be useful in a method of treating a cancer, wherein the cancer expressing one or more target antigens, for example but not limited to an HPV+, EGFR+, KRAS+, and/or HCC+ cancer, in an individual (e.g., a human) .

Also provided are compositions and kits comprising any of the isolated nucleic acid vaccines described herein, methods of preparing any of the isolated nucleic acid vaccines and the accompanying host cells described herein, and methods of use thereof for treating, preventing, vaccinating, or otherwise ameliorating diseases such as cancers (e.g., HPV+, EGFR+, KRAS+, and/or HCC+ cancers) .

I. Definitions

The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009) ; Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001) ; Maniatis et al., Molecular Cloning: A Laboratory Manual (1982) ; DNA Cloning: A Practical Approach, vol. I&II (D. Glover, ed. ) ; Oligonucleotide Synthesis (N. Gait, ed., 1984) ; Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985) ; Transcription and Translation (B. Hames & S. Higgins, eds., 1984) ; Animal Cell Culture (R. Freshney, ed., 1986) ; Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of infection and/or tumorigenesis in an individual or cell being treated prophylactically or during the course of clinical pathology. Desirable effects of treatment include ameliorating or reducing cancer and symptoms thereof, and eliciting an immune response to induce the amelioration or reduction of cancer, and symptoms thereof. For example, an individual is successfully “treated” if one or more symptoms associated with the cancer are prevented, mitigated, or eliminated, including, but not limited to, increasing the quality of life of those suffering from the cancer, decreasing the dose of other medications required to treat the pathology, and/or prolonging survival of individuals affected by the pathology.

As used herein, an “effective amount” refers to an amount of an agent or drug effective to vaccinate against or treat a cancer in a subject. The "therapeutically effective amount" can vary depending on the compound or the cell, the disease and its severity and the age, weight, etc., of the subject to be treated.

As used herein, an “individual” or a “subject” refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

The term “vaccine” is used in the broadest sense and specifically covers any biological preparation that provides active acquired immunity to a particular pathology.

As used herein, the term “vaccinate” refers to clinical intervention designed to administer a therapeutically effective amount of a vaccine to an individual in need thereof in order to prevent, prophylactically immunize, reduce severity, elicit an immune response, or treat an infection in an individual who has been or may be exposed to a pathogen such as viruses. For example, an individual who is effectively vaccinated may not contract or may contract only a mild pathology caused by an infection, and any associated symptoms including cancer, compared to an individual who is not vaccinated.

As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN^TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

As used herein, “percent (%) nucleic acid sequence identity” and “homology” with respect to a nucleic acid, DNA, or RNA sequence are defined as the percentage of nucleic acid nucleotides in a candidate sequence that are identical with the nucleic acid nucleotides in the specific DNA or RNA sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN^TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The term “cell” includes the primary subject cell and its progeny.

It will be understood by one of ordinary skill in the art that uracil and thymine can both be represented by ‘t’ , instead of ‘u’ for uracil and ‘t’ for thymine; in the context of a ribonucleic acid, it will be understood that ‘t’ is used to represent uracil unless otherwise indicated.

Unless otherwise indicated, the term "alkyl" by itself or as part of another term refers to a substituted or unsubstituted straight chain or branched, saturated or unsaturated hydrocarbon having the indicated number of carbon atoms (e.g., “-C₁-C₈ alkyl” or “-C₁-C₁₀” alkyl refer to an alkyl group having from 1 to 8 or 1 to 10 carbon atoms, respectively) . When the number of carbon atoms is not indicated, the alkyl group has from 1 to 32 carbon atoms. Representative straight chain “-C₁-C₈ alkyl” groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; while branched –C₃-C₈ alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; unsaturated -C₂-C₈ alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1 pentenyl, -2 pentenyl, -3-methyl-1-butenyl, -2 methyl-2-butenyl, -2, 3 dimethyl-2-butenyl, -1-hexyl, 2-hexyl, -3-hexyl, -acetylenyl, -propynyl, -1 butynyl, -2 butynyl, -1 pentynyl, -2 pentynyl and -3 methyl 1 butynyl. Sometimes an alkyl group is unsubstituted. An alkyl group can be substituted with one or more groups. In other aspects, an alkyl group will be saturated.

Unless otherwise indicated, "alkylene, " by itself of as part of another term, refers to a substituted or unsubstituted saturated, branched or straight chain or cyclic hydrocarbon radical of the stated number of carbon atoms, typically 1-10 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to: methylene (-CH₂-) , 1, 2-ethylene (-CH₂CH₂-) , 1, 3-propylene (-CH₂CH₂CH₂-) , 1, 4-butylene (-CH₂CH₂CH₂CH₂-) , and the like. In preferred aspects, an alkylene is a branched or straight chain hydrocarbon (i.e., it is not a cyclic hydrocarbon) .

Unless otherwise indicated, "aryl, " by itself or as part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of the stated number of carbon atoms, typically 6-20 carbon atoms, derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as “Ar” . Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is a phenyl group.

Unless otherwise indicated, an “arylene, ” by itself or as part of another term, is an aryl group as defined above which has two covalent bonds (i.e., it is divalent) and can be in the ortho, meta, or para orientations as shown in the following structures, with phenyl as the exemplary group:

as shown.

Unless otherwise indicated, a “heterocyclyl” or “heterocycle” by itself or as part of another term, refers to a monovalent substituted or unsubstituted aromatic or non-aromatic monocyclic or bicyclic ring system having the specified numbers of annular atoms with one to four heteroatom ring members independently selected from N, O, P or S, and derived by removal of one hydrogen atom from a ring atom of a parent ring system. One or more N, C or S atoms in the heterocycle can be oxidized. The ring that includes the heteroatom can be aromatic or nonaromatic. Heterocycles in which all the ring atoms are involved in aromaticity are referred to as heteroaryls and otherwise are referred to heterocycloalkyl.

Unless otherwise noted, the heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. As such a heteroaryl may be bonded through an aromatic carbon of its aromatic ring system, referred to as a C-linked heteroaryl, or through a non-double-bonded N atom (i.e., not =N-) in its aromatic ring system, which is referred to as an N-linked heteroaryl. Thus, nitrogen-containing heterocycles may be C-linked or N-linked and include pyrrole moieties, such as pyrrol-1-yl (N-linked) and pyrrol-3-yl (C-linked) , and imidazole moieties such as imidazol-1-yl and imidazol-3-yl (both N-linked) , and imidazol-2-yl, imidazol-4-yl and imidazol-5-yl moieties (all of which are C-linked) .

Unless otherwise indicated, a “heteroaryl, ” is an aromatic heterocycle in which the specified number denotes the total number of annular atoms of the cyclic ring system of the heterocycle. Representative examples of a heterocycle include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene) , furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, and isoxazolyl.

When explicitly given, the size of the ring system of a heterocycle or heteroaryl is indicated by the total number of atoms in the ring. For example, designation as a 5-or 6-membered heteroaryl indicates the total number or aromatic atoms (i.e., 5 or 6) in the heteroaromatic ring system of the heteroaryl but does not imply the number of aromatic heteroatoms or aromatic carbons in that ring system. Fused heteroaryls are explicitly stated or implied by context as such and are typically indicated by the number of aromatic atoms in each aromatic ring that are fused together to make up the fused heteroaromatic ring system. For example, a 5, 6-membered heteroaryl is an aromatic 5-membered ring fused to an aromatic 6-membered ring in which one or both rings have aromatic heteroatom (s) or where a heteroatom is shared between the two rings.

A heterocycle fused to an aryl or heteroaryl such that the heterocycle remains non-aromatic and is part of a larger structure through attachment with the non-aromatic portion of the fused ring system is an example of an optionally substituted heterocycle in which the heterocycle is substituted by ring fusion with the aryl or heteroaryl. Likewise, an aryl or heteroaryl fused to heterocycle or carbocycle that is part of a larger structure through attachment with the aromatic portion of the fused ring system is an example of an optionally substituted aryl or heterocycle in which the aryl or heterocycle is substituted by ring fusion with the heterocycle or carbocycle.

Unless otherwise indicated, “heterocyclylene” by itself or as part of another term, refers to a heterocyclic defined above wherein one of the hydrogen atoms of the heterocycle is replaced with a bond (i.e., it is divalent) . Unless otherwise indicated, a “heteroarylene, ” by itself or as part of another term, refers to a heteroaryl group defined above wherein one of the heteroaryl group’s hydrogen atoms is replaced with a bond (i.e., it is divalent) .

"Optionally substituted" means the groups recited are unsubstituted, or have one or more hydrogen atoms, typically one, each independently replaced with a substituent. Typical substituents include, but are not limited to a -X, -R’, -OH, -OR’, -SR’, , -N (R’) ₂, -N (R’) ₃, =NR’, -CX₃, -CN, -NO₂, -NR’C (=O) R’, -C (=O) R’, -C (=O) N (R’) ₂, -S (=O) ₂R’, -S (=O) ₂NR^’, -S (=O) R’, -OP (=O) (OR’) ₂, -P (=O) (OR’) ₂, -PO₃ ⁼, PO₃H₂, -C (=O) R’, -C (=S) R’, -CO₂R’, -CO₂ ^-, -C (=S) OR’, -C (=O) SR’, -C (=S) SR’, -C (=O) N (R’) ₂, -C (=S) N (R) ’ ₂, and -C (=NR) N (R’) ₂, where each X is independently selected from the group consisting of a halogen: -F, -Cl, -Br, and -I; and wherein each R’ is independently selected from the group consisting of -H, -C₁-C₂₀ alkyl, -C₆-C₂₀ aryl, and -C₃-C₁₄ heterocycle.

More typically substituents are selected from the group consisting of -X, -R’, -OH, -OR’, -SR’, -N (R’) ₂, -N (R’) ₃, =NR’, -NR’C (=O) R’, -C (=O) R’, -C (=O) N (R’) ₂, -S (=O) ₂R’, -S (=O) ₂NR’, -S (=O) R’, -C (=O) R’, -C (=S) R’, -C (=O) N (R’) ₂, -C (=S) N (R’) ₂, and -C (=NR) N (R’) ₂, wherein each X is independently selected from the group consisting of –F and -Cl, or are selected from the group consisting of -X, -R’, -OH, -OR’, -N (R’) ₂, -N (R’) ₃, -NR’C (=O) R’, -C (=O) N (R’) ₂, -S (=O) ₂R’, -S (=O) ₂NR’, -S (=O) R’, -C (=O) R’, -C (=O) N (R’) ₂, and -C (=NR) N (R’) ₂, wherein each X is –F; and wherein each R’ is independently selected from the group consisting of hydrogen, -C₁-C₂₀ alkyl, -C₆-C₂₀ aryl, and -C₃-C₁₄ heterocycle.

In some aspects, an alkyl substituent is selected from the group consisting -N (R’) ₂, -N (R’) ₃ and -C (=NR) N (R’) ₂, wherein R’ is selected from the group consisting of hydrogen and -C₁-C₂₀ alkyl. In other aspects, alkyl is substituted with a series of ethyleneoxy moieties to define a PEG Unit. Alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety as described above may also be similarly substituted.

It is understood that embodiments of the invention described herein include “consisting” and/or “consisting essentially of” embodiments.

The term “modulate” as used herein encompasses change in nature of quantity, including for example, to enhance, increase, stimulate, reduce, suppress, or inhibit.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X” .

As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

The term “about X-Y” used herein has the same meaning as “about X to about Y. ”

As used herein and in the appended claims, the singular forms “a, ” “or, ” and “the” include plural referents unless the context clearly dictates otherwise.

II. Nucleic Acid Based Vaccine Constructs

Nucleic Acid Vaccine Constructs

In one aspect, provided herein is an immunomodulating nucleic acid system (e.g., DNA or RNA based system) comprising one or more nucleic acid molecules encoding for a target antigen and one or more peptide-based elements configured for modulating, such as enhancing, immunogenicity of the target antigen upon co-expression with the target antigen.

In some embodiments, the immunomodulating nucleic acid system is configured for enhancing a T cell mediated immune response against the target antigen. In some embodiments, the immunomodulating nucleic acid system comprises a first coding sequence encoding a helper T cell epitope. In some embodiments, the immunomodulating nucleic acid system is configured for enhancing a B cell mediated immune response against the target antigen.

In some embodiments, the immunomodulating nucleic acid system comprises a second coding sequence encoding a protein that modulates an immune response against the target antigen, and such protein, functional fragment or functional derivative thereof is herein referred to as an “immunomodulator. ” In some embodiments, the immunomodulator stimulates an immune response against the target antigen. In some embodiments, the immunomodulator inhibits an immune response against the target antigen. In some embodiments, the immunomodulator enhance T cell mediated immune response against the target antigen. In some embodiments, the immunomodulator enhance B cell mediated immune response against the target antigen.

In some embodiments, the immunomodulating nucleic acid system comprises a third coding sequence encoding a trafficking peptide. In some embodiments, the trafficking peptide is configured for intracellularly transporting the target antigen towards proteosome upon expression. In some embodiments, the trafficking peptide is configured for intracellularly transporting the target antigen towards endosomes or lysosomes upon expression.

In some embodiments, the immunomodulating nucleic acid system comprises a fourth coding sequence encoding a signal peptide.

In some embodiments, the immunomodulating nucleic acid system comprises a fifth coding sequence encoding a ubiquitin domain. In some embodiments, the ubiquitin peptide is configured for marking the target antigen for intracellular degradation through the ubiquitin pathway.

In some embodiments, the immunomodulating nucleic acid system comprises a sixth and/or additional coding sequences encoding one or more peptide linkers connecting one or more immunomodulating peptides provided herein with the target antigen as a fusion protein. In some embodiments, the peptide linker is a spacer peptide. In some embodiments, the spacer peptide is configured to determine a suitable distance between the ubiquitin peptide and the target antigen, thereby marking the target antigen for efficient degradation.

In some embodiments, it is desirable to provide the target antigen and one or more of the immunomodulating peptides as separate protein or peptide-based entities. For example, in some embodiments, the target antigen is configured to be transported to one intracellular compartment for post-translational processing and the immunomodulating peptide is configured for transportation to a different population of cells to stimulate immune response of such cell population. Hence, in some embodiments, the peptide linker is a cleavable linker configured to be cleaved to release multiple domains or portions of a fusion protein as separate peptides upon expression of the fusion protein.

In some embodiments, the peptide linker is not configured for intracellular cleavage. For example, in some embodiments, the peptide linker is configured to provide a connection of suitable flexibility and length between two functional domains of a fusion protein. Hence, in some embodiments, the peptide linker is not cleavable.

In some embodiments, the immunomodulatory and target antigen (or target antigen linked to functional elements, such as MITD) expressed by one nucleic acid molecule are not included in the same fusion protein.

According to the present disclosure, the one or more coding sequences encoding the target antigen and the additional functional peptides can be arranged in a single expression cassette in the same nucleic acid molecule, or as multiple expression cassettes in at least two different nucleic acid molecules, as long as the functional peptides as provided herein can be co-expressed with the target antigen and are capable of enhancing immunogenicity of the target antigen as intended, upon co-expression with the target antigen. In some embodiments, the immunomodulating nucleic acid system comprises one isolated nucleic acid molecule (e.g., DNA or RNA) encoding a fusion protein comprising a target antigen and one or more of the functional peptides as provided herein. In some embodiments, the immunomodulating nucleic acid system comprises a vector containing the nucleic acid molecule. In some embodiments, the immunomodulating nucleic acid system comprises at least two isolated nucleic acid molecules (e.g., DNA or RNA) collectively encoding a target antigen and one or more functional peptides as provided herein, respectively. In some embodiments, the immunomodulating nucleic acid system comprises at least two vectors each containing one of the at least two nucleic acid molecules.

In some embodiments, the present invention provides an isolated nucleic acid (e.g., DNA or RNA) comprising a coding sequence encoding a target antigen. In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a helper T cell epitope. In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising the target antigen and the helper T cell epitope. In some embodiment, the T cell epitope is configured for enhancing a T cell mediated immune response towards the target antigen. In some embodiments, the helper T cell epitope described herein is a universal CD4 epitope. In some embodiments, the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827- ₈₄₁, pDT_331-350, TT_632-651, and PADRE. In some embodiments, the isolated nucleic acid comprises a coding sequence for helper T cell epitope which comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72-75 and 171. In some embodiments, the helper T cell epitope is PADRE.

In alternative embodiments, the isolated nucleic acid molecule comprises two coding sequences encoding the target antigen and the helper T cell epitope as separate polypeptides, respectively. In specific embodiments one polypeptide comprises (a) the target antigen, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. Each of the elements (a) the target antigen, (b) the helper T cell epitope can be selected independently from those described in Section II (A) (Exemplary Target Antigens) and Section II (C) (Helper T Cell Epitope) .

In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a trafficking peptide. In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising the target antigen and the trafficking peptide. In alternative embodiments, the isolated nucleic acid molecule comprises at least two coding sequences encoding the target antigen and the trafficking peptide as separate polypeptides, respectively.

In one aspect, the present invention provides an isolated nucleic acid (e.g., DNA or RNA) comprising a coding sequence encoding a fusion protein comprising a target antigen and a trafficking peptide, wherein the trafficking peptide is derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor. In some embodiments, the trafficking peptide is positioned C-terminal to the target antigen in the fusion protein. In some embodiments, the trafficking peptide is positioned N-terminal to the target antigen in the fusion protein. In some embodiments, the fusion protein further comprises a helper T cell epitope (such as an epitope of tetanus and diphtheria toxoids, for example PADRE) , optionally positioned between the target antigen and the trafficking peptide.

In specific embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide. In alternative specific embodiments, the isolated nucleic acid molecule comprises multiple coding sequences collectively encoding (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide in at least two separate polypeptides. In specific embodiments one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In alternative specific embodiments, both of the at least two polypeptides comprise the target antigen. Each of the elements (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide can select independently from those described in Section II (A) (Exemplary Target Antigens) , Section II (B) (Trafficking Peptide) , and Section II (C) (Helper T Cell Epitope) of the present disclosure.

In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the trafficking peptide, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In specific embodiments, one polypeptide comprises (a) the target antigen and (c) the trafficking domain, while the other polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide.

In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding an immunomodulator peptide. In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising the target antigen and the immunomodulator. In alternative embodiments, the isolated nucleic acid molecule comprises at least two coding sequences encoding the target antigen and the immunomodulator as separate polypeptides, respectively. In some embodiments, the immunomodulator is configured for enhancing immune response against the target antigen. In some embodiments, the immunomodulator is configured for maintaining an immune response against the target antigen. In some embodiments, the immunomodulator is configured for reducing an immune response against the target antigen.

In some embodiments, the isolated nucleic acid molecule further comprises a coding sequence encoding an immunomodulator, such as GM-CSF and/or STING, optionally connected with the fusion protein with a cleavable linker (e.g., a P2A linker) . In some embodiments, the fusion protein further comprises a signal peptide (such as a signal peptide derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor) .

In specific embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator. In alternative specific embodiments, the isolated nucleic acid molecule comprises multiple coding sequences collectively encoding (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator in at least two separate polypeptides. In specific embodiments, one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In alternative specific embodiments, both of the at least two polypeptides comprise the target antigen. Each of the elements (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator can be selected independently from those described in Section II (A) (Exemplary Target Antigens) , Section II (C) (Helper T Cell Epitope) and Section II (D) (Immunomodulator) of the present disclosure.

In specific embodiments, one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (c) the immunomodulator and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (c) the immunomodulator and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope, and (c) the immunomodulator, and does not contain the target antigen.

In alternative specific embodiments, both of the at least two polypeptides comprise the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, while the other polypeptide comprises (a) the target antigen, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (a) the target antigen and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (a) the target antigen, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen and (b) the helper T cell epitope. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen and (b) the helper T cell epitope. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen and (c) the immunomodulator.

In specific embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising (a) the target antigen, (b) the helper T cell epitope, (c) the immunomodulator, and (d) the trafficking peptide. In alternative specific embodiments, the isolated nucleic acid molecule comprises multiple coding sequences collectively encoding (a) the target antigen, (b) the helper T cell epitope, (c) the immunomodulator, and (d) the trafficking peptide in at least two separate polypeptides. In specific embodiments, one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In alternative embodiments, both of the at least two polypeptides comprise the target antigen. Each of the elements (a) the target antigen, (b) the helper T cell epitope, (c) the immunomodulator, and (d) the trafficking peptide can select independently from those described in Section II (A) (Exemplary Target Antigens) , Section II (B) (Trafficking Peptide) , Section II (C) (Helper T Cell Epitope) and Section II (D) (Immunomodulator) of the present disclosure.

In specific embodiments, one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (d) the trafficking peptide, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope, while the other polypeptide comprises (c) the immunomodulator and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (c) the immunomodulator and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen.

In alternative embodiments, both of the at least two polypeptides comprise the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (d) the trafficking peptide, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator.

In some embodiments, the isolated nucleic acid further comprises an enhancement component, e.g., a ubiquitin peptide that optionally further comprises a C-terminal extension peptide. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA, e.g., an mRNA, a self-amplifying RNA, or a circular RNA. The enhancement element and features of the RNA molecule can be selected independently from Sections (II) E (Enhancement Component) and Section II (I) (mRNA features) of the present disclosure.

In some embodiments, the one or more polypeptides encoded by the present immunomodulating nucleic acid system further comprises a signal peptide. Signal peptides that can be used in connection with the present disclosure can be selected from any signal peptides described in Section II (F) (Signal Peptide) of the present disclosure.

In specific embodiments, the immunomodulating nucleic acid system comprises an isolated nucleic acid (e.g., DNA or RNA) comprising: 1) a coding sequence encoding a fusion protein comprising a target antigen and a helper T cell epitope (such as an epitope of tetanus and diphtheria toxoids, for example PADRE) , and 2) a coding sequence encoding an immunomodulator, wherein the immunomodulator comprises GM-CSF and/or STING. In some embodiments, the isolated nucleic acid is DNA or RNA (e.g., mRNA) . In some embodiments, the helper T cell epitope is positioned C-terminal to the target antigen in the fusion protein. In some embodiments, the immunomodulator is connected with the fusion protein with a cleavable linker (e.g., a P2A linker) . In some embodiments, the fusion protein further comprises a trafficking peptide (such as a trafficking peptide derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor) . In some embodiments, the fusion protein further comprises a signal peptide (such as a signal peptide derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor) . In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA, e.g., an mRNA, a self-amplifying RNA, or a circular RNA. In some embodiments, the isolated nucleic acid further comprises an enhancement component, e.g., a ubiquitin peptide that optionally further comprises a C-terminal extension peptide.

In some embodiments, the coding sequence encoding the trafficking peptide described herein comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 76-80, 83-86, 172, and 201. In some embodiments, the trafficking peptide is derived from MHC class I or LAMP3. In some embodiments, the trafficking peptide comprises MHC class I trafficking domain (MITD) . In some embodiments, the coding sequence further encodes a signal peptide described herein comprising a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 64-71, 163-164, and 199.

In some embodiments, the helper T cell epitope described herein is a universal CD4 epitope. In some embodiments, the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE. In some embodiments, the isolated nucleic acid comprises a coding sequence for helper T cell epitope which comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72-75 and 171. In some embodiments, the helper T cell epitope is PADRE.

In some embodiments, the fusion protein further comprises an enhancement component. In some embodiments, the enhancement component comprises a ubiquitin peptide. In some embodiments, the ubiquitin peptide comprises an amino acid sequence set forth in SEQ ID NO: 120. In some embodiments, the ubiquitin peptide further comprises a C-terminal extension peptide. In some embodiments, the C-terminal extension peptide is at least about 25 amino acids long. In some embodiments, the C-terminal extension peptide comprises an amino acid sequence set forth in SEQ ID NO: 121. In some embodiments, the enhancement component is N-terminal to the target antigen.

In specific embodiments, the immunomodulating nucleic acid system comprises a nucleic acid (e.g., DNR or RNA) encoding a fusion protein. In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a helper T cell epitope (e.g., PADRE) , a trafficking peptide (e.g., MHC class I trafficking domain; MITD) , a cleavable linker (e.g., p2A) and a immunomodulator (e.g., STING or GM-CSF) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking domain; MITD) , a helper T cell epitope (e.g., PADRE) , a cleavable linker (e.g., p2A) and a immunomodulator (e.g., STING or GM-CSF) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a ubiquitin peptide fused to a target antigen. In specific embodiments, the ubiquitin peptide is connected to the target antigen by a spacer having at least 25 amino acids.

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a helper T cell epitope (e.g., P2, P16) , a trafficking peptide (e.g., MHC class I trafficking domain; MITD) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking domain; MITD) , and a helper T cell epitope (e.g., P2, P16) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a ubiquitin peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking domain; MITD) . In specific embodiments, the ubiquitin peptide is connected with the target antigen by a spacer peptide having at least 25 amino acids.

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking domain; MITD) , a cleavable linker (e.g., p2A) and a immunomodulator (e.g., STING or GM-CSF) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a helper T cell epitope (e.g., pp65) , and a trafficking peptide (e.g., MHC class I trafficking domain; MITD) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking domain; MITD) , and a helper T cell epitope (e.g., pp65) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a helper T cell epitope (e.g., PADRE) , and a trafficking peptide (e.g., MHC class I trafficking domain; MITD) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking domain; MITD) , and a helper T cell epitope (e.g., PADRE) ,

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide (e.g., LAMP3 signal peptide) , a target antigen, a helper T cell epitope (e.g., p2p16) , and a trafficking peptide (e.g., LAMP3 transmembrane domain) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide (e.g., LAMP3 signal peptide) , a target antigen, a trafficking peptide (e.g., LAMP3 transmembrane domain) , and a helper T cell epitope (e.g., p2p16) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., MHC class I trafficking domain) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., HLA-E trafficking domain or trafficking signal) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., LAMP1 trafficking domain or trafficking signal) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., LAMP3 trafficking domain or trafficking signal) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., HLA-DMB trafficking domain or trafficking signal) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a target antigen, a helper T cell epitope (e.g., PADRE) , a cleavable linker (e.g., P2A) and an immunomodulator (e.g., GM-CSF) .

In some embodiments the isolated nucleic acid is an mRNA. In some embodiments the mRNA further comprises a 5’ untranslated region (UTR) . In some embodiments, the 5’ UTR has at least 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 198. In some embodiments, the mRNA further comprises a 3’ untranslated region (UTR) . In some embodiments, the 3’ UTR has at least 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the mRNA further comprises a poly (A) sequence. In some embodiments, the poly (A) sequence that has a length of about 50 nucleotides or longer. In some embodiments, the mRNA further comprises a 5’ cap.

In some embodiments, the isolated nucleic acid (e.g., mRNA) comprises a chemical modification. In some embodiments, the chemical modification comprises pseudouridine, optionally 1-methylpseudouridine.

A. Exemplary Target Antigens

The present invention provides an isolated nucleic acid, for example an mRNA construct, comprising a coding sequence for a target antigen. The target antigen can comprise any one or more antigens known in the art to be associated with pathology, e.g., associated with a cancer. For example, the isolated nucleic acid can include a coding sequence for any one or more of the target antigens of MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA9, MAGEA10, MAGEA11, MAGEA12, BAGE, BAGE2, BAGE3, BAGE4, BAGE5, MAGEB1, MAGEB2, MAGEB5, MAGEB6, MAGEB3, MAGEB4, GAGE1, GAGE2A, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GAGE 8, SSX1, SSX2, SSX2b, SSX3, SSX4, CTAG1B, LAGE-lb, CTAG2, MAGEC1, MAGEC3, SYCP1, BRDT, MAGEC2, SPANXA1, SPANXB1, SPANXC, SPANXD, SPANXN1, SPANXN2, SPANXN3, SPANXN4, SPANXN5, XAGE1D, XAGE1C, XAGE1B, XAGE1, XAGE2, XAGE3, XAGE-3b, XAGE-4/RP 11-167P23.2, XAGE5, DDX43, SAGE1, ADAM2, PAGE5, CT16.2, PAGE1, PAGE2, PAGE2B, PAGE3, PAGE4, LIPI, VENTXP1, IL13RA2, TSP50, CTAGE1, CTAGE-2, CTAGE5, SPA17, ACRBP, CSAG1, CSAG2, DSCR8, MMAlb, DDX53, CTCFL, LUZP4, CASC5, TFDP3, JARID1B, LDHC, MORC1, DKKL1, SPOl l, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A2, CT45A3, CT45A4, CT45A5, CT45A6, HORMAD1, HORMAD2, CT47A1, CT47A2, CT47A3, CT47A4, CT47A5, CT47A6, CT47A7, CT47A8, CT47A9, CT47A10, CT47A11, CT47B 1, SLC06A1, TAG, LEMD1, HSPB9, CCDC110, ZNF165, SPACA3, Cxorf48, THEG, ACTL8, NLRP4, COX6B2, LOC348120, CCDC33, LOC196993, PASD1, LOC647107, TULP2, CT66/AA884595, PRSS54, RBM46, CT69/BC040308, CT70/BI818097, SPINLW1, TSSK6, ADAM29, CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA10, HIWI, MIWI, PIWI, PIWIL2, ARMC3, AKAP3, Cxorf6l, PBK, C2lorf99, OIP5, CEP290, CABYR, SPAG9, MPHOSPH1, ROPN1, PLAC1, CALR3, PRM1, PRM2, CAGE1, TTK, LY6K, IMP-3, AKAP4, DPPA2, KIAA0100, DCAF12, SEMG1, POTED, POTEE, POTEA, POTEG, POTEB, POTEC, POTEH, GOLGAGL2 FA, CDCA1, PEPP2, OTOA, CCDC62, GPATCH2, CEP55, FAM46D, TEX14, CTNNA2, FAM133A, LOC130576, ANKRD45, ELOVL4, IGSF11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PTPN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATA19, ODF1, ODF2, ODF3, ODF4, ATAD2, ZNF645, MCAK, SPAG1, SPAG6, SPAG8, SPAG17, FBX039, RGS22, cyclin Al, Cl5orf60, CCDC83, TEKT5, NR6A1, TMPRSS 12, TPPP2, PRSS55, DMRT1, ED AG, NDR, DNAJB8, CSAG3B, CTAG1A, GAGE12B, GAGE12C, GAGE 12D, GAGE12E, GAGE12F, GAGE12G, GAGE12H, GAGE 121, GAGE12J, GAGE 13, LOC728137, MAGEA2B, MAGEA9B/LOC728269, NXF2B, SPANXA2, SPANXB2, SPANXE, SSX4B, SSX5, SSX6, SSX7, SSX9, TSPY1D, TSPY1E, TSPY1F, TSPY1G, TSPY1H, TSPY1I, TSPY2, XAGE1E, XAGE2B/CTD-2267G17.3, a portion thereof, and/or variants thereof; see, e.g., WO2020097291. In some embodiments, the target antigen is selected from the group consisting of an HPV antigen, an EGFR, a KRAS antigen, an HCC antigen, a portion thereof, and any combination thereof.

For example, in some embodiments, the fusion protein comprises a target antigen comprising: (a) an E6 component comprising a Human Papillomavirus (HPV) E6 protein or an immunogenic variant or fragment thereof, and (b) an E7 component comprising an HPV E7 protein or an immunogenic variant or fragment thereof. In some embodiments, the E6 component comprises a wildtype HPV E6 protein or an immunogenic fragment thereof. In some embodiments, the E6 component comprises a variant HPV E6 protein or an immunogenic fragment thereof. In some embodiments, the variant HPV E6 sequence comprises one or more mutations that reduce binding to the E6 target protein (s) , particularly p53. In some embodiments, the variant HPV E7 sequence comprises one or more mutations that reduce binding to the E7 target protein (s) , particularly Rb. This can be accomplished, for example, by incorporating one or more point mutations in the target protein binding region that makes the protein expressed therefrom display reduced or no binding to its target protein (s) . In some embodiments, the HPV E6 and/or E7 gene (s) comprise (s) at least one, such as at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more point mutations that reduce binding to the target protein (s) . In some embodiments, the E6 component and the E7 component are fused directly to each other. As a result, in some embodiments, the fused HPV E6 and E7 sequence encodes a fusion protein wherein the fusion protein does not bind to p53 and/or Rb. In some embodiments, the E6 component and the E7 components are fused via a linker, such as a GS linker. In some embodiments, the E6 component is N-terminal to the E7 component. In some embodiments, the E6 component is C-terminal to the E7 component.

In some embodiments, the fusion protein comprises a wild-type E6 protein comprising an amino acid sequence set forth in SEQ ID NO: 103 or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises an E6 protein that is at least about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) amino acid sequence identity to the amino acid sequence of SEQ ID NO: 103, or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises a variant E6 protein comprising an amino acid sequence set forth in SEQ ID NO: 104, or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises a wild-type E7 protein comprising an amino acid sequence set forth in SEQ ID NO: 105, or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises an E7 protein that is at least about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) amino acid sequence identity to the amino acid sequence of SEQ ID NO: 105, or an immunogenic fragment thereof. In some embodiments, the fusion protein comprises variant E7 protein comprises an amino acid sequence set forth in SEQ ID NO: 106, or an immunogenic fragment thereof.

i. HPV

Human papilloma virus is a small, naked, double stranded DNA virus of approximately 7.9 kilobases that displays high species specificity. More than 200 types of HPV have been recognized based on DNA sequence data that identify genomic differences. Eighty-five HPV genotypes are well-characterized. An additional 120 isolates are partially characterized and identified as potential new genotypes (see, e.g., Burd (2003) , Clin Microbiol Rev; 16 (1) : 1-17, hereby incorporated by reference in its entirety) . Examples of HPV strains include, but are not limited to, any of HPV types 2a, 3, 7, 10, 11, 13, 16, 18, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 77, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, etc. While approximately 90%of HPV infections resolve spontaneously within two years, certain strains are high risk for the development of HPV+ cancers. For example, see Table 1 below for disease association based on exemplary HPV types (Burd (2003)) . Although minor variations do occur, all HPV genomes described have at least seven early genes (i.e., E1 to E7) and two late genes (i.e., L1 and L2) . L1 and L2 are the major and minor capsid proteins responsible for host infection, but these proteins lose expression after successful viral infection of the host. Most prophylactic HPV vaccines target L1 and/or L2 but have no therapeutic efficacy for HPV+ cancer due to the reduction in L1 and L2 expression over time. Similarly, E1 and E2 genes are involved in viral replication and transcriptional control, respectively, and tend to be disrupted by viral integration. However, E6 and E7 are involved in viral transformation via the targeted degradation of tumor suppressor gene p53 and Rb, respectively, which results in stimulation of cellular DNA synthesis and cell proliferation. Moreover, E6 and E7 gene are constitutively expressed by the HPV+ cancer cells. As both p53 and Rb are host cell tumor suppressor proteins, the activity of the proteins encoded by HPV E6 and E7 genes ultimately can lead to cell transformation and tumorigenesis, such as invasive cervical carcinoma in women that frequently results in death.

Table 1. HPV strain and associated diseases in humans.

Of the HPV strains that have been identified as low or high risk for the development of HPV+ cancers, strain HPV16 is the most commonly detected worldwide. HPV16 and HPV18 together account for approximately 70%of cervical cancer cases. Clinical trials of vaccines targeting HPV16 protein epitopes (i.e., L1, L2, E6, and E7) demonstrated cross-reactivity with other HPV strains, including multiple strains listed in Table 1 above, providing evidence of cross-reactivity, epitope spreading, and de novo immune stimulation (see, e.g., Nakagawa et al. (2015) , Clin Vaccine Immunol; 22 (7) : 679-687, hereby incorporated by reference in its entirety) . Thus, in some embodiments, there is provided an isolated nucleic acid comprising a coding sequence encoding a fusion protein comprising a target antigen and a trafficking peptide, wherein the target antigen further comprises cross-reactivity with E6 and/or E7 proteins or fragments thereof from one or more HPV strains, such as HPV strains described herein, including in Table 1 above. In some embodiments, there is provided an isolated nucleic acid comprising a coding sequence encoding a fusion protein comprising a target antigen and a helper T cell epitope, wherein the target antigen further comprises cross-reactivity with E6 and/or E7 proteins or fragments thereof from one or more HPV strains, such as HPV strains described herein, including in Table 1 above. In some embodiments, there is provided an isolated nucleic acid comprising a coding sequence encoding a fusion protein comprising a target antigen and a immunomodulator peptide wherein the target antigen further comprises cross-reactivity with E6 and/or E7 proteins or fragments thereof from one or more HPV strains, such as HPV strains described herein, including in Table 1 above.

In some embodiments, the target antigen is selected from the group consisting of an HPV antigen, an EGFR antigen, a KRAS antigen, an HCC antigen, a portion thereof, and any combination thereof. For example, in some embodiments, the target antigen is an HPV16 antigen. In some embodiments, the HPV16 antigen comprises E6 and/or E7 components. In some embodiments, the E6 component is the full-length protein of 158 amino acids and/or the E7 component is the full-length protein of 98 amino acids. In some embodiments, the E6 and/or E7 components are an immunogenic fragment comprising a portion of the full-length protein that elicits an immune response. For example, previously identified immunogenic fragments of HPV16 E6 and E7 proteins include E6 peptides VYDFAFRDL and DKKQRFHNI and E7 peptides RAHYNIVTF and LCVQSTHVD (see Fernandes de Oliveira et al. (2015) , PloS One; 10 (9) : e0138686) . Therefore, in some embodiments, the E6 immunogenic fragment comprises a peptide of about 5 amino acids to about 158 amino acids in length, such as any of about 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, and 158 amino acids long. In some embodiments, the E7 immunogenic fragment comprises a peptide of about 5 amino acids to about 98 amino acids in length, such as any of about 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, and 98 amino acids long. In some embodiments, the E6 immunogenic fragment encodes a peptide that is at least about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) amino acid sequence identity to a portion of the amino acid sequence of SEQ ID NO: 103. In some embodiments, the E6 immunogenic fragment comprises mutation E18R, F47R, and/or D49A, wherein the amino acid positions are relative to SEQ ID NO: 103. In some embodiments, the E6 immunogenic fragment comprising the mutation E18R, F47R, and/or D49A is encoded by a nucleic acid, wherein the nucleic acid positions are relative to a nucleic acid selected from the group consisting of SEQ ID NOs: 18-26. In some embodiments, the fusion protein comprises a variant E6 protein comprising an amino acid sequence set forth in SEQ ID NO: 104, or an immunogenic fragment thereof. In some embodiments, the E7 immunogenic fragment encodes a peptide that is at least about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) amino acid sequence identity to a portion of the amino acid sequence of SEQ ID NO: 105. In some embodiments, the E7 immunogenic fragment comprises mutation C24G and/or E26Q, wherein the amino acid positions are relative to SEQ ID NO: 105. In some embodiments, the E7 immunogenic fragment comprising the mutation C24G and/or E26Q is encoded by a nucleic acid, wherein the nucleic acid positions are relative to a nucleic acid selected from the group consisting of SEQ ID NOs: 27-35. In some embodiments, the fusion protein comprises variant E7 protein comprises an amino acid sequence set forth in SEQ ID NO: 106, or an immunogenic fragment thereof. In some embodiments, the E6 protein and the E7 protein are fused via a linker, such as a GS linker. In some embodiments, the linker comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 109-112 and 130-145. In some embodiments, linker comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the E6 protein is N-terminal to the E7 protein. In some embodiments, the E6 protein is C-terminal to the E7 protein.

ii. EGFR

Epidermal growth factor receptor (EGFR) is a cell membrane glycoprotein that binds to epidermal growth factor, thereby inducing receptor dimerization and tyrosine autophosphorylation that leads to increased cell proliferation and decreased apoptosis. Activating mutations in the EGFR gene are associated with a number of different cancers, including but not limited to non-small cell lung cancer, breast cancer, metastatic colorectal cancer, and glioblastoma. Specific activating EGFR mutations are either short, in-frame nucleotide deletions, in-frame duplications/insertions or single-nucleotide substitutions clustered around the adenosine triphosphate (ATP) binding pocket of the tyrosine kinase (TK) domain. In-frame deletions in exon 19 around the LeuArgGluAla motif (del19) at residues 746-750 (the most common being del E746_A750) , and exon 21 L858R point mutation, are the best characterized mutations, together representing 85-90%of all EGFR mutations in NSCLC.

iii. KRAS

RAS is one of the most frequently mutated oncogenes in human cancer, but the frequency and distribution of RAS gene mutations are not uniform. Kirsten rat sarcoma viral oncogene homologue (KRAS) is the isoform most frequently mutated, which constitutes 86%of RAS mutations. KRAS is associated with a series of highly fatal cancers, including pancreatic ductal adenocarcinoma (PDAC) , non-small-cell lung cancer (NSCLC) , and colorectal cancer (CRC) . KRAS mutations are also present in biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia, and breast cancer. KRAS functions as a finely regulated molecular switch that controls multiple signaling cascades by cycling between activated and inactivated conformations. KRAS can be activated by growth factors, chemokines, Ca2+ or receptor tyrosine kinase (RTK) . KRAS mutations are dominated by single-base missense mutations, 98%of which are found at amino acid residue positions 12 (G12) , 13 (G13) , or 61 (Q61) (see, e.g., Huang et al. (2021) , Signal Transduct Target Ther. 6 (1) : 386) . KRAS mutations also occur in codons 63, 117, 119, and 146 but with less frequency.

iv. HCC

Hepatocellular carcinoma (HCC) accounts for approximately 90%of the incidence of all primary liver cancers. Both environmental and genetic risk factors contribute to the etiology of HCC. The most notable environmental and potentially preventable risk factors include oncogenic virus infection with hepatitis B virus (HBV) , hepatitis C virus (HCV) , alcohol abuse, and metabolic syndrome related to obesity and diabetes mellitus. HCC incidence has doubled in the last three decades in the United States, due in part to untreated HCV infections and increasingly obesity-related non-alcoholic fatty liver disease (NAFLD) progressing to non-alcoholic steatohepatitis (NASH) . Tumor-specific antigens (TSA) or tumor-associated antigens (TAA) in HCC include, but are not limited to, α-fetoprotein, hTERT, glypican-3 (GPC3) , p53, melanoma antigen gene A (MAGE-A) , squamous cell carcinoma antigen recognized by T cells (SART) , and NY-ESO-1. More recently, the oncogenic phosphatase PRL3 was confirmed as a TAA, as it was shown to be expressed in tumors, but not in patient-matched normal tissue, across 11 cancers. In particular, four tumor antigens have been identified that display high expression in HCC and no, or weak, expression in surrounding tumor-free liver tissue: Annexin-A2, GPC-3, MAGE-C1 and MAGE-C2.

B. Trafficking peptide

Without being bound by theory, it is contemplated that sorting proteins to endosomes and lysosomes is mediated by sorting signals presented within the proteins. Many endosomal-lysosomal sorting signals have been characterized, and most of these signals are contained within the cytosolic domains of transmembrane proteins. See Bonifacino and Traub; Annu. Rev. Biochem. 2003. 72: 395–447 for a review, the content of which is incorporated herein by reference in its entirety. In general, the signals consist of short, linear arrays of amino acid residues. These arrays are not exactly conserved sequences but degenerate motifs of four to seven residues of which two or three are often critical for function. The critical residues are generally bulky and hydrophobic, although charged residues are also important determinants of specificity for some signals. Accordingly, in some embodiments, the trafficking domain of the polypeptide encoded by the present therapeutic nucleic acid can be derived from a protein sequence that contains the sorting signal.

Without being bound by any theory, it is contemplated that two major classes of endosomal-lysosomal sorting signals are referred to as “tyrosine-based” and “dileucine based” signals, respectively owing to the identity of their most critical residues. Particularly, the tyrosine-based sorting signals conform to the NPXY orconsensus motifs, and the dileucine-based signals conforms to [DE] XXXL [LI] or DXXLL consensus motifs, where X stands for any amino acid, stands for an amino acid residue with a bulky hydrophobic side chain, [DE] stands for D or E, and [LI] stands for L or I. Other sorting signals include acidic clusters, lysosomal avoidance signals, NPFX (1, 2) D-type signals and ubiquitin-based signals. See Table 1 below. Bonifacino and Traub; Annu. Rev. Biochem. 2003. 72: 395–447.

Table A Endosomal/lysosomal sorting signals

Motifs in this and other Tables B-H below are denoted using the PROSITE syntax (www. expasy. ch/prosite/) . Amino acid residues are designated according to the single letter code as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan, and Y, tyrosine. X stands for any amino acid andstands for an amino acid residue with a bulky hydrophobic side chain. Abbreviations: PTB, phosphotyrosine-binding; Dab2, disabled-2; AP, adaptor protein; VHS domain present in Vps27p, Hrs, Stam; GGAs, Golgi-localized, γ-ear-containing, ARF-binding proteins; PACS-1, phosphofurin acidic cluster sorting protein 1; TIP47, tail-interacting protein of 47 kDa; SHD1, Sla1p homology domain 1; UBA, ubiquitin associated; UBC, ubiquitin conjugating; UIM, ubiquitin interaction motif.

Tables B to G list trafficking signals for various proteins of human or non-human origins. According to the present disclosure, the trafficking domain described herein can be derived from a protein selected from (but is not limited to) those proteins listed in Tables B to G. In some embodiments, the trafficking domain comprises a portion of the one or more proteins listed in Tables B to G. In some embodiments, the trafficking domain comprises the trafficking signal sequence listed for the corresponding protein as shown in Tables B to G.

Table B Endocytic machinery motifs and their recognition domains

Motifs are denoted as indicated in the legend to Table 1. EH, eps15 homology.

Table C NPXY-type signals

Numbers in parentheses indicate motifs that are present in more than one copy within the same protein. The signals in this and other tables should be considered examples. Not all of these sequences have been shown to be active in sorting; some are included because of their conservation in members of the same protein family. Key residues are indicated in bold type. Numbers of amino acids before (i.e., amino-terminal) and after (i.e., carboxy-terminal) the signals are indicated. Abbreviations: Tm, transmembrane; LDL, low density lipoprotein; LRP1, LDL receptor related protein 1; APP, _-amyloid precursor protein; APLP1, APP-like protein 1.

Table D-type signals

See legends to Tables 1–3 for explanation of signal format.

Table E [DE] XXX [LI] -type signals

See legends to Tables 1–3 for explanation of signal format.

Table F DXXLL-type dileucine-based signals

See legends to Tables 1–3 for explanation of signal format. Serine and threonine residues are underlined.

Table G Acidic cluster signals

See legends to Tables 1–3 for explanation of signal format. Serine and threonine residues are underlined. *The number in parentheses is the motif number.

Trafficking peptides described herein can be derived from a protein (or polypeptide) selected from (but is not limited to) , MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.

In some embodiments, the trafficking peptide comprises a portion of the one or more polypeptides. In some embodiments, the trafficking peptide comprises the trafficking signal of the one or more polypeptides as described in Tables B to G herein. In some embodiments, the trafficking peptide comprises one or more variations (e.g., substitutions, deletions, and/or additions) in the wild-type sequence of the one or more polypeptides. In some embodiments, the trafficking peptide comprises one or more variations (e.g., substitutions, deletions, and/or additions) in or to the wild-type sequence of the trafficking signal of the one or more polypeptides as described in Tables B to G herein. In some embodiments, the trafficking peptide comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of the one or more polypeptides. In some embodiments, the trafficking peptide comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of the trafficking signal of the one or more polypeptides as described in Tables B to G herein.

Sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present within the cytosolic domains of the proteins. Most signals consist of short, linear sequences of amino acid residues. Signals are referred to as either tyrosine-based sorting signals or dileucine-based signals, each of which can be identified based on specific consensus motifs. All of these signals are recognized by components of protein coats peripherally associated with the cytosolic face of membranes. Phosphorylation events regulate signal recognition. In addition to peptide motifs, ubiquitination of cytosolic lysine residues also serves as a signal for sorting at various stages of the endosomal-lysosomal system. Non-limiting examples of trafficking peptides can be found in, e.g., J.S. Bonifacino & L.M. Traub (2003) , Annu Rev Biochem 72: 395-447, hereby incorporated by reference in its entirety. These peptides can be utilized as described herein by identifying the trafficking peptides based on the presence of the relevant consensus motifs and then isolating the nucleotides that encode said peptides and inserting these nucleotides into any of the nucleic acid constructs described herein by any method known in the art, such as by standard cloning techniques such as restriction enzyme digestion, PCR amplification, homologous recombination, or by nucleic acid synthesis. Nucleotide sequences encoding said peptides may be inserted 5′or 3′of the target antigen to promote the trafficking of the target antigen through the endosomal-lysosomal pathway of the antigen presenting cells.

CD1d: The CD1 family is an antigen presenting protein related to MHC class I but technically distinct from both MHC class I and II. The CD1 family mainly presents bacterial and pathogenic lipid and glycolipid antigens to T cells, specifically the natural killer T cell (NKT) subtype that produces IL-4 and IFNγ. CD1d plays a critical role in facilitating late endosomal trafficking of the processed antigen to the plasma membrane for presentation. See, e.g., Lawton et al., Immunol. Cell Biol., 2004; de Araujo et al., J Immunol Res., 2021; Miura et al., J. Virol., 2010; Jawawardena-Wolf et al., Immunity, 2001. HLA-E: HLA-E is a non-canonical MHC class I molecule that binds to CD94/NKG2A/B/C receptor on NK cells. Binding to A/B receptors prevents target cell lysis by NK cells, while C receptor binding triggers expansion of NK cells to aid in fighting infection. HLA-E is also capable of presenting antigens to CD8+T cells. HLA-E can simultaneously stimulate NK-and CD8+ T cell-mediated immune response, prevent rejection of transplanted cells by NK self-recognition mechanism, and resist the pathogen-mediated downregulation of HLA-I molecules that allows infection to evade immune response. Furthermore, HLA-E molecules are preferentially recycled and reused for antigen presentation rather than degraded, and HLA-E molecules have a longer residence in late and recycled endosomes. See, e.g., Braud et al., Nature, 1998; Rolle et al., J Clin Invest., 2014.; and He et al., JEM, 2023.

LAMP1: LAMP1 is a glycoprotein found on various membranes within a cell, for example lysosome membranes. See, e.g., Watts, FEBS Open Bio., 2022; Cheng et al., J Cell Biol., 2018.

LAMP3: LAMP3 (DC-LAMP) is a glycoprotein found on the surface of lysosomes similar to LAMP1 and LAMP2. LAMP3 is specific only to mature dendritic cells and plays a key role in the maturation of DCs. LAMP3 specifically localizes to the MHC class II compartment immediately before translocation of MHC-II molecules to the cell surface, and helps with antigen processing and loading onto the MHC-II molecule. See, e.g., Barois et al., Traffic, 2002; de Saint-Vis et al., Immunity, 1998.

HLA-DMB: New MHC-II molecules leave the ER and are directed to the endocytic pathway by the Invariant chain (Ii) chaperone, which is subsequently degraded to CLIP (class II-associated Ii-chain peptide) while still bound to MHC II. HLA-DMB is a non-classical MHC-II molecule that displaces CLIP and helps load a processed antigen onto the target MHC-II molecule for antigen presentation. See, e.g., Barois et al., Traffic, 2002; Santambrogio, Front. Immunol., 2022.

CLASS I MHC: MHC class I is involved in antigen presentation. Antigen presentation in APCs is accomplished by loading antigen peptides onto MHC-II molecules, however it is known that MHC class I molecules also reside in and travel through the cellular compartments involved in MHC class II antigen processing and presentation. CLASS I MHC has been shown to improve antigen presentation and immune response in a manner dependent on MHC-I function. See, e.g., Kreiter et al., J. Immunol., 2008.

LAMP-2a: LAMP2a is associated with lysosomal function, specifically with chaperone-mediated autophagy. See, e.g., Kaushik and Cuervo, Nat. Rev. Mol. Cell Biol., 2018.

CD63: CD63 is a surface glycoprotein also found on membranes of intracellular vesicles, for example of immature dendritic cells, alongside MHC class II molecules. See, e.g., Piper and Katzmann, Annu. Rev. Cell Dev. Biol., 2010.

GMP-17: Granule membrane protein 17 (i.e., NKG7 or GIG-1) is a membrane protein typically localized to cytotoxic granules and highly expressed in NK cells and CD8+ T cells. GMP-17 expression was recently found to be induced in activated CD4+ T cells stimulated by IL-27. See, e.g., Ng et al., Nat. Immunol., 2020; Malarkannan, Nat. Immunol., 2020.

CD1b: CD1b is a glycoprotein expressed on APCs such as dendritic cells that binds to self-and non-self-lipid antigens for presentation. CD1b is specifically capable of binding and presenting microbial lipid antigens of various alkyl chain lengths. See, e.g., Gras et al., Nat. Comm., 2016.

CD1c: CD1c is a glycoprotein expressed on APCs such as dendritic cells that binds self-and non-self-lipid antigens for presentation. CD1c labels a distinct subpopulation of DCs that secrete high levels of IL-12 and potently prime cytotoxic T-cell responses. See, e.g., Nizzoli et al., Blood, 2013; Heger et al., Front. Immunol., 2020.

Cystinosin: Cystinosin is a lysosomal membrane protein that primarily transports cystine from lysosomes in a H+-dependent manner. Cystine is a disulfide form of the amino acid cysteine that forms from lysosomal protein hydrolysis, and mutations in the Cystinosin gene cause the lysosomal storage disease cystinosis. See, e.g., Kalatzis et al., EMBO J., 2001.

CTLA-4: Cytotoxic T cell antigen 4 is a surface membrane protein expressed by activated T cells and binds to the B7 ligand to negatively regulate T cell function and proliferation. See, e.g., Bashyam, J. Exp. Med., 2007; Salvatori et al., npj Vaccines, 2022.

CD4: CD4 is a receptor for helper T cells that assists the T cell receptor (TCR) in binding to antigen-presenting MHC class II molecules on APCs. See, e.g., Miceli and Parnes, Seminars in Immunology, 1991.

NPC1: Niemann-Pick disease type C1 is a membrane protein that mediates intracellular cholesterol trafficking. Mutations in NPC1 cause Niemann-Pick disease, where lipids accumulate in late endosomes and lysosomes.

CIMPR: Cation-independent mannose 6-phosphate receptor (CIMPR, or insulin-like growth factor 2 receptor (IGF2R) ) binds to IGF2 at the cell surface for transport to early endosomes for the release of IGF2 and attenuation of IGF2 signaling. CIMPR also shuttles mannose 6-phosphate containing lysosomal enzymes through the endosomal system. See, e.g., Bohnsack et al., J. Biol. Chem., 2009; and Ghosh et al., Nat. Rev. Mol. Cell Biol., 2003.

LRP3: low density lipoprotein (LDL) receptor-related protein 3 is a probable surface receptor for internalization of lipophilic molecules and signal transduction, however its precise role in this process remains unclear. See, e.g., Ishii et al., Genomics, 1998.

Furin: Furin is a protease in the trans-Golgi network that cleaves a number of target proteins for activation. In T cells, Furin helps maintain peripheral tolerance by cleaving the anti-inflammatory cytokine TGF-β1 and generally preventing overproduction of cytokines. Furin is involved in proteolytic maturation of substrate proteins in the secretory pathway. See, e.g., Pesu et al., Nature, 2008.

VAMP4: Vesicle-associated membrane protein 4 is involved in membrane fusion during vesicle transport, specifically by mediating the transport of vesicles from the trans-Golgi network to endosomes. See, e.g., Steegmaier et al., Mol. Biol. Cell, 1999.

VMAT1: Vesicular monoamine transporter 1 (SLC181) is an integral membrane protein in neuronal and endocrine cells that allows for monoamine neurotransmitter transport into secretory vesicles to then be discharged into extracellular space by exocytosis. See, e.g., Eiden et al., Pflugers Archiv., 2004.

VMAT2: Vesicular monoamine transporter 2 (SLC182) is an integral membrane protein in neuronal and endocrine cells that allows for monoamine neurotransmitter transport into secretory vesicles to then be discharged into extracellular space by exocytosis. See, e.g., Eiden et al., Pflugers Archiv., 2004.

PAM: Peptidyl-glycine alpha-amidating monooxygenase helps to form active neuroendocrine peptides by catalyzing their alpha-amidation. See, e.g., Driscoll et al., Mol. Pharmacol., 1999.

CPD: Carboxypeptidase D is a trans-Golgi resident enzyme that cleaves C-terminal arginine or lysine residues to aid in biosynthesis of neuropeptides and peptide hormones. See, e.g., Song and Fricker, Enzymology, 1995.

PC7: Proprotein convertase 7 is a secretory pathway enzyme that helps process secreted proteins to active state. PC7 can reach the cell surface through canonical ER/Golgi-dependent secretory pathway, i.e., predominantly through its pro-segment. The PC7 transmembrane domain also regulates trafficking to the surface through an unconventional pathway that is not dependent on COPII vesicles. See, e.g., Rousselet et al., J. Biol. Chem., 2011.

Beta-secretase (BACE) : BACE is an aspartic-acid protease important in the formation of myelin sheaths in peripheral nerve cells. BACE is transported through the secretory pathway to the cell surface. See, e.g., Capell et al., J. Biol. Chem., 2000.

Sortilin: Sortilin is a membrane glycoprotein responsible for mediating protein transport between the Golgi, endosomes, lysosomes, and plasma membrane. See, e.g., Nielsen et al., EMBO J., 2001.

GLUT4: Glucose transporter type 4 is a plasma membrane protein that facilitates passive diffusion of circulating glucose down a concentration gradient into muscle and fat cells in an insulin-dependent manner. See, e.g., James et al., Nature, 1989.

TRP-1 (TYRP1) : Tyrosinase-related protein 1 is a transmembrane protein involved in the processing and maturation of melanin. TRP-1 is trafficked through the melanosomes of melanocytes. See, e.g., Rzepka et al., Postepy Hig. Med. Dosw., 2016; and Kameyama et al., Pigment Cell Res., 1995.

LDL receptor: Low-density lipoprotein receptor is a cell surface receptor that mediates endocytosis of low-density lipoprotein (LDLs) by recognition of apolipoprotein B100. See, e.g., Sudhof et al., Science, 1985.

LRP1: low density lipoprotein (LDL) receptor-related protein 1 is involved in receptor mediated endocytosis and plays a role in many related biological processes. See, e.g., Etique et al., Biomed Research International, 2013.

Megalin (LRP2) : Megalin is a mediator of ligand endocytosis, leading to the degradation of these target molecules within lysosomes. See, e.g., Eshbach et al., Annu. Rev. Physiol., 2017.

Integrin beta-1: Integrin beta-1 is a cell surface protein that functions as a collagen receptor with Integrin alpha 1 and Integrin alpha 2. See, e.g., Hynes, Cell, 1992.

APLP1: The amyloid-like protein 1 is a membrane-associated glycoprotein that modulates glucose and insulin homeostasis. APLP1 is cleaved by secretases within the secretory pathway. See, e.g., Bayer et al., Mol. Psychiatry, 2000.

APP: The amyloid-beta precursor protein is an integral membrane protein that functions as a cell surface receptor involved in synapse formation and neural plasticity, among other functions. APP is trafficked to the cell surface through the secretory system. See, e.g., Turner et al., Progress in Neurobiology, 2003.

Insulin receptor: The insulin receptor (IR) is a transmembrane protein that binds circulating insulin in order to promote glucose homeostasis and initiate cellular uptake of glucose molecules. See, e.g., McKern et al., Nature, 2006.

EGF receptor: Epidermal growth factor receptor (EGFR) is a cell surface receptor for epidermal growth factor (EGF) . Activating mutations in EGFR have been associated with a number of cancers, as described in Subsection II. A. ii.: EGFR above.

In some embodiments, the coding sequence encoding the trafficking peptide comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 76-80, 83-86, 172, and 201. In some embodiments, the coding sequence encoding the trafficking peptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 76-80, 83-86, 172, and 201. In some embodiments, the trafficking peptide comprises a polypeptide comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 119, and 122-125. In some embodiments, the trafficking peptide comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 119, and 122-125. In some embodiments, the trafficking peptide is derived from MHC class I or LAMP3. In some embodiments, the trafficking peptide is positioned C-terminal to the target antigen in the fusion protein.

In some embodiments, the coding sequence encoding the MHC class I trafficking domain comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 76-79, 172, and 201. In some embodiments, the coding sequence encoding the MHC class I trafficking domain comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 76-79, 172 and 201. In some embodiments, the MHC class I trafficking domain comprises a polypeptide comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 119.

In some embodiments, the coding sequence encoding the LAMP3 trafficking peptide comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 80 and 85. In some embodiments, the coding sequence encoding the LAMP3 trafficking peptide comprises a nucleic acid selected from the group consisting of SEQ ID NOs: 80 and 85. In some embodiments, the LAMP3 trafficking peptide comprises a polypeptide comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 122. In some embodiments, the LAMP3 trafficking peptide comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 and 122.

C. Helper T Cell Epitope

In some embodiments, the fusion protein further comprises a helper T cell epitope. In some embodiments, the helper T cell epitope is a universal CD4 epitope. In some embodiments, the helper T cell epitope is an epitope of tetanus and diphtheria toxoids. In some embodiments, the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632- ₆₅₁, and PADRE.

The P2 helper T cell epitope is a signal peptide from the tetanus toxoid amino acid residues 830-844 that functions as a universal CD4+ T-cell activator. The P16 helper T cell epitope is a signal peptide from tetanus toxoid that also functions as a universal CD4+ T-cell activator. These two epitopes can be connected into the P2P16 epitope, for example via a linker peptide such as a flexible linker or a cleavable linker as described in Subsection F: Linker Sequences below.

The TT_827-841 helper T cell epitope is a signal peptide from the tetanus toxoid amino acid residues 827-841 that have been shown to stimulate T-helper cell activity. The pDT_331-350 helper T cell epitope is a region of diphtheria toxin (DTX) of amino acid residues 331-350 that is recognized by human CD4+ T cells and may help to activate the helper T cell response to a target antigen. The TT_632-651 helper T cell epitope is a signal peptide from the tetanus toxoid amino acid residues 632-651 that has been shown to stimulate helper T cell activity.

The pan-DR epitope (PADRE) is a 13 amino acid synthetic peptide that stimulates CD4+ T cell activation by binding to fifteen of the sixteen most common HLA-DR types. In proliferation assays, PADRE has been shown to induce up to a 100-fold more potent helper T cell response than other universal helper T cell epitopes such as tetanus toxin-derived epitopes.

In some embodiments, the helper T cell epitope comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72-75 and 171. In some embodiments, the helper T cell epitope comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72-75 and 171. In some embodiments, the helper T cell epitope encodes a polypeptide sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 126-128 and 147-152. In some embodiments, the helper T cell epitope encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 126-128 and 147-152.

In some embodiments, the helper T cell epitope is a pan DR-binding epitope (PADRE) . In some embodiments, the coding sequence encoding the helper T cell epitope that is PADRE comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO: 75 or 171. In some embodiments, the coding sequence encoding the helper T cell epitope that is PADRE comprises a nucleic acid sequence of SEQ ID NO: 75 or 171. In some embodiments, the coding sequence encoding the helper T cell epitope that is PADRE comprises an amino acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 128. In some embodiments, the coding sequence encoding the helper T cell epitope that is PADRE comprises an amino acid sequence of SEQ ID NO: 128.

Thus, in some embodiments, the coding sequence encoding the helper T cell epitope comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72-75 and 171. In some embodiments, the helper T cell epitope comprises a polypeptide comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 126-128 and 147-152. In some embodiments, the helper T cell epitope is PADRE. In some embodiments, the coding sequence encoding the helper T cell epitope comprises a nucleic acid sequence comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO: 75 or 171. In some embodiments, the helper T cell epitope comprises a polypeptide comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 128. In some embodiments, the helper T cell epitope is positioned between the target antigen and the trafficking peptide. For example, the fusion protein can comprise, from N-terminus to C-terminus: a signal domain peptide, an optional ubiquitin peptide (either comprising or not comprising the C-terminal extension peptide) , a target antigen, an optional linker, an optional helper T cell epitope, a trafficking peptide, an optional 2A (e.g., P2A) cleavable linker, and an optional immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: an optional signal peptide, an optional ubiquitin peptide (either comprising or not comprising the C-terminal extension peptide) , a target antigen, an optional linker, a helper T cell epitope, an optional trafficking peptide, a 2A (e.g., P2A) cleavable linker, and an immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: a signal peptide, a target antigen, an optional linker, an optional helper T cell epitope, a trafficking peptide, an optional 2A (e.g., P2A) cleavable linker, and an optional immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: an optional signal peptide, a target antigen, an optional linker, a helper T cell epitope, an optional trafficking peptide, a 2A (e.g., P2A) cleavable linker, and an immunomodulator.

D. Immunomodulator

In some embodiments, the isolated nucleic acid, e.g., mRNA, comprises a coding sequence encoding one or more immunomodulators. In some embodiments, the immunomodulator is selected from the group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, a portion thereof, and any combination thereof.

GM-CSF: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the differentiation of granulocytes, macrophages, eosinophils, and erythrocytes from hematopoietic precursors, specifically myeloid-biased common myeloid progenitors (CMPs) . GM-CSF is also critical for the maturation of hematopoietic progenitors, specifically circulating monocytes, to dendritic cells (DCs) . GM-CSF therefore promotes DC maturation. See, e.g., Banchereau and Palucka, Nature Reviews Immunology, 2005; Greter et al., Immunity, 2012. In some embodiments, the GM-CSF is mouse GM-CSF (mGM-CSF) . In some embodiments, the GM-CSF is human GM-CSF (hGM-CSF) . In some embodiments, the GM-CSF is a wild-type polypeptide. In some embodiments, the GM-CSF polypeptide is full-length. In some embodiments, the GM-CSF polypeptide is a naturally occurring isoform. In some embodiments, the GM-CSF is a naturally occurring variant polypeptide. In some embodiments, the GM-CSF is a mutated polypeptide comprising one or more variations (e.g., substitutions, deletions, and/or additions) with respect to the amino acid sequence set forth in SEQ ID NO: 108 or 181. In some embodiments, the GM-CSF polypeptide is truncated.

STING: STING (stimulator of interferon genes) is an Endoplasmic Reticulum (ER) -localized membrane protein that upregulates the expression of interferon-related genes and production of type I interferons in response to pathogenic nucleic acids released from viruses, bacteria, and/or parasites. DCs upregulate antigen presentation in response to interferon signaling and inflammation generally, indicating that constitutive STING activity may help stimulate DC function. Therefore, expression of STING stimulates DCs to produce interferons, thereby upregulating their own maturation and antigen-presenting functions. See, e.g., Nakhaei et al., J. Mol. Cell Biol., 2009; and Jneid et al., Science Immunology, 2023. In some embodiments, the STING is mouse STING (mSTING) . In some embodiments, the STING is human STING (hSTING) . In some embodiments, the STING is a wild-type polypeptide. In some embodiments, the STING polypeptide is full-length. In some embodiments, the STING polypeptide is a naturally occurring isoform. In some embodiments, the STING is a naturally occurring variant polypeptide. In some embodiments, the STING is a mutated polypeptide comprising one or more variations (e.g., substitutions, deletions, and/or additions) with respect to the amino acid sequence set forth in SEQ ID NO: 107 or a corresponding murine polypeptide. In some embodiments, the STING polypeptide is truncated.

FLT3L: Fms-related tyrosine kinase 3 ligand is a cytokine involved in regulation of hematopoietic progenitor function in mouse and hematopoietic stem cell homeostasis in human. FLT3L is also critical to development of both classic and plasmacytoid dendritic cells, and intratumoral FLT3L has been shown to induce accumulation and antigen presentation of DCs along with an in situ vaccine and a TLR3 agonist. See, e.g., Shortman and Naik, Nature Review Immunology, 2007; and Hammerich et al., Nature Medicine, 2019.

c-FLIP: Cellular FLICE-inhibitory protein is a master anti-apoptotic regulator that functions by suppressing TNF-α, Fas-L, and TNF-related (TRAIL) -induced apoptosis. C-FLIP can also activate other pro-survival signaling proteins such as Akt, ERK, and NF-κB. It is found to be upregulated in many tumor types to prevent cancer cell death. See, e.g., Safa, Exp. Oncol., 2012.

IKKβ: Inhibitor of NF-κB kinase subunit beta phosphorylates inhibitor of NF-κB (IκB) , targeting it for degradation and thereby driving nuclear translocation of NF-κB to act as a transcription factor. Among other roles, active NF-κB mediates maturation of DCs to become fully functional. See, e.g., Voigt et al., Nature Comm., 2020; and Creusot, Nature Medicine, 2011.

RIPK1: Receptor interacting protein 1 plays a critical role in TNF receptor-mediated activation of NF-κB, which would benefit DC maturation and function. Furthermore, RIPK1 has also been shown to play a role in activating TNF-induced apoptosis and necrosis under certain conditions. See, e.g., Lin, Necrotic Cell Death, 2014.

Btk: Bruton’s tyrosine kinase activates NF-κB. B cell receptor (BCR) -dependent NF-κB signaling requires functional Btk. See, e.g., Mohamed et al., Immunol. Rev., 2009.

TAK1: Mitogen-activated protein 3 kinase 7 has a powerful pro-survival role in activating the IKK-NF-κB pathway to block apoptosis, promote cell proliferation, and stimulate inflammatory responses. TAK1 may also play a role in mediating necroptosis with RIPK1 and RIPK3. Ultimately, TAK1 may therefore help to activate NF-κB in DCs to improve DC survival. See, e.g., Paul, Oncogene, 1999; Mihaly et al., Cell Death and Differentiation, 2014.

TAK-TAB1: Binding partner of TAK1 is needed for TAK1 functional activation of NF-κB. TAK-TAB1 therefore may be similar to TAK1 by helping to activate NF-κB in DCs to improve DC survival. See, e.g., Xu and Lei, Front. Immunol., 2021.

TBK1: TANK-binding kinase 1 is a non-canonical IKK kinase that phosphorylates IκB to activate NF-κB, similar to IKKB.

MyD88: Myeloid differentiation primary response 88 is a universal adapter protein that activates NF-κB in response to toll-like receptor (TLR) activity. See, e.g., Lord et al., Oncogene, 1990.

IRAK1: Interleukin receptor-associated kinase 1 is activated downstream of toll-like receptor (TLR) or interleukin-1 receptor (IL-1R) activation by inflammatory signaling to upregulate and activate NF-κB. See, e.g., Cao et al., Science, 1996; and Muzio et al., Science, 1997.

IRAK2: Interleukin receptor-associated kinase 2 is activated downstream of TLR or IL-1R activation by inflammatory signaling to upregulate and activate NF-κB.

TAB2: TGF-beta activated kinase 1 (MAP3K7) binding protein 2 is required for the IL-1 induced activation of NF-κB and MAPK by forming a kinase complex with TRAF6, MAP3K7, and TAB1.

TAB3: TGF-beta activated kinase 1 (MAP3K7) binding protein 3 forms a complex with MAP3K7 and either TRAF2 or TRAF6 in response to TNF or IL-1 signaling to activate NF-κB.

TRAF6: TNF receptor associated factor 6 mediates signal transduction from TNF and IL-1 to activate IKK in response to inflammation for downstream NF-κB activation.

TRAM: Translocation associated membrane protein 2 is a component of the translocon gated channel that controls the posttranslational processing of nascent secretory and membrane proteins at the ER.

MKK3: Mitogen-activated protein 2 kinase 3 activates MAPK14/p-38MAPK in response to mitogenic and environmental stress. MAPK activation drives cell differentiation and proliferation.

MKK4: Mitogen-activated protein 2 kinase 4 activates the MAPK pathway in response to mitogenic and environmental stress. MAPK activation drives cell differentiation and proliferation.

MKK6: Mitogen-activated protein 2 kinase 6 activates the MAPK pathway in response to mitogenic and environmental stress. MAPK activation drives cell differentiation and proliferation.

Type I IFN: Type I interferons are cytokines that drive inflammation and can induce T cell responses. Type I IFNs can stimulate DC maturation and function, and preliminary studies have shown that cancer vaccines employing IFNα/β improve DC-mediated neoantigen-specific T cell activation and NK cell activation. See, e.g., Sprooten et al., International Review of Cell and Molecular Biology, 2019.

In some embodiments, the immunomodulator is GM-CSF. In some embodiments, the GM-CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) . In some embodiments, the GM-CSF is a full-length GM-CSF polypeptide. In some embodiments, the murine full-length mGM-CSF polypeptide comprises at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, the mGM-CSF comprises a truncated mGM-CSF or a mutant mGM-CSF comprising an amino acid sequence comprising one or more variations compared to the amino acid sequence set forth in SEQ ID NO: 108, wherein the truncated mGM-CSF or mutant mGM-CSF is capable of stimulating macrophage differentiation and proliferation, and/or activating antigen presenting cells (APCs) . In some embodiments, the mGM-CSF is encoded by a nucleic acid comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO: 63, 174, 175, and 202. In some embodiments, the human full-length hGM-CSF polypeptide comprises at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 181. In some embodiments, the hGM-CSF comprises a truncated hGM-CSF or a mutant hGM-CSF comprising an amino acid sequence comprising one or more variations compared to the amino acid sequence set forth in SEQ ID NO: 181, wherein the truncated hGM-CSF or mutant hGM-CSF is capable of stimulating macrophage differentiation and proliferation, and/or activating antigen presenting cells (APCs) . In some embodiments, the hGM-CSF is encoded by a nucleic acid comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 177-178.

In some embodiments, the immunomodulator is STING. In some embodiments, the STING is human STING (hSTING) or murine STING (mSTING) . In some embodiments, the STING is human STING (hSTING) (V155M) . In some embodiments, the hSTING comprises a polypeptide comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 107. In some embodiments, the hSTING is encoded by a nucleic acid comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO: 62.

Thus, in some embodiments, the immunomodulator comprises a polypeptide comprising at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence selected from the group consisting of SEQ ID NO: 107, 108, and 181. In some embodiments, the coding sequence encoding the immunomodulator comprises a nucleic acid comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 62, 63, 174, 175, 177, 178, and 202. In some embodiments, the immunomodulator is connected to the C-terminus of the fusion protein. In some embodiments, the immunomodulator is connected to the N-terminus of the fusion protein. In some embodiments, the immunomodulator is connected to the fusion protein via a cleavable linker, such as a 2A peptide (e.g., P2A, F2A, T2A, and E2A) . In some embodiments, the fusion protein further comprises a signal peptide. For example, the fusion protein can comprise, from N-terminus to C-terminus: a signal domain peptide, an optional ubiquitin peptide (either comprising or not comprising the C-terminal extension peptide) , a target antigen, an optional linker, an optional helper T cell epitope, a trafficking peptide, an optional 2A (e.g., P2A) cleavable linker, and an optional immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: an optional signal peptide, an optional ubiquitin peptide (either comprising or not comprising the C-terminal extension peptide) , a target antigen, an optional linker, a helper T cell epitope, an optional trafficking peptide, a 2A (e.g., P2A) cleavable linker, and an immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: a signal peptide, a target antigen, an optional linker, an optional helper T cell epitope, a trafficking peptide, an optional 2A (e.g., P2A) cleavable linker, and an optional immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: an optional signal peptide, a target antigen, an optional linker, a helper T cell epitope, an optional trafficking peptide, a 2A (e.g., P2A) cleavable linker, and an immunomodulator.

E. Enhancement Component

In some embodiments, the coding sequence of the isolated nucleic acid encodes an enhancement component that improves immunogenicity. The acquired immune system is primed and activated by antigen presenting cells, most commonly dendritic cells, that phagocytose antigens, partially degrade the antigens, and then process and present the antigens in complex with major histocompatibility complex I or II (MHC) proteins to train lymphocytes (e.g., B cells and T cells) to recognize and eliminate any cells that present the target antigen. Antigen processing can proceed through two distinct pathways: proteasomal degradation and phagolysosomal degradation. Antigens found in the dendritic cell cytoplasm can be targeted by chaperone proteins to the proteasome for degradation. Antigens that are taken up via phagocytosis into the phagosome can be targeted for processing by the fusion of phagosome and lysosome for partial degradation. Various enhancement components can be included in a vaccine construct in order to promote more efficient and/or effective antigen processing via one or both of these processing pathways and/or antigen presentation to promote vaccine immunogenicity. For example, these could include enhancements that increase antigen protein stability and enhancements that target the antigen proteins to the proteasome or to the phagolysosome for processing.

i. Ubiquitin

The ubiquitin pathway is well described for targeting proteins to the proteasome. Ubiquitin molecules can be found in multiple peptide forms, such as polyubiquitin chains that are branched or linear and that are heterotypic or homotypic chains. For example, homotypic K48-linked ubiquitin drives proteasomal degradation, whereas branched ubiquitin can act to promote a diverse range of cellular functions (see, e.g., Kolla et al., (2022) , Trends Biochem Sci; 47 (9) : 759-771, hereby incorporated by reference in its entirety) . Polyubiquitin chains attached to a target protein function differently based on the Lysine residue of the ubiquitin that is linked: Lys-6-linked may be involved in DNA repair; Lys-11-linked is involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle regulation; Lys-29-linked is involved in proteotoxic stress response and cell cycle; Lys-33-linked is involved in kinase modification; Lys-48-linked is involved in protein degradation via the proteasome; Lys-63-linked is involved in endocytosis, DNA-damage responses, and signaling processes leading to activation of the transcription factor NF-kappa-B. Linear polymer chains formed via attachment by the initiator Methionine lead to cell signaling. Ubiquitin typically is conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed.

Ubiquitin peptides are encoded by one of four genes. UBA52 and RPS27A genes code for a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively. UBB and UBC genes code for a polyubiquitin precursor with exact head to tail repeats, the number of which are species dependent.

In some embodiments, the ubiquitin peptide comprises an amino acid sequence comprising about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 120. In some embodiments, the ubiquitin peptide comprises an amino acid sequence of SEQ ID NO: 120. In some embodiments, the ubiquitin peptide is encoded by a nucleic acid comprising about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 81. In some embodiments, the ubiquitin peptide is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 81. In some embodiments, the ubiquitin peptide comprises an amino acid sequence that is encoded by any one of the UBA52, RPS27A, UBB, or UBC genes. In some embodiments, the ubiquitin peptide comprises an amino acid sequence of about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence that is encoded by any one of the UBA52, RPS27A, UBB, or UBC genes.

ii. C-terminal extension peptide

An alternative transcript of a ubiquitin peptide encoded by the UBB gene has been identified wherein the peptide can further include a 19 amino acid C-terminal extension that inhibits proteasomal degradation activity, thereby stabilizing the protein to which this ubiquitin peptide is attached. However, when extended to 25 amino acids, the alternative ubiquitin peptide attached at the N-terminus of a larger protein can target efficiently the larger protein for proteasomal degradation (see Vergoef, et al. (2009) , FASEB J; 23 (1) : 123-133, hereby incorporated by reference in its entirety) . Within the greater context of eliciting an immune response to a vaccine, efficient proteasomal processing and degradation of target antigens increases the likelihood of vaccine efficacy. Therefore, enhancements to the vaccine construct such as the fusion of the alternative ubiquitin peptide to the vaccine construct can improve overall efficacy and likelihood of successful vaccination and/or treatment.

In some embodiments, the isolated nucleic acid further comprises a ubiquitin peptide at the 5’ terminus of the coding region. In some embodiments, the ubiquitin peptide further comprises a C-terminal extension peptide of at least about 25 amino acids in length (e.g., about any of 25, 26, 27, 28, 29, or 30 or more amino acids in length) . In some embodiments, the ubiquitin peptide further comprises a C-terminal extension peptide of exactly 25 amino acids in length. In some embodiments, the ubiquitin peptide comprises an amino acid sequence encoded at the 5’ terminus of the coding sequence that comprises the amino acid sequence of about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 120. In some embodiments, the ubiquitin peptide comprises an amino acid sequence encoded at the 5’ terminus of the coding sequence that comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the extension peptide comprises the amino acid sequence of about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 121. In some embodiments, the extension peptide comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the ubiquitin peptide comprises an amino acid sequence that is encoded by any one of the UBA52, RPS27A, UBB, or UBC genes. In some embodiments, the ubiquitin peptide comprises an amino acid sequence of about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence that is encoded by any one of the UBA52, RPS27A, UBB, or UBC genes. For example, the fusion protein can comprise, from N-terminus to C-terminus: a signal domain peptide, an optional ubiquitin peptide (either comprising or not comprising the C-terminal extension peptide) , a target antigen, an optional linker, an optional helper T cell epitope, a trafficking peptide, an optional 2A (e.g., P2A) cleavable linker, and an optional immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: an optional signal peptide, an optional ubiquitin peptide (either comprising or not comprising the C-terminal extension peptide) , a target antigen, an optional linker, a helper T cell epitope, an optional trafficking peptide, a 2A (e.g., P2A) cleavable linker, and an immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: a signal peptide, a target antigen, an optional linker, an optional helper T cell epitope, a trafficking peptide, an optional 2A (e.g., P2A) cleavable linker, and an optional immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: an optional signal peptide, a target antigen, an optional linker, a helper T cell epitope, an optional trafficking peptide, a 2A (e.g., P2A) cleavable linker, and an immunomodulator.

F. Signal peptide

In some embodiments, the fusion proteins further comprise a signal peptide. As used herein, the term “signal peptide” is a term of art which refers to short peptides that may be present at the N-terminus or the C-terminus of a newly synthesized protein, that may function to properly translocate the protein. In some embodiments, the signal peptide assists with translocating the fusion protein encoded by the coding region of the isolated nucleic acid. In some embodiments, the signal peptide translocates the fusion peptide to Golgi apparatus, the Endoplasmic Reticulum (ER) , the endosome, the lysosome, the proteasome, etc. to promote the processing of the fusion protein for antigen presentation in antigen presenting cells (APCs) . Many signal peptides of prokaryotic and eukaryotic proteins are known in the art. For review, see e.g., Owji et al. Eur J Cell Biol. 2018 Aug; 97 (6) : 422-441.

In some embodiments, the signal peptide is derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor. In some embodiments, the signal peptide is derived from the same protein as the trafficking peptide. In some embodiments, the signal peptide comprises an amino acid sequence comprising at least about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-118. In some embodiments, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-118. In some embodiments, the signal peptide is encoded by a nucleic acid sequence comprising at least about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 64-71 and 163-164. In some embodiments, the signal peptide is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 64-71, 163-164, and 199.

In some embodiments, the signal peptide is at the N-terminus of the fusion protein encoded by the coding region of the isolated nucleic acid (e.g., mRNA) . In some embodiments, the signal peptide is at the C-terminus of the fusion protein encoded by the coding region of the isolated nucleic acid (e.g., mRNA) .

For example, the fusion protein can comprise, from N-terminus to C-terminus: a signal peptide, an optional ubiquitin peptide (either comprising or not comprising the C-terminal extension peptide) , a target antigen, an optional linker, an optional helper T cell epitope, a trafficking peptide, an optional 2A (e.g., P2A) cleavable linker, and an optional immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: an optional signal peptide, an optional ubiquitin peptide (either comprising or not comprising the C-terminal extension peptide) , a target antigen, an optional linker, a helper T cell epitope, an optional trafficking peptide, a 2A (e.g., P2A) cleavable linker, and an immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C- terminus: a signal peptide, a target antigen, an optional linker, an optional helper T cell epitope, a trafficking peptide, an optional 2A (e.g., P2A) cleavable linker, and an optional immunomodulator. In some embodiments, the fusion protein can comprise, from N-terminus to C-terminus: an optional signal peptide, a target antigen, an optional linker, a helper T cell epitope, an optional trafficking peptide, a 2A (e.g., P2A) cleavable linker, and an immunomodulator.

G. mRNA Coding Sequence

In some embodiments, the isolated nucleic acid is an mRNA. The mRNA can further comprise a 5’ untranslated region (UTR) . In some embodiments, the 5’ UTR has at least 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the 5’ UTR has at least 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 198. In some embodiments, the isolated nucleic acid mRNA further comprises a 3’ untranslated region (UTR) . In some embodiments, the 3’ UTR has at least 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the mRNA further comprises a poly (A) sequence. In some embodiments, the poly (A) sequence has a length of about 50 nucleotides or longer. In some embodiments, the mRNA further comprises a 5’ cap.

In some embodiments, the coding sequence encoding the fusion protein is codon optimized. In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 436 of SEQ ID NO: 2.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 523 of SEQ ID NO: 3.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-2, 423 of SEQ ID NO: 4.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 709 of SEQ ID NO: 5.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 304 of SEQ ID NO: 6.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 295 of SEQ ID NO: 7.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-2, 396 of SEQ ID NO: 8.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 220 of SEQ ID NO: 10.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 208 of SEQ ID NO: 11.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 178 of SEQ ID NO: 12.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 175 of SEQ ID NO: 13.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 178 of SEQ ID NO: 14.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 781 of SEQ ID NO: 156.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 781 of SEQ ID NO: 157.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 781 of SEQ ID NO: 158.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 790 of SEQ ID NO: 161.

In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to nucleotides 72-1, 790 of SEQ ID NO: 162.

In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 10-14, 156-158, and 161-162. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 10-14, 156-158, and 161-162. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 10-14, 156-158, and 161-162.

In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence that encodes the fusion protein comprising an amino acid sequence having at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid selected from the group consisting of SEQ ID NOs: 90-96, 98-102, and 179-180. In some embodiments, the isolated nucleic acid encodes a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-96, 98-102, and 179-180.

Thus, in some embodiments, the coding sequence encoding the fusion protein is codon optimized. In some embodiments, the coding sequence of the isolated nucleic acid encoding the fusion protein comprises a nucleic acid sequence having a least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to: nucleotides 72-1, 436 of SEQ ID NO: 2; nucleotides 72-1, 523 of SEQ ID NO: 3; nucleotides 72-2, 423 of SEQ ID NO: 4; nucleotides 72-1, 709 of SEQ ID NO: 5; nucleotides 72-1, 304 of SEQ ID NO: 6; nucleotides 72-1, 295 of SEQ ID NO: 7; nucleotides 72-2, 396 of SEQ ID NO: 8; nucleotides 72-1, 220 of SEQ ID NO: 10; nucleotides 72-1, 208 of SEQ ID NO: 11; nucleotides 72-1, 178 of SEQ ID NO: 12; nucleotides 72-1, 175 of SEQ ID NO: 13; nucleotides 72-1, 178 of SEQ ID NO: 14; nucleotides 72-1, 781 of SEQ ID NO: 156; nucleotides 72-1, 781 of SEQ ID NO: 157; nucleotides 72-1, 781 of SEQ ID NO: 158; nucleotides 72-1, 790 of SEQ ID NO: 161; or nucleotides 72-1, 790 of SEQ ID NO: 162. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleic acid selected from the group consisting of SEQ ID NOs: 2-8, 10-14, 156-158, and 161-162. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 10-14, 156-158, and 161-162. In some embodiments, the isolated nucleic acid encodes a polypeptide having at least 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-96, 98-102, and 179-180. In some embodiments, the isolated nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-96, 98-102, and 179-180.

H. Linker Sequences

The polypeptides encoded by the isolated nucleic acids described herein in some embodiments comprise linker sequences, which can be present, for example, between portions of the fusion protein or between the fusion protein and the immunomodulators. The linkers can be peptide linkers of any length. In some embodiments, the peptide linker is from about 1 to about 10 amino acids long, from about 2 to about 15 amino acids long, from about 3 to about 12 amino acids long, from about 4 to about 10 amino acids long, from about 5 to about 9 amino acids long, from about 6 to about 8 amino acids long, from about 1 to about 20 amino acids long, from about 21 to about 30 amino acids long, from about 1 to about 30 amino acids long, from about 2 to about 20 amino acids long, from about 10 to about 30 amino acids long, from about 2 to about 19 amino acids long, from about 2 to about 18 amino acids long, from about 2 to about 17 amino acids long, from about 2 to about 16 amino acids long, from about 2 to about 10 amino acids long, from about 2 to about 14 amino acids long, from about 2 to about 13 amino acids long, from about 2 to about 12 amino acids long, from about 2 to about 11 amino acids long, from about 2 to about 9 amino acids long, from about 2 to about 8 amino acids long, from about 2 to about 7 amino acids long, from about 2 to about 6 amino acids long, or from about 2 to about 5 amino acids long. In some embodiments, the peptide linker is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the peptide linker is any of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In some embodiments, the peptide linker is about 2 to about 30 amino acids long, such as about 2 to about 15 amino acids long, about 15 amino acids long, or about 6 amino acids long.

In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G) n, glycine-serine polymers (including, for example, (GS) n, (GSGGS) n (SEQ ID NO: 143) , (GGGS) n (SEQ ID NO: 144) , or (GGGGS) n (SEQ ID NO: 145) , where n is an integer of at least one) , glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 3: 73-142 (1992) ) . In some embodiments, the linker comprises amino acid residues selected form the group consisting of glycine, serine, arginine, and alanine. Exemplary flexible linkers include, but are not limited to, Gly-Gly, Gly-Gly-Ser-Gly (SEQ ID NO: 137) , Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 138) , Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 139) , Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 140) , Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 141) , Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 142) , Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 131) , Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 130) , Gly-Arg-Ala-Gly-Gly-Gly-Gly-Ala-Gly-Gly-Gly-Gly (SEQ ID NO: 133) , Gly-Arg-Ala-Gly-Gly-Gly (SEQ ID NO: 134) , GGGSGGGGSGGGSGGGGS (SEQ ID NO: 109) , GGSGGGGSGGKK (SEQ ID NO: 110) , KKLGSSGGGGSPGGGSS (SEQ ID NO: 111) , GSGGSGGGGSGG (SEQ ID NO: 112) , GGGGS (SEQ ID NO: 132) , and the like. In some embodiments, the linker is GGGSGGGGSGGGSGGGGS (SEQ ID NO: 109) . In some embodiments, the linker comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 109-112 and 130-145. Exemplary nucleotides encoding flexible linkers can include, but are not limited to, nucleic acid sequences set forth in SEQ ID NOs: 36-61, 153-155, 165-169, 182-184, and 203-205. The ordinarily skilled artisan will recognize that design of an isolated nucleic acid (e.g., mRNA) vaccine encoding a fusion polypeptide can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired antigenic structure.

In some embodiments, the linker is a stable linker (not cleavable by protease, especially MMPs) . In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a protease substrate cleavage sequence, for example, an MMP substrate cleavage sequence. Substrate sequences that can be cleaved by MMPs have been extensively studied. In some embodiments, the protease cleavage site is recognized by MMP-2, MMP-9, or a combination thereof. In some embodiments, the linker is a 2A peptide, for example any one or more of a P2A, F2A, T2A, or E2A cleavable linker. In some embodiments, the linker is a P2A linker. In some embodiments, the P2A linker is encoded by a nucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 87, 88, and 200. In some embodiments, the P2A linker comprises an amino acid sequence of SEQ ID NO: 129.

I. mRNA Features

The isolated nucleic acids provided herein may comprise sequences or features in addition to the coding sequence encoding a fusion protein. These features may be used, for example, to improve antigenicity of the fusion protein encoded by the isolated nucleic acid (e.g., mRNA) and to increase stability of the isolated nucleic acid. Exemplary features may include, but are not limited to, a nucleic acid sequence encoding a signal peptide, 5’ and 3’ untranslated regions (UTRs) , poly (A) tails, 5’ caps, N-terminal ubiquitin peptides attached to a 25 amino acid C-terminal extension peptide, and/or chemical modifications of the isolated nucleic acid (e.g., mRNA) .

i. 5’ and 3’ Untranslated Region (UTR)

In some embodiments, the isolated nucleic acid (e.g., mRNA) comprises one or more untranslated regions (UTRs) . The UTR of the isolated nucleic acid (e.g., mRNA) may be involved in various regulatory aspects of gene expression. It should be understood that the UTRs (e.g., the 5’ UTRs and/or the 3’ UTRs) provided herein are examples, and that the isolated nucleic acid (e.g., mRNA) may comprise any UTR from any gene. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide synthetic (e.g., artificial UTRs) which are not variants of wild-type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5’ or 3’ UTR may be inverted, shortened, lengthened, or made chimeric with one or more other 5’ UTRs or 3’ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3’ or 5’ UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, or swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3’ or 5’) comprise a variant UTR.

In some embodiments, a double, triple, or quadruple UTR, such as a 5’ or 3’ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. It is also within the scope of the present invention to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. The UTRs provided herein may also include translation enhancer elements (TEE) .

In some embodiments, the mRNA comprises a 5’ UTR. The 5’ UTRs provided herein may be recognized by the ribosome, thereby allowing the ribosome to bind and initiate translation of the mRNA (e.g., translation of the coding sequence and/or nucleic acid encoding the mRNA) . In some embodiments, the 5’ UTR is upstream from the coding sequence of the mRNA.

In some embodiments, the 5’ UTR is from an organism or is synthetic. In some embodiments, the 5’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 15. In some embodiments, the 5’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 15.

In some embodiments, the 5’ UTR is from an organism or is synthetic. In some embodiments, the 5’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 198. In some embodiments, the 5’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 198.

In some embodiments, the mRNA comprises a 3’ UTR. The 3’ UTRs provided herein may be involved in translation termination (e.g., translation of the coding sequence and/or nucleic acid encoding the mRNA) and can also be important for post-transcriptional modifications. In some embodiments, the 3’ UTR is downstream from the coding sequence of the mRNA. In some embodiments, the 3’ UTR immediately follows the translation stop codon of the coding sequence of the mRNA. In some embodiments, the mRNA comprises one or more stop codons before the 3’ UTR.

In some embodiments, the 3’ UTR is from an organism or is synthetic. In some embodiments, the 3’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

In some embodiments, the isolated nucleic acid (e.g., mRNA) comprises a 5’ UTR and a 3’ UTR, such as any of the 5’ UTRs and 3’ UTRs described or provided herein. In some embodiments, the 5’ UTR and the 3’ UTR are derived from the same species. In some embodiments, the 5’ UTR and the 3’ UTR are not derived from the same species. In some embodiments, the 5’ UTR is synthetic, and the 3’ UTR is not synthetic. In some embodiments, the 5’ UTR is not synthetic, and the 3’ UTR is synthetic. In some embodiments, both the 5’ UTR and the 3’ UTR are synthetic. In some embodiments, neither the 5’ UTR nor the 3’ UTR are synthetic.

In some embodiments, the 5’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity the nucleic acid sequence set forth in SEQ ID NO: 15, and the 3’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the 5’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 15, and the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

In some embodiments, the 5’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity the nucleic acid sequence set forth in SEQ ID NO: 198, and the 3’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity the nucleic acid sequence set forth in SEQ ID NO: 16. In some embodiments, the 5’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 198, and the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 16.

ii. Additional Features

In some embodiments, the isolated nucleic acid, e.g., mRNA comprises one or more additional features, such as but not limited to a poly (A) sequence, one or more chemical modifications, a 5’ cap, or a combination thereof.

In some embodiments, the isolated nucleic acid, e.g., mRNA comprises a poly (A) sequence (e.g., a polyadenylation sequence) . Poly (A) sequences consist of multiple adenosine monophosphates in succession. In some embodiments, the poly (A) sequence is crucial for translation of the mRNA. In some embodiments, the poly (A) sequence is downstream of the coding sequence of the mRNA. In some embodiments, the poly (A) sequence is downstream of a 3’ UTR of the mRNA. In some embodiments, the poly (A) sequence has a length of about 50 nucleotides or longer, such as about 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 110 nucleotides, 120 nucleotides, 130 nucleotides, 140 nucleotides, 150 nucleotides, or longer. In some embodiments, the poly (A) sequence has a length of about 100 nucleotides or shorter, such as about 90 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides, or shorter. In some embodiments, the poly (A) sequence has a length of about 100 to 200 nucleotides. In some embodiments, the poly (A) sequence has a length of about 150 nucleotides.

In some embodiments, the isolated nucleic acid, e.g., mRNA comprises a chemical modification. In some embodiments, at least about 10% (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or all) of the nucleotides on the isolated nucleic acid are chemically modified. In some embodiments, the chemical modification occurs in the coding sequence, an intron, the 3’ UTR, or the 5’ UTR of the mRNA. In some embodiments, the chemical modification includes a modification to an adenosine, cytidine, guanosine, and/or a uridine base. In some embodiments, at least any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%adenosine base of the isolated nucleic acid comprises a chemical modification. In some embodiments, at least any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%cytidines of the isolated nucleic acid comprise a chemical modification. In some embodiments, at least any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%guanosine base of the isolated nucleic acid comprises a chemical modification. In some embodiments, at least any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%uridine base of the isolated nucleic acid comprises a chemical modification. In some embodiments, the adenosine is converted to an inosine, or methylated to N¹-methyladenosine, N⁶-methyladenosine, or N⁶, N⁶-dimethyladenosine. In some embodiments, the cytidine is converted to uridine, acetylated to N⁴-acetylcytidine, or methylated to 3-methylcytidine or 5-methylcytidine. In some embodiments, the 5-methylcytidine is further converted to 5- hydroxymethylcytidine. In some embodiments, the guanosine is methylated to 7-methylguanosine or oxidized to 7, 8-dihydro-8-oxoguanosine. In some embodiments, the ribose sugars of all nucleotides can be 2′-O-methylated. In some embodiments, the uridine is be converted to pseudouridine (Ψ) . In some embodiments, each uridine of the (e.g. ) mRNA is converted to a pseudouridine. In some embodiments, the (e.g. ) mRNA comprises an N¹-methylpseudouridine chemical modification. In some embodiments, each uridine of the (e.g. ) mRNA is converted to an N¹-methylpseudouridine.

In some embodiments, the isolated nucleic acid, e.g., mRNA comprises a 5’ cap. In some embodiments, the 5’ cap comprises a 7-methylguanosine (m⁷G) moiety, a trimethylated m^2′2′7G moiety, or an NAD⁺. In some embodiments, the 5’ cap is added to the (e.g. ) mRNA via a 5’ –5’ triphosphate linkage to the first transcribed nucleotide of the mRNA.

III. Methods of making the isolated nucleic acid

The isolated nucleic acid (e.g., mRNA) described herein can be synthesized by methods known in the art, for example, through in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription can be obtained, for example, by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. In some embodiments, the RNA may have modified nucleosides, including, for example, pseudouridine, optionally 1-methylpseudouridine.

Further, isolated nucleic acids such as mRNA produced by in vitro transcription can be purified to remove contaminants, for example dsRNA that activates an immunogenic type I interferon response. This interferon response leads to inhibition of translation and degradation of cellular and ribosomal RNA, which decreases the vaccine efficiency. Methods such as reverse-phase fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) can be used to purify the isolated nucleic acid (e.g., mRNA) construct, thereby dramatically increasing protein production by up to 1,000-fold in human dendritic cells, which are responsible for antigen processing and presentation to lymphocytes to elicit an immune response and effectively vaccinate an individual (see, e.g., Pardi et al. (2018) , Nat Rev Drug Discov; 17: 261-279, hereby incorporated by reference in its entirety) .

IV. Pharmaceutical Compositions, Unit Dosages, Articles of Manufacture, and Kits

Further provided by the present application are compositions (e.g., pharmaceutical compositions) comprising any of the isolated nucleic acids, such as a messenger ribonucleic acid (mRNA) , described herein, wherein the isolated nucleic acid comprises a coding sequence encoding a fusion protein described herein, and optionally a pharmaceutically acceptable carrier.

The composition (e.g., pharmaceutical composition) of the present invention may comprise one or more components to, for example, increase stability of the isolated nucleic acid (e.g., mRNA) , increase cell transfection of the isolated nucleic acid (e.g., mRNA) , permit sustained or delayed release of the isolated nucleic acid (e.g., mRNA) , change the biodistribution of the isolated nucleic acid (e.g., mRNA) , increase the translation of encoded fusion protein in vivo, and/or alter the release profile of the encoded fusion protein in vivo.

In some embodiments, the composition (e.g., pharmaceutical composition) may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art (see for example Remington's The Science and Practice of Pharmacy, 21^st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006) .

Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and/or combinations thereof. Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly (vinyl-pyrrolidone) (crospovidone) , sodium carboxymethyl starch (sodium starch glycolate) , carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose) , methylcellulose, pregelatinized starch (starch 1500) , microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicatesodium lauryl sulfate, quaternary ammonium compounds, and/or combinations thereof.

Surface active agents and/or emulsifiers may include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin) , colloidal clays (e.g., bentonite [aluminum silicate] and [magnesium aluminum silicate] ) , long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol) , carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer) , carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose) , sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolauratepolyoxyethylene sorbitan [60] , polyoxyethylene sorbitan monooleatesorbitan monopalmitatesorbitan monostearatesorbitan tristearateglyceryl monooleate, sorbitan monooleate ), polyoxyethylene esters (e.g., polyoxyethylene monostearate [45] , polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., ) , polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [30] ) , poly (vinyl-pyrrolidone) , diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, 68, 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or combinations thereof.

A binding agent may be starch (e.g., cornstarch and starch paste) ; gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol) ; natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly (vinyl-pyrrolidone) , magnesium aluminum silicateand larch arabogalactan) ; alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binding agent.

Preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite. Chelating agents include ethylenediaminetetraacetic acid (EDTA) , citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, benzyl alcohol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA) , butylated hydroxytoluene (BHT) , ethylenediamine, sodium lauryl sulfate (SLS) , sodium lauryl ether sulfate (SLES) , sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTmethylparaben, 115, NEOLONE^TM, KATHON^TM, and/or

Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES) , magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and/or combinations thereof. Lubricating agents may selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

Examples of oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.

In some aspects, the isolated nucleic acid or the composition (e.g., pharmaceutical composition) described herein may comprise lipidoids, liposomes, lipid nanoparticles (LNPs) , polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the isolated nucleic acid, such as mRNA (e.g., for transplantation into an individual) , hyaluronidase, nanoparticle mimics, and combinations thereof. In some embodiments, the compositions (e.g., pharmaceutical compositions) of the invention can include one or more excipients provided in a ratio to optimize the properties of the isolated nucleic acid, e.g., mRNA. In some embodiments, the isolated nucleic acid (e.g., mRNA) of the present invention may be formulated in a pharmaceutical composition using self-assembled nucleic acid nanoparticles. In some embodiments, the composition (e.g., pharmaceutical composition) comprises at least one isolated nucleic acid (e.g., mRNA, such 1, 2, 3, 4 or 5 mRNA) .

In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0. In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 6.5. In some embodiments, the pharmaceutical composition can also be made to be isotonic with blood by the addition of a suitable tonicity modifier.

The (pharmaceutical) compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the isolated nucleic acid (e.g., mRNA) with an excipient and/or one or more other accessory ingredients. In some embodiments, the pharmaceutical composition may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the isolated nucleic acid (e.g., mRNA) . The amount of the isolated nucleic acid (e.g., mRNA) may generally be equal to the dosage of the isolated nucleic acid (e.g., mRNA) which would be administered to an individual and/or a convenient fraction of such a dosage including, but not limited to, one-half or one-third of such a dosage.

In some embodiments, the relative amounts of the isolated nucleic acid (e.g., mRNA) , the pharmaceutically acceptable excipient, and/or any additional ingredients in the pharmaceutical composition may vary, depending upon the identity, size, and/or condition of the individual being administered the pharmaceutical composition as well as the route by which the pharmaceutical composition is to be administered. In some embodiments, the pharmaceutical composition may comprise between 0.1%and 99% (w/w) of the isolated nucleic acid (e.g., mRNA) .

A. Lipid nanoparticles (LNPs)

In some embodiments, the composition (e.g., pharmaceutical composition) comprises an LNP. In some embodiments, the modified isolated nucleic acid, e.g., RNA is formulated in the LNP, such as those described in International Publication No. WO2012170930, herein incorporated by reference in its entirety. In some embodiments, the particle size of the LNP may be increased and/or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the mRNA when administered to an individual.

In some embodiments, the LNP comprises between about 30 molar percent to about 55 molar percent of a cationic lipid. In some embodiments, the LNP comprises greater than about 30 molar percent of a cationic lipid, such as greater than any of about 35 molar percent, 40 molar percent, 45 molar percent, 50 molar percent, 55 molar percent, or greater, of a cationic lipid. In some embodiments, the LNP comprises less than about 55 molar percent of a cationic lipid, such as less than any of about 50 molar percent, 45 molar percent, 40 molar percent, 35 molar percent, 30 molar percent, or less, of a cationic lipid.

In some embodiments, the LNP comprises between about 5 molar percent to about 40 molar percent of a phospholipid. In some embodiments, the LNP comprises greater than about 5 molar percent of a phospholipid, such as greater than any of about 10 molar percent, 15 molar percent, 20 molar percent, 25 molar percent, 30 molar percent, 35 molar percent, 40 molar percent, or greater, of a phospholipid. In some embodiments, the LNP comprises less than about 40 molar percent of a phospholipid, such as less than any of about 35 molar percent, 30 molar percent, 25 molar percent, 20 molar percent, 15 molar percent, 10 molar percent, 5 molar percent, or less, of a phospholipid.

In some embodiments, the LNP comprises between about 20 molar percent to about 50 molar percent of a sterol. In some embodiments, the LNP comprises greater than about 20 molar percent of a sterol, such as greater than any of about 25 molar percent, 30 molar percent, 35 molar percent, 40 molar percent, 45 molar percent, 50 molar percent, or greater, of a sterol. In some embodiments, the LNP comprises less than about 50 molar percent of a sterol, such as less than any of about 45 molar percent, 40 molar percent, 35 molar percent, 30 molar percent, 25 molar percent, 20 molar percent, or less, of a sterol.

In some embodiments, the LNP comprises a cationic lipid, a phospholipid, a sterol, and a polymer conjugated lipid, such as any of the cationic lipids, phospholipids, sterols, and polymer conjugated lipids described herein. In some embodiments, the LNP comprises i) between about 30 molar percent to about 55 molar percent of a cationic lipid, ii) between about 5 molar percent to about 40 molar percent of a phospholipid.

In some embodiments, the LNP comprises a total lipid to modified RNA weight ratio of about 10: 1 to about 30: 1, such as any of about 10: 1 to about 20: 1, about 15: 1 to about 25: 1, and about 20: 1 to about 30: 1. In some embodiments, the LNP comprises a total lipid to modified RNA weight ratio of greater than about 10: 1, such as greater than any of about 15: 1, 20: 1, 25: 1, 30: 1, or greater. In some embodiments, the LNP comprises a total lipid to modified RNA weight ratio of less than about 30: 1, such as less than any of about 25: 1, 20: 1, 15: 1, 10: 1, or less. In some embodiments, the total lipid to mRNA weight ratio may be adjusted depending on the other components of the pharmaceutical composition, the individual to be administered, and/or the route of administration. The amount of mRNA in an LNP, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy) .

In some embodiments, the LNPs disclosed herein further comprise a therapeutic payload. The payload can be any substance or compound that has a therapeutic or prophylactic effect. In some embodiments, the therapeutic payload is a small molecule, a cytotoxin, a radioactive ion, a chemotherapeutic compound, a vaccine, or a compound that elicits an immune response.

In some embodiments, the LNPs disclosed herein comprise a nucleic acid. In some embodiments, the nucleic acid is a DNA. In some embodiments, the DNA is catalytic DNA, plasmid DNA, aptamer, or complementary DNA (cDNA) . In some embodiments, the nucleic acid is an RNA. In some embodiments, the RNA is a messenger RNA (mRNA) , circular RNA (circRNA) , cyclization precursor RNA, self-amplifying RNA (saRNA) , antisense oligonucleotide, microRNA (miRNA) , miRNA inhibitor (e.g., antagomir or antimir) , messenger-RNA-interfering complementary RNA (micRNA) , multivalent RNA, dicer substrate RNA (dsRNA) , small hairpin RNA (shRNA) , antisense RNA, transfer RNA (tRNA) , asymmetrical interfering RNA (aiRNA) , a ribozyme, an aptamer, or a vector. In some embodiments, the RNA is an mRNA hybrid. In some embodiments, the nucleic acid is an mRNA. In some embodiments, the mRNA encodes a protein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the LNPs comprise an RNAi agent or RNAi-inducing agent.

The terms “circRNA” or “circular RNA” are used interchangeably and refer to a polyribonucleotide that forms a circular structure through covalent bonds. Circular RNA (circRNA) is a type of single-stranded RNA which forms a 3’-5’ covalently closed loop. CircRNA, or circular RNA, can be produced through various mechanisms. One primary method is backs plicing, a non- canonical splicing process mediated by the spliceosome. In this process, a downstream splice donor site i s joined to an upstream splice acceptor site, forming a covalently closed loop. Additionally, circRNAs ca n also be generated through chemical ligation, enzymatic ligation, and ribozyme methods. These alternat ive approaches enable the formation of circular RNA structures, expanding the variety of circRNAs that can be synthesized for research and therapeutic applications. Unlike linear mRNAs, circRNAs do not require a 5’-cap or 3’-poly (A) tail for their stability. The closed ring structure of circRNAs protects them from exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and having a longer half-life compared to their linear mRNA counterparts.

Self-replicating RNA (saRNA) , such as that derived from viral replicons, is useful for expression of proteins. It results in the production of numerous copies of the original RNA, leading to high and sustained expression levels of the protein of interest.

i. Cationic Lipids

In one embodiment, the cationic lipid contained in the compositions, nanoparticle compositions, or nanoparticles described herein is a cationic lipid described in International Patent Publication No. WO2021204175, the entirety of which is incorporated herein by reference.

In one embodiment, the cationic lipid is a compound of Formula (01-I) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

G1 and G2 are each independently a bond, C2-C12 alkylene, or C2-C12 alkenylene, wherein one or more -CH2-in the alkylene or alkenylene is optionally replaced by -O-;

L1 is –OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NraC (=O) R1, -C (=O) NRbRc, -NraC (=O) NRbRc, -OC (=O) NRbRc, -NraC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (Orb) (Orc) , - (C6-C10 arylene) -R1, - (6-to 10-membered heteroarylene) -R1, or R1;

L2 is –OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NreRf, -NRdC (=O) NreRf, -OC (=O) NreRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (Ore) (Orf) , - (C6-C10 arylene) -R2, - (6-to 10-membered heteroarylene) -R2, or R2;

R1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;

Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;

Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

G3 is C2-C24 alkylene, C2-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene;

R3 is -N (R4) R5;

R4 is C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4-to 8-membered heterocyclyl, or C6-C10 aryl; or R4, G3 or part of G3, together with the nitrogen to which they are attached form a cyclic moiety;

R5 is C1-C12 alkyl or C3-C8 cycloalkyl; or R4, R5, together with the nitrogen to which they are attached form a cyclic moiety;

x is 0, 1 or 2; and

wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

In one embodiment, the cationic lipid is a compound of Formula (01-II) :

is a single bond or a double bond;

L2 is –OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NreRf, -NRdC (=O) NreRf, -OC (=O) NreRf, -NRdC (=O) OR2, - SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (Ore) (Orf) , - (C6-C10 arylene) -R2, - (6-to 10-membered heteroarylene) -R2, or R2;

R1 and R2 are each independently C6-C32 alkyl or C6-C32 alkenyl;

Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;

Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

G4 is a bond, C1-C23 alkylene, C2-C23 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene;

R3 is -N (R4) R5;

R4 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, 4-to 8-membered heterocyclyl, or C6-C10 aryl; or R4, G3 or part of G3, together with the nitrogen to which they are attached form a cyclic moiety;

x is 0, 1 or 2; and

In one embodiment, the compound is a compound of Formula (01-I-B) , (01-I-B’) , (01-I-B” ) , (01-I-C) , (01-I-D) , or (01-I-E) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, G1 and G2 are each independently C3-C7 alkylene. In one embodiment, G1 and G2 are each independently C5 alkylene. In one embodiment, G3 is C2-C4 alkylene. In one embodiment, G3 is C2 alkylene. In one embodiment, G3 is C4 alkylene.

In one embodiment, R3 has one of the following structures:

In one embodiment, R1, R2, Rc and Rf are each independently branched C6-C32 alkyl or branched C6-C32 alkenyl. In one embodiment, R1, R2, Rc and Rf are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl. In one embodiment, R1, R2, Rc and Rf are each independently -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl. In one embodiment, R1, R2, Rc and Rf are each independently -R7-CH (R8) (R9) , wherein R7 is C0-C1 alkylene, and R8 and R9 are independently C4-C8 alkyl.

In one embodiment, the compound is a compound in Table 2, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

Table 2:

In one embodiment, the cationic lipid contained in the compositions, nanoparticle compositions, or nanoparticles provided herein is a cationic lipid described in International Patent Publication No. WO 2023/138611, the entirety of which is incorporated herein by reference. In one embodiment, the cationic lipid is a compound of Formula (02-I) :

G1 and G2 are each independently C2-C12 alkylene or C2-C12 alkenylene, wherein one or more -CH2-in G1 and G2 is optionally replaced by -O-, -C (=O) O-, or -OC (=O) -;

each L1 is independently –OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NraC (=O) R1, -C (=O) NRbRc, -NraC (=O) NRbRc, -OC (=O) NRbRc, -NraC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (Orb) (Orc) , -NraP (=O) (Orb) (Orc) ;

each L2 is independently –OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NreRf, -NRdC (=O) NreRf, -OC (=O) NreRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (Ore) (Orf) , -NRdP (=O) (Ore) (Orf) ;

R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;

Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;

G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by a C3-C8 cycloalkylene or C3-C8 cycloalkenylene;

R3 is -N (R4) R5, -OR6, or -SR6;

R4 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;

R5 is H, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;

R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C10 aryl;

x is 0, 1, or 2; and

wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

In one embodiment, the cationic lipid is a compound of Formula (02-II) :

R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;

Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;

R3 is -N (R4) R5, -OR6, or -SR6;

x is 0, 1, or 2; and

In one embodiment, the compound is a compound of Formula (02-V-A) , (02-V-B) , (02-V-C) , (02-V-D) , (02-V-E) , (02-V-F) :

wherein z is an integer from 2 to 12,

x0 is an integer from 1 to 11;

y0 is an integer from 1 to 11;

x1 is an integer from 0 to 9;

y1 is an integer from 0 to 9;

x2 is an integer from 2 to 5;

x3 is an integer from 1 to 5;

x4 is an integer from 0 to 3;

y2 is an integer from 2 to 5;

y3 is an integer from 1 to 5; and

y4 is an integer from 0 to 3;

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, z is an integer from 2 to 6. In one embodiment, z is 2, 4, or 5. In one embodiment, x0 and y0 are independently 2 to 6. In one embodiment, x0 and y0 are independently 4 or 5. In one embodiment, x1 and y1 are independently 2 to 6. In one embodiment, x1 and y1 are independently 4 or 5. In one embodiment, x2 and y2 are independently an integer from 2 to 5. In one embodiment, x2 and y2 are independently 3 or 5. In one embodiment, x3 and y3 are both 1. In one embodiment, x4 and y4 are independently 0 or 1.

In one embodiment, each L1 is independently -OR1, -OC (=O) R1 or -C (=O) OR1, and each L2 is independently –OR2, -OC (=O) R2 or -C (=O) OR2. In one embodiment, R1 and R2 are independently straight C6-C10 alkyl, or -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl.

In one embodiment, the compound is a compound of formula (02-VI-A) , (02-VI-B) , (02-VI-C) , (02-VI-D) , (02-VI-E) , or (02-VI-F) :

wherein z is an integer from 2 to 12;

y is an integer from 2 to 12;

x0 is an integer from 1 to 11;

x1 is an integer from 0 to 9;

x2 is an integer from 2 to 5;

x3 is an integer from 1 to 5; and

x4 is an integer from 0 to 3;

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, z is an integer from 2 to 6. In one embodiment, z is 2, 4 or 5. In one embodiment, x0 is 4 or 5. In one embodiment, x1 is 4 or 5. In one embodiment, x2 is an integer from 2 to 5. In one embodiment, x2 is 3 or 5. In one embodiment, x3 is 0 or 1. In one embodiment, y is an integer from 2 to 6. In one embodiment, y is 5.

In one embodiment, each L1 is independently -OR1, -OC (=O) R1 or -C (=O) OR1, and L2 is -OC (=O) R2 or -C (=O) OR2, -NRdC (=O) R2, or -C (=O) NreRf. In one embodiment, R1 is straight C6-C10 alkyl or -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In one embodiment, R2 and Rf are each independently straight C6-C18 alkyl, C6-C18 alkenyl, or -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In one embodiment, Rd and Re are each independently H.

In one embodiment, the compound is a compound in Table 3, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

Table 3:

In one embodiment, the cationic lipid contained in the compositions, nanoparticle compositions, or nanoparticles described herein is a cationic lipid described in International Patent Publication No. WO2022152109, the entirety of which is incorporated herein by reference.

In one embodiment, the cationic lipid is a compound of Formula (03-I) :

G1 and G2 are each independently a bond, C2-C12 alkylene, or C2-C12 alkenylene, wherein one or more -CH2-in G1 and G2 is optionally replaced by -O-;

each L1 is independently –OC (=O) R1, -C (=O) OR1, -OC (=O) OR1, -C (=O) R1, -OR1, -S (O) xR1, -S-SR1, -C (=O) SR1, -SC (=O) R1, -NraC (=O) R1, -C (=O) NRbRc, -NraC (=O) NRbRc, -OC (=O) NRbRc, -NraC (=O) OR1, -SC (=S) R1, -C (=S) SR1, -C (=S) R1, -CH (OH) R1, -P (=O) (Orb) (Orc) , -NraP (=O) (Orb) (Orc) , - (C6-C10 arylene) -R1, - (6-to 10-membered heteroarylene) -R1, - (4-to 8-membered heterocyclylene) -R1, or R1;

each L2 is independently –OC (=O) R2, -C (=O) OR2, -OC (=O) OR2, -C (=O) R2, -OR2, -S (O) xR2, -S-SR2, -C (=O) SR2, -SC (=O) R2, -NRdC (=O) R2, -C (=O) NreRf, -NRdC (=O) NreRf, -OC (=O) NreRf, -NRdC (=O) OR2, -SC (=S) R2, -C (=S) SR2, -C (=S) R2, -CH (OH) R2, -P (=O) (Ore) (Orf) , -NRdP (=O) (Ore) (Orf) , - (C6-C10 arylene) -R2, - (6-to 10-membered heteroarylene) -R2, - (4-to 8-membered heterocyclylene) -R2, or R2;

R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;

Rc and Rf are each independently C1-C24 alkyl or C2-C24 alkenyl;

G3 is C2-C12 alkylene or C2-C12 alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by C3-C8 cycloalkylene, C3-C8 cycloalkenylene, C3-C8 cycloalkynylene, 4-to 8-membered heterocyclylene, C6-C10 arylene, or 5-to 10-membered heteroarylene;

R3 is hydrogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C3-C8 cycloalkynyl, 4-to 8-membered heterocyclyl, C6-C10 aryl, or 5-to 10-membered heteroaryl; or R3, G1 or part of G1, together with the nitrogen to which they are attached form a cyclic moiety; or R3, G3 or part of G3, together with the nitrogen to which they are attached form a cyclic moiety;

R4 is C1-C12 alkyl or C3-C8 cycloalkyl;

x is 0, 1, or 2;

n is 1 or 2;

m is 1 or 2; and

wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

In one embodiment, the compound is a compound of Formula (03-II-A) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (03-II-B) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (03-II-C) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (03-II-D) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, G1 and G2 are each independently C2-C12 alkylene. In one embodiment, G1 and G2 are each independently C5 alkylene. In one embodiment, G3 is C2-C6 alkylene.

In one embodiment, R3 is C1-C12 alkyl, C2-C12 alkenyl, or C3-C8 cycloalkyl. In one embodiment, R3 is C3-C8 cycloalkyl. In one embodiment, R3 is unsubstituted. In one embodiment, R4 is substituted C1-C12 alkyl. In one embodiment, R4 is –CH2CH2OH.

In one embodiment, L1 is –OC (=O) R1, -C (=O) OR1, -NraC (=O) R1, or -C (=O) NRbRc; and L2 is –OC (=O) R2, -C (=O) OR2, -NRdC (=O) R2, or -C (=O) NreRf. In one embodiment, R1, R2, Rc, and Rf are each independently straight C6-C18 alkyl, straight C6-C18 alkenyl, or -R7-CH (R8) (R9) , wherein R7 is C0-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In one embodiment, R1, R2, Rc, and Rf are each independently straight C7-C15 alkyl, straight C7-C15 alkenyl, or -R7-CH (R8) (R9) , wherein R7 is C0-C1 alkylene, and R8 and R9 are independently C4-C8 alkyl or C6-C10 alkenyl. In one embodiment, Ra, Rb, Rd, and Re are each independently H.

In one embodiment, the compound is a compound in Table 4, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

Table 4:

In one embodiment, the cationic lipid contained in the particles or compositions provided herein is a cationic lipid described in International Patent Application No. WO2022247755A1, the entirety of which is incorporated herein by reference.

In one embodiment, the cationic lipid is a compound of Formula (04-I) :

G1 and G2 are each independently a bond, C2-C12 alkylene, or C2-C12 alkenylene;

R1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;

Ra, Rb, Rd, and Re are each independently H, C1-C24 alkyl, or C2-C24 alkenyl;

Rc and Rf are each independently C1-C32 alkyl or C2-C32 alkenyl;

R0 is C1-C12 alkyl, C2-C12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;

G3 is C2-C12 alkylene or C2-C12 alkenylene;

R5 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl;

x is 0, 1, or 2;

s is 0 or 1; and

wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene, is independently optionally substituted.

In one embodiment, the cationic lipid is a compound of Formula (04-III) :

R1 and R2 are each independently C5-C32 alkyl or C5-C32 alkenyl;

G3 is C2-C12 alkylene or C2-C12 alkenylene;

G4 is C2-C12 alkylene or C2-C12 alkenylene;

R3 is -N (R4) R5 or -OR6;

R5 is C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C10 aryl, or 4-to 8-membered heterocycloalkyl; or R4, R5, together with the nitrogen to which they are attached form a cyclic moiety;

R6 is hydrogen, C1-C12 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, or C6-C10 aryl; and wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, and cyclic moiety is independently optionally substituted.

In one embodiment, the compound is a compound of Formula (04-IV) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, G3 is C2-C4 alkylene. In one embodiment, G4 is C2-C4 alkylene.

In one embodiment, R0 is C1-C6 alkyl. In one embodiment, R3 is -OH. In one embodiment, R3 is -N (R4) R5. In one embodiment, R4 is C3-C8 cycloalkyl. In one embodiment, R4 is unsubstituted. In one embodiment, R5 is –CH2CH2OH.

In one embodiment, L1 is –OC (=O) R1, -C (=O) OR1, -C (=O) R1, -C (=O) NRbRc, or R1; and L2 is –OC (=O) R2, -C (=O) OR2, -C (=O) R2, -C (=O) NreRf, or R2. In one embodiment, R1 and R2 are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl. In one embodiment, R1 and R2 are each independently -R7-CH (R8) (R9) , wherein R7 is C1-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl or C2-C10 alkenyl. In one embodiment, R1 is straight C6-C24 alkyl and R2 is branched C6-C24 alkyl. In one embodiment, R1 is straight C6-C24 alkyl and R2 is -R7-CH (R8) (R9) , wherein R7 is C1-C5 alkylene, and R8 and R9 are independently C2-C10 alkyl.

In one embodiment, the compound is a compound in Table 5, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

Table 5:

It is understood that any embodiment of the compounds provided herein, as set forth above, and any specific substituent and/or variable in the compound provided herein, as set forth above, may be independently combined with other embodiments and/or substituents and/or variables of the compounds to form embodiments not specifically set forth above. In addition, in the event that a list of substituents and/or variables is listed for any particular group or variable, it is understood that each individual substituent and/or variable may be deleted from the particular embodiment and/or claim and that the remaining list of substituents and/or variables will be considered to be within the scope of embodiments provided herein.

It is understood that in the present description, combinations of substituents and/or variables of the depicted formulae are permissible only if such contributions result in stable compounds.

B. Administration

The compositions (e.g., pharmaceutical compositions) may be suitable for a variety of modes of administration described herein, including for example systemic or localized administration. In some embodiments, the composition (e.g., pharmaceutical composition) is administered locally to the tumor. In some embodiments, the composition (e.g., pharmaceutical composition) is administered systemically. In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for intramuscular administration. In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for intratumoral administration. In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for intradermal administration. In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for topical administration. In some embodiments, the composition (e.g., pharmaceutical composition) is formulated for administration by topical ointment.

The compositions (e.g., pharmaceutical compositions) to be used for in vivo administration are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Sterility is readily accomplished by filtration through sterile filtration membranes. In some embodiments, the composition is free of pathogens. For parenteral administration, the composition (e.g., pharmaceutical composition) can be in the form of liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the composition (e.g., pharmaceutical composition) can be in a solid form and re-dissolved or suspended, e.g., in water, immediately prior to use. Lyophilized compositions are also included. Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets, as described herein.

In some embodiments, the composition (e.g., pharmaceutical composition) is formulated in accordance with routine procedures as a composition (e.g., pharmaceutical composition) adapted for parenteral administration, such as for intramuscular, intratumoral, or intradermal administration. In some embodiments, the composition (e.g., pharmaceutical composition) is formulated in accordance with routine procedures as a composition adapted for topical administration.

In some embodiments, the composition (e.g., pharmaceutical composition) is formulated in accordance with routine procedures as a composition (e.g., pharmaceutical composition) adapted for injection intramuscularly, intratumorally, intradermally, intravenously, intraperitoneally, subcutaneously, or intravitreally. In some embodiments, the composition (e.g., pharmaceutical composition) is formulated in accordance with routine procedures as a composition (e.g., pharmaceutical composition) adapted for topical application. Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered, it can be reconstituted as needed accordingly.

In some embodiments, the composition (e.g., pharmaceutical composition) is suitable for administration to a human. In some embodiments, the composition (e.g., pharmaceutical composition) is suitable for administration to a rodent (e.g., mice, rats) or non-human primates (e.g., Cynomolgus monkey) . In some embodiments, the composition (e.g., pharmaceutical composition) is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the composition (e.g., pharmaceutical composition) is contained in a multi-use vial. In some embodiments, the composition (e.g., pharmaceutical composition) is contained in bulk in a container. In some embodiments, the composition (e.g., pharmaceutical composition) is cryopreserved.

Also provided are unit dosage forms of any of the vaccines described herein, or compositions (such as pharmaceutical compositions) thereof. These unit dosage forms can be stored in a suitable packaging in single or multiple unit dosages and may also be further sterilized and sealed. In some embodiments, the composition (e.g., pharmaceutical composition) is administered as a single dose. In some embodiments, the composition (e.g., pharmaceutical composition) is administered as multiple doses. In some embodiments, the composition (e.g., pharmaceutical composition) is administered no more than once a year.

In some embodiments, the composition (e.g., pharmaceutical composition) is administered in combination with one or more anti-cancer therapies. In some embodiments, the one or more anti-cancer therapies are selected from a chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery. In some embodiments, the one or more anti-cancer therapies comprises anti-PD-1, anti-PDL-1, and/or anti-PDL-2 monoclonal antibodies or biologically active fragments thereof. In some embodiments, the one or more anti-cancer therapies further comprises anti-TGF-βRII antibodies or biologically active fragments thereof. In some embodiments, the individual is human.

In some cases, a subject method involves administering to an individual in need thereof an effective amount of a composition (e.g., pharmaceutical composition) described herein. In some embodiments, an “effective amount” of a subject composition (e.g., pharmaceutical composition) is an amount that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to prevent target antigen-expressing cancer (e.g., HPV+ cancer) or to reduce symptoms of said cancer in the individual by at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, or greater than 10-fold, compared to the individual in the absence of treatment with the composition (e.g., pharmaceutical composition) .

C. Combination Therapy

Exemplary routes of administration of any composition (e.g., pharmaceutical composition) described herein and one or more anti-cancer therapies include, but are not limited to, oral, intravenous, intracavitary, intratumoral, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and epidermal routes, or be delivered into lymph glands, body spaces, organs or tissues known to contain cancer cells. In some embodiments, the composition (e.g., pharmaceutical composition) is administered in combination with one or more anti-cancer therapies. In some embodiments, the composition (e.g., pharmaceutical composition) and the one or more anti-cancer therapies are administered by the same route. In some embodiments, the composition (e.g., pharmaceutical composition) and the one or more anti-cancer therapies are administered by different routes. In some embodiments, the composition (e.g., pharmaceutical composition) is administered by intramuscular injection and the one or more anti-cancer therapies are administered by subcutaneous injection or intravenous infusion. In some embodiments, the composition (e.g., pharmaceutical composition) and the one or more anti-cancer therapies are administered simultaneously. In some embodiments, the composition (e.g., pharmaceutical composition) and the one or more anti-cancer therapies are administered sequentially. In some embodiments, the one or more anti-cancer therapies are selected from a chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery. In some embodiments, the one or more anti-cancer therapies comprise an immunotherapy or therapeutic antibody. In some embodiments, the immunotherapy or therapeutic antibody comprises anti-PD-1, anti-PDL-1, and/or anti-PDL-2 monoclonal antibodies or biologically active fragment thereof. In some embodiments, the anti-PD-1, anti-PDL-1, and/or anti-PDL-2 monoclonal antibodies or biologically active fragment thereof are selected from one or more of: Atezolizumab, Cemiplimab, Dostarlimab, Nivolumab, Pidilizumab, Pembrolizumab, Lambrolizumab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Toripalimab, Retifanlimab, AMP-224, AMP-514, STI-A1110, Durvalumab, Avelumab, KY-1003, MCLA-145, AUR-012, STI-A1010, STI-A1014, KN035, Cosibelimab, AUNP12, BMS-936559, BMS-986189, and rHIgM12B7. In some embodiments, the anti-PD-1, anti-PDL-1, and/or anti-PDL-2 monoclonal antibody or biologically active fragment thereof is Atezolizumab. In some embodiments, the immunotherapy or therapeutic antibody comprises an inhibitor of TGF-β signaling, such as a small molecule-based, nucleic acid-based, or antibody or antibody fragment-based inhibitor of TGF-β, TGF-βRI, and/or TGF-βRII. In some embodiments, the inhibitor of TGF-β signaling is selected from one or more of: Vactosertib, Galunisertib, Bintrafusp alfa, AVID200, Luspatercept, Fresolimumab, LY3022859, Belagenpumatucel-L, SAR438459, SRK181-mIgG1, Trabedersen, AP11014, and AP15012. In some embodiments, the inhibitor of TGF-β signaling comprises an inhibitor of TGF-βRII. In some embodiments, the inhibitor of TGF-βRII comprises an antibody or antibody fragment. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F (ab’) ₂, Fv, scFv, or other antigen- binding subsequences of the full-length antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variants or derivatives thereof. In some embodiments, the one or more anti-cancer therapies comprises an anti-TGF-βRII antibody or biologically active fragment thereof.

In some embodiments, the composition (e.g., pharmaceutical composition) is administered in combination with one or more anti-cancer therapies. In some embodiments, the one or more anti-cancer therapies are selected from a chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery. In some embodiments, the immunotherapy is an immunomodulatory agent that modulates an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and ligands thereof. In some embodiments, the immunomodulatory agent, and/or a second immunomodulatory agent, and/or a third immunomodulatory agent is an immune-stimulating agent. In some embodiments, the immune-stimulating agent is an activator of OX40, 4-1BB or CD40.

“Immunomodulatory agent” refers to an agent that when present, alters, suppresses or stimulates the body's immune system. Immunomodulatory agents can target specific molecules, such as the checkpoint molecules. For example, immunomodulatory agents can include compounds that inhibit the activity of an immune checkpoint such as an antagonist (e.g., antagonistic antibody) of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR, or TIM3. The immunomodulatory agent can be of any one of the molecular modalities known in the art, including, but not limited to, aptamer, mRNA, siRNA, microRNA, shRNA, peptide, antibody, anticalin, Spherical nucleic acid, TALEN, Zinc Finger Nuclease, CRISPR/Cas9, and small molecule.

In some embodiments, the immunomodulatory agent comprises an immune checkpoint inhibitor that is a natural or engineered ligand of an inhibitory immune checkpoint molecule, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2) , ligands of TIM3 (e.g., Galectin-9) , ligands of A2a Receptor (e.g., adenosine, Regadenoson) , ligands of LAG3 (e.g., MHC class I or MHC class II molecules) , ligands of BTLA (e.g., HVEM, B7-H4) , ligands of KIR (e.g., MHC class I or MHC class II molecules) , ligands of PD-1 (e.g., PDL-1, PDL-2) , ligands of IDO (e.g., NKTR-218, Indoximod, NLG919) , ligands of CD47 (e.g., SIRP-alpha receptor) , and ligands of CSF1R. In some embodiments, the immune checkpoint inhibitor is an antibody, inhibitory small molecule, or antisense oligonucleotide. In some embodiments, the immune checkpoint inhibitor is an antibody that targets an inhibitory immune checkpoint protein. In some embodiments, the immunomodulatory agent is an antibody selected from the group consisting of anti-CTLA-4 (e.g., Ipilimumab, Tremelimumab, KAHR-102) , anti-TIM3 (e.g., F38-2E2, ENUM005) , anti-LAG3 (e.g., BMS-986016, IMP701, IMP321, C9B7W) , anti-KIR (e.g., Lirilumab, IPH2101, IPH4102) , anti-PD-1 (e.g., Cemiplimab, Dostarlimab, Nivolumab, Pidilizumab, Pembrolizumab, Lambrolizumab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Toripalimab, Retifanlimab, AMP-224, AMP-514, STI-A1110) , anti-PD-L1/anti-PD-L2 (e.g., Atezolizumab, Durvalumab, Avelumab, KY-1003, MCLA-145, AUR-012, STI-A1010, STI-A1014, KN035, Cosibelimab, AUNP12, BMS-936559, BMS-986189, rHIgM12B7) , anti-CD73 (e.g., AR-42 (OSU-HDAC42, HDAC-42, AR42, AR 42, OSU-HDAC 42, OSU-HDAC-42, NSC D736012, HDAC-42, HDAC 42, HDAC42, NSCD736012, NSC-D736012) , MEDI-9447) , anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9) , anti-CD47 (e.g., CC-90002, TTI-621, VLST-007) , anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1, anti-B7-H4, anti-CD52 (such as alemtuzumab) , anti-IL-10, anti-IL-35, anti-TGF-β (such as Fresolumimab) , anti-TGF-βRI or II (e.g., M7824, LY3022859) , anti-CSF1R (e.g., FPA008) , anti-NKG2A (e.g., monalizumab) , anti-MICA (e.g., IPH43) , and anti-CD39. In some embodiments, the antibody is an antagonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F (ab’) 2, and Fv, scFv, or other antigen-binding subsequences of the full-length antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variants or derivatives thereof.

The immunomodulatory agents can be used singly or in combination. For example, any number (such as any of 1, 2, 3, 4, 5, 6, or more) of immune checkpoint inhibitors can be used simultaneously or sequentially. Sequential administration of immunomodulatory agents can be separated by hours, days, or weeks. The administration route (s) for two or more immunomodulatory agents can be the same or different. For example, one immunomodulatory agent can be administered intratumorally, and a second immunomodulatory agent can be administered intravenously; or two immunomodulatory agents can be administered both intratumorally.

For example, in some embodiments, there is provided a method of treating cancer expressing one or more target antigens (e.g., an HPV+ cancer) in an individual (such as a human) , comprising administering to the individual i) an effective amount of a composition (e.g., pharmaceutical composition) comprising: an isolated nucleic acid comprising a coding sequence encoding a fusion protein comprising a target antigen and a trafficking peptide; or 1) a coding sequence encoding a fusion protein comprising a target antigen and a helper T cell epitope, and 2) a coding sequence encoding an immunomodulator, wherein the immunomodulator comprises GM-CSF and/or STING; and ii) an effective amount of an immunomodulatory agent (such as an immune checkpoint inhibitor, for example an anti-PD-1 or anti-PDL-1 antibody) . In some embodiments, the composition (e.g., pharmaceutical composition) and the immunomodulatory agent are administered sequentially. In some embodiments, the composition (e.g., pharmaceutical composition) and the immunomodulatory agent are administered simultaneously.

The present application further provides articles of manufacture comprising the compositions (such as pharmaceutical compositions) described herein in suitable packaging. Suitable packaging for compositions (such as pharmaceutical compositions) described herein are known in the art, and include, for example, vials (such as sealed vials) , spray bottles, vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) , and the like. These articles of manufacture further may be sterilized and/or sealed.

The present application also provides kits comprising compositions (such as pharmaceutical compositions) described herein and further may comprise instruction (s) on methods of using the composition, such as uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, topical applicators, and/or package inserts with instructions for performing any methods described herein.

V. Methods of Treating, Preventing, and/or Vaccinating

One aspect of the present application provides a method of treating, preventing, vaccinating, and/or stimulating an immune response against a disease such as a cancer (e.g., HPV+, EGFR+, KRAS+, or HCC+ cancer) in an individual (such as a human) , comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein. In some embodiments, the method comprises a method of prophylactically immunizing an individual. In some embodiments, the method comprises a method of preventing an individual from contracting a cancer. In some embodiments, the method comprises a method of reducing the severity or lethality of a cancer in an individual. In some embodiments, the method further comprises a method of treating an individual having a cancer. In some embodiments, the method comprises a method of eliciting an immune response in an individual. In some embodiments, the individual is human.

In some embodiments, there is provided a method of stimulating an immune response against a heterologous antigen in an individual, comprising administering to the individual an effective amount of a composition (e.g., pharmaceutical composition) comprising an isolated nucleic acid (e.g., an mRNA) comprising a coding sequence encoding a fusion protein comprising a target antigen and a trafficking peptide, wherein the trafficking peptide is derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor. In other embodiments, there is provided a method of stimulating an immune response against a heterologous antigen in an individual, comprising administering to the individual an effective amount of a composition (e.g., pharmaceutical composition) comprising an isolated nucleic acid (e.g., an mRNA) comprising: 1) a coding sequence encoding a fusion protein comprising a target antigen and a helper T cell epitope, and 2) a coding sequence encoding an immunomodulator, wherein the immunomodulator comprises GM-CSF and/or STING. In some embodiments, the composition is effective to induce cytotoxic and/or helper T lymphocyte activity in the individual. In some embodiments, the amount of the composition is effective to induce the production of antibodies in the individual. In some embodiments, the composition (e.g., pharmaceutical composition) is administered parenterally, such as by intramuscular or intradermal administration. In some embodiments, the composition (e.g., pharmaceutical composition) is administered intratumorally. In some embodiments, the composition (e.g., pharmaceutical composition) is administered topically. In some embodiments, the composition (e.g., pharmaceutical composition) is administered once. In some embodiments, the composition (e.g., pharmaceutical composition) is administered more than once, for example with an interval of about 2 weeks to about 1 year. In some embodiments, the composition (e.g., pharmaceutical composition) is administered with an interval of about any of every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, or 1 year. In some embodiments, at least two doses of the composition are administered to the individual. In some embodiments, the at least two doses are administered at least one week apart. In some embodiments, the individual is human.

In some embodiments, there is provided a method of treating a cancer (such as an HPV+ cancer, an EGFR+cancer, a KRAS+ cancer, or an HCC+ cancer) in an individual (such as a human) , comprising administering to the individual an effective amount of a composition (e.g., pharmaceutical composition) comprising an isolated nucleic acid (e.g., an mRNA) comprising a coding sequence encoding a fusion protein comprising a target antigen and a trafficking peptide, wherein the trafficking peptide is derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor. In other embodiments, there is provided a method of treating a cancer (such as an HPV+ cancer, an EGFR+ cancer, a KRAS+ cancer, or an HCC+ cancer) in an individual (such as a human) , comprising administering to the individual an effective amount of a composition (e.g., pharmaceutical composition) comprising an isolated nucleic acid (e.g., an mRNA) comprising: 1) a coding sequence encoding a fusion protein comprising a target antigen and a helper T cell epitope, and 2) a coding sequence encoding an immunomodulator, wherein the immunomodulator comprises GM-CSF and/or STING. In some embodiments, the composition (e.g., pharmaceutical composition) is administered parenterally, such as by intramuscular or intradermal administration. In some embodiments, the composition (e.g., pharmaceutical composition) is administered intratumorally. In some embodiments, the composition (e.g., pharmaceutical composition) is administered topically. In some embodiments, the composition (e.g., pharmaceutical composition) is administered once. In some embodiments, the composition (e.g., pharmaceutical composition) is administered more than once, for example with an interval of about 2 weeks to about 1 year. In some embodiments, the composition (e.g., pharmaceutical composition) is administered with an interval of about any of every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, or 1 year. In some embodiments, at least two doses of the composition (e.g., pharmaceutical composition) are administered to the individual. In some embodiments, the at least two doses are administered at least one week apart. In some embodiments, the individual is human.

In some embodiments, the method is used to treat, prevent, or vaccinate an individual (such as human) who has previously been vaccinated against a target antigen-expressing virus or cancer. Any of the methods of treatment, prevention, or vaccination provided herein may be used to treat, prevent, or vaccinate an individual (such as a human) who has not previously been vaccinated against a target antigen-expressing cancer. In some embodiments, the method is used to prophylactically immunize an individual (such as a human) . In some embodiments, the method is used to treat a cancer in an individual (such as a human) . In some embodiments, the method further ameliorates or reduces the cancer and associated symptoms in an individual (such as a human) . In some embodiments, the method is used to elicit an immune response (such as activation of lymphocytes, such as B cells or T cells, including CD4+ T cells and/or CD8+ T cells; and myeloid cells, including but not limited to monocytes, macrophages, neutrophils, granulocytes, mast cells, dendritic cells, and/or eosinophils) in an individual (such as a human) . In some embodiments, the method is used as a prophylactic vaccine against a cancer. In some embodiments, the method is used as a first-or second-line therapy to ameliorate or otherwise reduce the severity or lethality of the cancer and associated symptoms thereof. In some embodiments, the individual is human.

The methods described herein are suitable for vaccinating against a variety of target antigens and/or stimulating an immune response against a variety of target antigens, including viral antigens that may be present on one or more viral species and/or strains, for example but not limited to HPV strains. The methods are applicable to all such species and strains, for example all HPVs, including but not limited to any of HPV types 2a, 3, 7, 10, 11, 13, 16, 18, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 77, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, etc. In one aspect, there is provided a pharmaceutical composition comprising the isolated nucleic acid (e.g., mRNA) construct described herein. For example, in some embodiments, the pharmaceutical composition vaccinates against multiple strains of a virus species, such as HPV selected from the group consisting of HPV types 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 70.

The methods described herein may be used as a vaccination, a preventative, or a treatment to reduce, ameliorate, or eliminate disease, wherein the treatment may act as a first therapy, second therapy, third therapy, or combination therapy with other types of anti-cancer therapies known in the art, such as therapeutic agents selected from the group consisting of a chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery and mixtures thereof or the like, in an adjuvant setting or a neoadjuvant setting. For example, the chemotherapeutic agent can be further selected from the group consisting of an auristatin, a vinca alkaloid, a podophyllotoxin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a puromycin a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, a dolastatin, etc. Likewise, the immunotherapy can be further selected from the group consisting of an immune checkpoint inhibitor, an immunotherapeutic antibody or biologically active fragment thereof (e.g. anti-PD-1, anti-PDL-1, anti-PDL-2, anti-CTLA-4, anti-TIM-3, anti-LAG-3) , a cytokine (e.g. IL-2, IL-7, IL-15) , an inhibitor of cytokine signaling (e.g. anti-TGF-βRII) a chemokine (e.g. CXCL9, CXCL10, CXCL11, CXCL16) , an inhibitor of chemokine signaling/fugetaxis (e.g. CXCL12 inhibitor or antibody depletion) , CAR-T therapy, CAR-NK therapy, adoptive cellular transfer of tumor infiltrating lymphocytes (TILs) , etc. In one aspect, there is provided a method of treating or preventing a cancer in an individual, comprising administering to the individual a therapeutically effective amount of the composition (e.g., pharmaceutical composition) described herein. In some embodiments, the method of treating or preventing a cancer in an individual comprises a method of prophylactically immunizing an individual against a cancer. In some embodiments, the method of treating or preventing a cancer in an individual comprises a method of preventing an individual from contracting a cancer. In some embodiments, the method of treating or preventing a cancer in an individual comprises a method of reducing the severity or symptoms of a cancer in an individual. In some embodiments, the method of treating or preventing a cancer in an individual further comprises a method of treating an individual having a cancer. In some embodiments, the method of treating or preventing a cancer in an individual comprises a method of eliciting an immune response in an individual. In some embodiments, the individual is human.

Exemplary routes of administration of any of the nucleic acid-LNP vaccines described herein or compositions (e.g., pharmaceutical compositions) thereof include, but are not limited to, oral, intravenous, intracavitary, intratumoral, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and epidermal routes, or be delivered into lymph glands, body spaces, organs or tissues known to be virally infected cells. In some embodiments, the composition (e.g., pharmaceutical composition) is administered by intramuscular, intratumoral, intradermal, or topical administration, such as by injection, cream, ointment, or spray. In some embodiments, the composition (e.g., pharmaceutical composition) is administered parenterally, such as by intramuscular, intratumoral, or intradermal administration. In some embodiments, the composition (e.g., pharmaceutical composition) is administered by intramuscular administration.

The dosing regimen of the vaccine (or pharmaceutical composition thereof) administered to the individual (such as human) may vary with the particular vaccine composition, the method of administration, and the particular type and stage of viral infection being treated. In some embodiments, that effective amount of the vaccine is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the composition (e.g., pharmaceutical composition) is administered to the individual. In some embodiments, the individual is human.

EMBODIMENTS

1. An isolated nucleic acid comprising a coding sequence encoding a fusion protein comprising a target antigen and a trafficking peptide, wherein the trafficking peptide is derived from one or more polypeptides selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.

2. The isolated nucleic acid of embodiment 1, wherein the fusion protein further comprises a helper T cell epitope.

3. The isolated nucleic acid of embodiment 1 or embodiment 2, wherein the coding sequence further comprises an immunomodulator.

4. The isolated nucleic acid of embodiment 3, wherein the immunomodulator is selected from the group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, and any combination thereof.

5. The isolated nucleic acid of embodiment 3 or embodiment 4, wherein the immunomodulator is GM-CSF and/or STING.

6. An isolated nucleic acid comprising:

1) a coding sequence encoding a fusion protein comprising a target antigen and a helper T cell epitope, and

2) a coding sequence encoding an immunomodulator, wherein the immunomodulator comprises GM-CSF and/or STING.

7. The isolated nucleic acid of any one of embodiments 1-6, wherein the isolated nucleic acid is DNA or RNA.

8. The isolated nucleic acid of any one of embodiments 1-7, wherein the RNA is mRNA, self-amplifying RNA, or circular RNA.

9. The isolated nucleic acid of any one of embodiments 3-8, wherein the GM-CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) .

10. The isolated nucleic acid of any one of embodiments 3-9, wherein the GM-CSF is a full-length GM-CSF polypeptide.

11. The isolated nucleic acid of embodiment 10, wherein the full-length GM-CSF polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 108 or 181.

12. The isolated nucleic acid of any one of embodiments 3-9, wherein the GM-CSF comprises a truncated GM-CSF or a mutant GM-CSF comprising an amino acid sequence comprising one or more variations compared to the amino acid sequence set forth in SEQ ID NO: 108 or 181, and wherein the truncated GM-CSF or mutant GM-CSF is capable of stimulating macrophage differentiation and proliferation, and/or activating antigen presenting cells (APCs) .

13. The isolated nucleic acid of any one of embodiments 3-12, wherein the STING is human STING (hSTING) (V155M) .

14. The isolated nucleic acid of embodiment 13, wherein the STING comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 107.

15. The isolated nucleic acid of any one of embodiments 3-14, wherein the immunomodulator is connected to the fusion protein via a cleavable linker.

16. The isolated nucleic acid of embodiment 15, wherein the cleavable liner is a 2A peptide.

17. The isolated nucleic acid of embodiment 15 or embodiment 16, wherein the 2A peptide is selected from the group consisting of P2A, F2A, T2A, and E2A.

18. The isolated nucleic acid of any one of embodiments 3-17, wherein the immunomodulator is connected to the C-terminus of the fusion protein.

19. The isolated nucleic acid of any one of embodiments 3-17, wherein the immunomodulator is connected to the N-terminus of the fusion protein.

20. The isolated nucleic acid of any one of embodiments 2-19, wherein the helper T cell epitope is a universal CD4 epitope.

21. The isolated nucleic acid of any one of embodiments 2-20, wherein the helper T cell epitope is an epitope of tetanus and diphtheria toxoids.

22. The isolated nucleic acid of any one of embodiments 2-21, wherein the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE.

23. The isolated nucleic acid of any one of embodiments 2-22, wherein the coding sequence encoding the helper T cell epitope comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72-75 and 171.

24. The isolated nucleic acid of any one of embodiments 2-23, wherein the coding sequence encoding the helper T cell epitope that is a pan DR-binding epitope (PADRE) comprises a nucleic acid sequence of SEQ ID NO: 75 or 171.

25. The isolated nucleic acid of any one of embodiments 2-22, wherein the helper T cell epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126-128 and 147-152.

26. The isolated nucleic acid of any one of embodiments 2-22 and 25, wherein the helper T cell epitope is a pan DR-binding epitope (PADRE) , and wherein the PADRE peptide comprises the amino acid sequence of SEQ ID NO: 128.

27. The isolated nucleic acid of any one of embodiments 6-26, further comprising a coding sequence encoding a trafficking peptide.

28. The isolated nucleic acid of embodiment 27, wherein the trafficking peptide is derived from one or more polypeptides selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.

29. The isolated nucleic acid of any one of embodiments 1-28, wherein the coding sequence encoding the trafficking peptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 76-80, 83-86, and 172.

30. The isolated nucleic acid of any one of embodiments 1-5 and 27-29, wherein the trafficking peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 119, and 122-125.

31. The isolated nucleic acid of any one of embodiments 1-30, wherein the trafficking peptide is MHC class I trafficking domain or LAMP3 transmembrane domain (LAMP3 TM) .

32. The isolated nucleic acid of any one of embodiments 1-31, wherein the trafficking peptide is positioned C-terminal to the target antigen in the fusion protein.

33. The isolated nucleic acid of any one of embodiments 1-32, further comprising a signal peptide.

34. The isolated nucleic acid of embodiment 33, wherein the coding sequence encoding the signal peptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 64-71, 163-164, and 199.

35. The isolated nucleic acid of embodiment 33 or embodiment 34, wherein the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 113-118.

36. The isolated nucleic acid of any one of embodiments 1-35, wherein the target antigen is selected from the group consisting of an HPV antigen, EGFR, KRAS, an HCC antigen, a portion thereof, and any combination thereof.

37. The isolated nucleic acid of any one of embodiments 1-36, wherein the coding sequence encoding the fusion protein is codon optimized.

38. The isolated nucleic acid of any one of embodiments 1-37, further comprising a coding sequence for a ubiquitin peptide.

39. The isolated nucleic acid of embodiment 38, wherein the coding sequence encoding the ubiquitin peptide comprises the nucleic acid sequence set forth in SEQ ID NO: 81.

40. The isolated nucleic acid of claim 38, wherein the ubiquitin peptide comprises the amino acid sequence set forth in SEQ ID NO: 120.

41. The isolated nucleic acid of any one of embodiments 1-40, further comprising a 5’ untranslated region (UTR) and a 3’ UTR.

42. The isolated nucleic acid of embodiment 41, wherein the 5’ untranslated region (UTR) comprises the nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 198.

43. The isolated nucleic acid of embodiment 41, wherein the 3’ untranslated region (UTR) comprises the nucleic acid sequence set forth in SEQ ID NO: 16.

44. The isolated nucleic acid of any one of embodiments 1-43, further comprising a 5’ CAP.

45. The isolated nucleic acid of any one of embodiments 1-44, further comprising a poly (A) sequence.

46. The isolated nucleic acid of embodiment 45, wherein the poly (A) sequence has a length of about 50 nucleotides or longer.

47. The isolated nucleic acid of any one of embodiments 1-46, wherein the isolated nucleic acid comprises a chemical modification.

48. The isolated nucleic acid of embodiment 47, wherein the chemical modification comprises pseudouridine, optionally 1-methylpseudouridine.

49. The isolated nucleic acid of any one of claims 1-48, wherein the coding sequence encoding the fusion protein comprises a nucleic acid sequence having a least 80%identity to:

nucleotides 72-1, 436 of SEQ ID NO: 2;

nucleotides 72-1, 523 of SEQ ID NO: 3;

nucleotides 72-2, 423 of SEQ ID NO: 4;

nucleotides 72-1, 709 of SEQ ID NO: 5;

nucleotides 72-1, 304 of SEQ ID NO: 6;

nucleotides 72-1, 295 of SEQ ID NO: 7;

nucleotides 72-2, 396 of SEQ ID NO: 8;

nucleotides 72-1, 220 of SEQ ID NO: 10;

nucleotides 72-1, 208 of SEQ ID NO: 11;

nucleotides 72-1, 178 of SEQ ID NO: 12;

nucleotides 72-1, 175 of SEQ ID NO: 13;

nucleotides 72-1, 178 of SEQ ID NO: 14;

nucleotides 72-1, 781 of SEQ ID NO: 156;

nucleotides 72-1, 781 of SEQ ID NO: 157;

nucleotides 72-1, 781 of SEQ ID NO: 158;

nucleotides 72-1, 790 of SEQ ID NO: 161; or

nucleotides 72-1, 790 of SEQ ID NO: 162.

50. The isolated nucleic acid of any one of claims 1-49, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80%identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 10-14, 156-158, and 161-162.

51. The isolated nucleic acid of claim 50, wherein the isolated nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-8, 10-14, 156-158, and 161-162.

52. The isolated nucleic acid of any one of claims 1-49, wherein the isolated nucleic acid encodes an amino acid sequence having at least 80%identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-96, 98-102, and 179-180.

53. The isolated nucleic acid of claim 52, wherein the isolated nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-96, 98-102, and 179-180.

54. A vector comprising the isolated nucleic acid of any one of embodiments 1-53.

55. An antigen polypeptide encoded by the isolated nucleic acid of any one of embodiments 1-53 or the vector of embodiment 54.

56. A composition comprising the isolated nucleic acid of any one of embodiments 1-53, the vector of embodiment 54, or the antigen polypeptide of embodiment 55, and a pharmaceutically acceptable carrier.

57. The composition of embodiment 56, wherein the isolated nucleic acid or the vector is formulated in a lipid nanoparticle (LNP) .

58. The composition of embodiment 57, wherein the LNP comprises a cationic lipid.

59. The composition of embodiment 57 or embodiment 58, wherein the LNP comprises a phospholipid.

60. The composition of any one of embodiments 57-59, wherein the LNP comprises a sterol.

61. The composition of embodiment 59 or embodiment 60, wherein the LNP comprises a polymer conjugated lipid.

62. The composition of any one of embodiments 59-61, wherein the LNP comprises:

30-55%cationic lipid,

5-40%phospholipid,

20-50%sterol, and

a polymer conjugated lipid.

63. A method of stimulating an immune response against a heterologous antigen in an individual, comprising administering to the individual an effective amount of the composition of any one of embodiments 56-62.

64. The method of embodiment 63, wherein the amount of composition is effective to induce cytotoxic and/or helper T lymphocyte activity in the individual.

65. The method of embodiment 63 or embodiment 64, wherein the amount of the composition is effective to induce production of antibodies in the individual.

66. A method of treating or preventing a cancer in an individual, comprising administering to the individual a therapeutically effective amount of the composition of any of embodiments 56-62.

67. The method of embodiment 66, wherein the cancer expresses one or more of the target antigens selected from the group consisting of an HPV antigen, EGFR, KRAS, an HCC antigen, and a portion thereof.

68. The method of embodiment 66 or embodiment 67, wherein the composition is administered locally to the tumor.

69. The method of any one of embodiments 66-68, wherein the composition is administered systemically.

70. The method of any one of embodiments 66-69, wherein the composition is administered in combination with one or more anti-cancer therapies.

71. The method of embodiment 70, wherein the one or more anti-cancer therapies are selected from a chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery.

72. The method of embodiment 71, wherein the one or more anti-cancer therapies comprises anti-PD-1, anti-PDL-1, and/or anti-PDL-2 monoclonal antibodies or biologically active fragments thereof.

73. The method of embodiment 71, wherein the one or more anti-cancer therapies comprises anti-TGF-βRII antibodies or biologically active fragments thereof.

74. The method of any one of embodiment 63-73, wherein at least two doses of the composition are administered to the individual.

75. The method of embodiment 74, wherein the at least two doses are administered at least one week apart.

76. The method of any one of embodiments 63-75, wherein the individual is human.

EXAMPLES

The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation. For the embodiments in which details of the experimental methods are not described, such methods are carried out according to conventional conditions such as those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Fourth Edition (New York: Cold Spring Harbor Laboratory Press, 2012) , or as suggested by the manufacturers.

Example 1: Design of mRNA expression cassettes for target antigen presentation in dendritic cells

Efficiency of antigen presentation and downstream activation of target T cells can be influenced by specific molecules that regulate dendritic cell (DC) maturation and function. The purpose of this Example is to describe exemplary mRNA expression cassettes that include immunomodulators, trafficking peptides, and pan-CD4+ T-effector epitopes that, when expressed with target antigens, increase efficiency of antigen presentation by DCs and thus increase T cell response. These domains may be expressed with target antigens through direct fusions of open reading frames, via non-cleavable linkers (e.g., Gly-Ser linker domain) , or via cleavable linkers (e.g., P2A linker) .

The mRNA expression cassettes include coding sequences for a target antigen flanked by a signal peptide (SP) and MHC class I trafficking domain, a pan-CD4+ T-effector epitope, and/or an immunomodulator. SP/MHC class I trafficking domain traffic the target antigen (s) through the endosomal pathway for presentation at the cell surface by MHC class II molecules. Pan-CD4+ T-effector epitopes improve T_H cell activation by binding to the T cell receptor (TCR) with the target antigens. Immunomodulators improve antigen presentation by activating DC functionality (e.g., maturing DCs through NF-κB activation) (FIG. 1A) .

The exemplary mRNAs ABO-01 through ABO-14 include a fusion protein of i) HPV16 E6 mutated for binding to p53 and ii) HPV16 E7 mutated for binding to Rb as an exemplary target antigen (FIG. 1B) . The HPV16 E6 and HPV16 E7 open reading frames are connected by a GS non-cleavable linker. This fusion protein is flanked by either a ubiquitin construct and/or a signal peptide (SP) and MHC class I trafficking domain (FIG. 1B) . The ubiquitin construct can stimulate proteasomal degradation of target antigen, resulting in antigen presentation by MHC class I molecules. The SP/MHC class I trafficking domain combination can target the antigen to the endosomal pathway for processing and presentation by MHC class II molecules. Immunomodulator coding sequences included on exemplary mRNAs include human STING (ABO-04) and murine GM-CSF (ABO-05) (FIG. 1B) . Pan-CD4+ T cell epitope coding sequences included in exemplary mRNA constructs can be, for example, P2P16 65aa (ABO-02) , pp65 (ABO-06) , or PADRE (ABO-07) (FIG. 1B) . Trafficking peptide coding sequences included in exemplary mRNA constructs can be, for example, MHC class I trafficking domain (ABO-02 to ABO-07, ABO-10) , HLA-E (ABO-11) , LAMP1 (ABO-12) , LAMP3-TM (ABO-08, ABO-13) or HLA-DMB (ABO-14) (FIG. 1B) . Exemplary sequences are provided in Tables 6-7 below.

Table 6. Exemplary nucleic acid sequences.

Table 7. Exemplary amino acid sequences.

The mRNA expression cassettes are packaged with lipid nanoparticles (LNPs) , thereby creating the final vaccine composition used for in vivo testing, as described below. Exemplary LNPs are described in Subsection V.A. Lipid nanoparticles (LNPs) above. Exemplary methods of preparation for the cationic lipids described in Subsection V.A. Lipid nanoparticles (LNPs) above can be found in, e.g., WO2021204175A1, WO2023138611A1, WO2022152109A1, and WO2022247755A1. Exemplary methods for the preparation of lipid nanoparticles described in Subsection V.A. Lipid nanoparticles (LNPs) above and mRNA synthesis can be found in, e.g., patent WO2023098842A1.

Example 2: Addition of immunomodulator domains to mRNA expression cassettes increase murine immune response after vaccination via intramuscular injection

Antigen presentation by dendritic cells (DCs) induces an adaptive immune response by binding to receptors on effector T cells, thereby stimulating T cell proliferation and activation. These functions can be assessed by analyzing the level of cytokine production, e.g., IL-2 and IFN-γ, respectively. Immunomodulators are molecules that stimulate the maturation and function of DCs (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF) and STING, which both promote DC maturation) . The mRNA construct ABO-04 includes a human STING domain, and ABO-05 includes a murine GM-CSF domain, both of which were assessed to identify whether either can improve DC antigen presentation and increase effector T cell immune response during vaccination.

C57BL/6 mice were immunized via intramuscular injection at three timepoints (day 0, day 7, and day 14) with 10 μg of a vaccine composition or PBS sham vaccination. The tested vaccine compositions included ABO-01 through ABO-08, wherein each group of n=3 mice received one vaccine for a total of 8 vaccination groups and one negative control group. Three days following the final immunization timepoint, spleens were extracted, resuspended to single cells, and plated in ELISpot wells. Antigen-specific effector T cell response in the spleen was then determined by staining for IFN-γ secretion using an ELISpot assay, such that increased response to any of the vaccine compositions is indicated by increased number of IFN-γ+ spots in each well relative to the PBS sham control.

Results showed that inclusion of the immunomodulators, hSTING in ABO-04 or mGM-CSF in ABO-05, as part of the mRNA constructs containing the SP and MHC Class I trafficking domain increased the in vivo effector T cell response compared to the in vivo response to mRNA constructs containing only SP and MHC Class I trafficking domain (i.e., ABO-02) (FIGs. 2A-2B) . These results demonstrate a clear improvement in the level of adaptive immune response and a decrease in tumor burden in vivo when vaccinated with the vaccine composition that contains mRNA constructs with immunomodulator domains.

Example 3: Inclusion of immunomodulator domains in mRNA expression cassettes improves protection against tumorigenesis in vivo

The primary goal of an antigen-specific effector T cell response in cancer immunotherapy is to prevent, reduce, or cure tumorigenesis and/or tumor burden. The vaccine compositions that included the immunomodulator domain were tested for the degree of protect conferred by vaccination against tumorigenesis in vivo.

Cells from an HPV+ cancer cell line that expresses the HPV16 E6/E7 target antigen, called TC-1, were implanted into C57BL/6 mice to induce HPV+ tumorigenesis. Once TC-1-implanted C57BL/6 mice developed tumors of approximately 6 mm³ in size, they were immunized three times (day 0, day 7, and day 14) by intramuscular injection (i.m. ) with 10 μg vaccine composition or PBS sham vaccination, as described in Example 2 above. FIG. 3A provides a schematic overview of the experimental design.

In the in vivo TC-1 tumor experiment described above, control mice were immunized by intramuscular injection (i.m. ) with 50 μL 1x PBS (FIG. 3B) or 10 μg of ABO-01 vaccine (FIG. 3C) . No mice receiving PBS control showed complete response, while ABO-01 vaccination resulted in complete response in only 1/8 mice. Vaccination with 1μg, 5μg, and 10 μg of ABO-04 (human STING) showed a complete response in 4/8, 6/8, and 6/8 mice, respectively (FIG. 3D) . Vaccination with 10 μg of ABO-05 (murine GM-CSF) showed a complete response in 6/8, 5/8, and 5/8 mice, from three independent experiments (FIG. 3E) . These results demonstrated that the inclusion of STING (ABO-04) or GM-CSF (ABO-05) in the mRNA constructs led to a significant improvement in the rate of complete tumor regression compared to the vaccination efficacy of ubiquitin-fused HPV16 mRNA (ABO-01) in TC-1 tumor-bearing mice.

Example 4: Identification of pan T_H epitopes that can promote T cell activity

Pan T_H epitopes derived from the signal peptides of tetanus and diphtheria toxoids can help stimulate activation of CD4+ T_H cells during antigen presentation. The purpose of this Example is to determine the peptides that induce T cell activity for inclusion mRNA expression cassettes.

Approximately 3-5×10⁵ peripheral blood mononuclear cells (PBMCs) from 10 human donors were plated on ELISpot wells that had been previously blocked for non-specific binding with complete culture media. Individual wells with PBMC were then treated with 15 μM of each peptide listed in Table 8 or 10 μg/mL conA as a positive control. After 18 hours of incubation at 37℃, ELISpot plates were washed in PBS containing 0.05%fetal bovine serum (FBS) and then incubated with 100 μL biotinylated anti-IFN-γsecondary antibody for 2 hours at room temperature. ELISpot plates were then washed another 5 times and treated with 100 μL Streptavidin-HRP for 1 hour at room temperature. ELISpots were again washed 5 times and then developed with 100 μL TMB substrate solution for 5-10 minutes for detection of HRP staining. Plates were thoroughly washed with water and dried, and IFN-γ+ spots were counted with either an ELISpot reader or manually under a light microscope. IFN-γ+ spots from each PBMC donor were normalized to antigen control to account for background staining.

The addition of the pan T_H epitopes TT_827-841pDT_331–350, TT_632–651, P16, and pan DR-binding epitope (PADRE) all increased effector T cell activation compared to P2lP16 and P2kP16 peptides (FIG. 4) . These results indicate the levels of immunogenicity of pan T_H cell epitopes that can be utilized to improve anti-tumor vaccine efficacy as described herein.

Table 8. Exemplary pan-helper T cell epitope sequences.

Example 5: Alteration of trafficking peptides in mRNA vaccine compositions increases murine immune response after intramuscular vaccination

The process of antigen presentation by DCs is controlled by trafficking and processing of antigens through the secretory and endosomal pathways within DCs, as well as the surface molecules to be presented. The purpose of this Example is to determine whether the use of trafficking peptides other than MHC Class I trafficking domain can increase the effector T cell response to the exemplary HPV16 E6/E7 antigen.

The immune response following vaccination with ABO-09 through ABO-14 (FIG. 1B) was analyzed by IFN-γ ELISpot as described in Example 2 above.

Results showed that addition of the exemplary trafficking peptides, HLA-E (ABO-11) , LAMP1 (ABO-12) , LAMP3 (ABO-13) , and HLA-DMB (ABO-14) to the previously described mRNA constructs containing no trafficking peptides (e.g., ABO-09; FIG. 1B) improved splenic T cell response to vaccination (FIG. 5A-5B) . Splenic T cell response was equivalent to exemplary mRNA constructions containing SP and MHC Class I trafficking domain (ABO-10) , except for ABO-13, which showed that the inclusion of LAMP3 led to surprisingly significantly increased T cell response (FIG. 5B) . These results show that the inclusion of trafficking peptides in the vaccine mRNA construct can promote antigen presentation for effective vaccination.

Example 6: Design of mRNA expression cassettes for HPV antigen presentation in dendritic cells

This Example describes five additional exemplary mRNA expression cassettes that include immunomodulators, trafficking peptides, and pan-CD4+ T-effector epitopes that, when expressed with HPV target antigens (i.e., HPV16 E6 and E7 peptide antigens) , increase efficiency of antigen presentation by DCs and thus increase T cell response. These domains may be expressed with HPV antigens through direct fusions of open reading frames, via non-cleavable linkers (e.g., Gly-Ser linker domain) , or via cleavable linkers (e.g., P2A linker) .

The mRNA expression cassettes include coding sequences for an HPV16 E6 and E7 fusion polypeptide antigen flanked by an N-terminal signal peptide (SP) and C-terminal MHC class I trafficking domain, a pan-CD4+ helper T (T_H) cell epitope, and/or an immunomodulator. SP/MHC class I trafficking domain traffic the HPV16 E6/E7 antigen through the endosomal pathway for presentation at the cell surface by MHC class II molecules. Pan-CD4+ helper T cell epitopes improve T_H cell activation by binding to the T cell receptor (TCR) with the HPV16 E6/E7 antigens. Immunomodulators improve antigen presentation by activating DC functionality (e.g., maturing DCs through NF-κB activation) (FIG. 6A) .

The exemplary mRNAs ABO-15, ABO-16, ABO-17, ABO-20, and ABO-21 encodes a fusion protein with an HPV16 E6/E7 target antigen that includes: i) HPV16 E6 peptide mutated for binding to p53, and ii) HPV16 E7 peptide mutated for binding to Rb. This HPV target antigen peptide is flanked by a signal peptide (SP) and MHC class I trafficking domain, as shown in FIG. 6B. The SP/MHC class I trafficking domain combination can target the HPV antigen to the endosomal pathway for processing and presentation by MHC class II molecules. Immunomodulator coding sequences included in the exemplary mRNA sequences include murine GM-CSF (mGM-CSF; ABO-15 through ABO-17) and human GM-CSF (hGM-CSF; ABO-20 and ABO-21) . ABO-15 through ABO-17 each includes mGM-CSF and comprise a uniquely optimized codon sequence of the HPV16 E6/E7 target antigen that demonstrates high translational efficiency while coding for the same HPV16 E6/E7 amino acid sequence as other similarly designed constructs described herein. ABO-20 and ABO-21 each includes hGM-CSF and a uniquely optimized codon sequence of the HPV16 E6/E7 target antigen that demonstrates high translational efficiency while coding for the same HPV16 E6/E7 amino acid sequence as other similarly designed constructs described herein. ABO-15, ABO-16, ABO-17, ABO-20 and ABO-21 also each contains the pan-CD4+ T-effector epitope, PADRE, to improve helper T cell activation (FIG. 6B) . Exemplary sequences are provided in Tables 9-10 below.

Table 9. Exemplary nucleic acid sequences.

Table 10. Exemplary amino acid sequences.

The mRNA expression cassettes are packaged with lipid nanoparticles (LNPs) , thereby creating the final vaccine composition.

Example 7: Codon optimization-generated mRNA expression cassettes with robust protein expression in transfected cells after RNA modification

Cells engineered to express target antigens, such as HPV16 antigens, require high levels of protein expression to stimulate a robust immune response. This includes high expression of both the HPV16 antigen itself and other factors that can stimulate an adaptive immune response, for example immunomodulators like GM-CSF or STING. mRNA vaccines can be produced and introduced with certain modifications such as pseudouridinylation (Ψ) to allow for the delivery of the vaccine payload to be expressed in different host cells, and these modifications may impact the expression level of the associated antigen peptide and/or modulate the innate immune response in vivo. Accordingly, mRNA vaccine constructs must be carefully constructed to ensure high expression levels of the antigenic peptide and proper functioning as a vaccine.

The exemplary mRNA vaccine constructs ABO-15 through ABO-17 with pseudouridine (Ψ) modification were tested on protein expression levels. The human cell line expi293F was grown in vitro then transfected with 4μg (FIG. 7A) or 1μg (FIG. 7B) of one of the exemplary mRNA vaccine constructs ABO-15 through ABO-17 containing Ψ modification. After a 24-hour incubation period, cells were harvested, protein was extracted from whole cell lysates, and Western blots were performed to determine expression levels of the HPV16 E7 peptide, which was normalized to the housekeeping control, α-tubulin. FIG. 7A showed expression of the E7 peptide across each modified mRNA construct.

The effect of RNA modifications on immunomodulator peptide expression levels was also tested similarly. In short, the human cell line expi293F was grown in vitro, then transfected with 4μg or 1μg of one of the exemplary mRNA vaccine constructs ABO-15 through ABO-17 containing Ψ modification. After a 24-hour incubation period, the supernatant from the culture of the transfected cells was harvested and an ELISA was performed to determine the amount of secreted murine GM-CSF (mGM-CSF) . FIG. 8 showed that among the pseudouridine-modified constructs, ABO-16 showed the highest level of mGM-CSF secretion.

These experiments were repeated using exemplary mRNA vaccine constructs with domains for human GM-CSF (hGM-CSF) , i.e., ABO-20 and ABO-21. The human cell line expi293F was grown in vitro then transfected with 4μg or 1μg of one of the exemplary mRNA vaccine constructs, ABO-20 and ABO-21, containing Ψ modification. After a 24-hour incubation period, the supernatant from the culture of the transfected cells was harvested, and an ELISA was performed to determine the amount of secreted hGM-CSF. FIG. 9 showed that Ψ-modified ABO-20 and ABO-21 secreted comparable level of hGM-CSF, similar to the mGM-CSF results shown in FIG. 8.

Taken together, the exemplary mRNA constructs and the vaccine compositions that include these constructs show strong immunogenicity and anti-tumor activity, including robust expression of the encoded antigenic proteins in transfected human cells. The degree of immunogenicity can be fine-tuned based on the selection of domains, as shown using the tested exemplary mRNA constructs ABO-02 through ABO-17, ABO-20, and ABO-21, for maximal vaccine efficacy.

Example 8: Intramuscular HPV 16 mRNA-LNP vaccination which added PADRE-mGM-CSF (ABO-15) or mGM-CSF (ABO-05) modulator significantly induced tumor complete regression

As illustrated in FIG. 10A, to generate the tumor model, TC-1 cells were implanted into C57BL/6 mice. The commercially available TC-1 cell line was derived from primary lung epithelial cells of C57BL/6 mice and express the HPV16-E6 and HPV16-E7 proteins. Then TC-1-implanted C57BL/6 mice were immunized three times (d0, d7, d14) by intramuscular injection (i.m. ) with 5 μg HPV16 E6E7 mRNA-LNP. At the start of treatment (d0) , the average tumor size was approximately 6 mm³. TC-1 cells were re-implanted into immunized C57BL/6 mice that demonstrated complete TC-1 tumor regression. The results measured in tumor volume up to d35 are summarized in the table below and plotted in FIG. 10B.

*CR 2/8 means that out of 8 mice in the test group, 2 showed complete tumor regression.

As shown, vaccination with 5 μg of ABO-15 (with PADRE-mGM-CSF-MITD modulator) per mouse was compared and showed a complete response in 2/8 mice. Vaccination with 5 μg of ABO-05 (with mGM-CSF-MITD modulator) per mouse were compared and showed a complete response rate of 0/8, respectively. This result showed that ABO-15 vaccination significantly induced tumor complete regression than ABO-05 in TC-1 tumor-bearing mice. Through comparative analysis of in vivo efficacy, it was demonstrated that the PADRE component in backbone enhances anti-tumor activity.

Example 9: Enhancement of KRAS antigen immunogenicity by backbone elements.

To demonstrate the enhancement of KRAS antigen immunogenicity by backbone elements, the different arrangements and combinations of elements mRNA/LNP were designed to intramuscular injected HLA-A*1101 transgenic mice three times (d0, d7, d14) , 5 mice each group. At day 17, all mice were sacrificed and the spleens were collected for single cell suspension preparation. The cell suspension were stimulation by the peptide libraries of G12A, G12C, G12D, G13D, WT and PADRE peptide for 24 hours in ELISPOT plates. Then antigen-specific T cells response were measured by IFN-γ secretion.

Table 11. Exemplary amino acid sequences.

At the same dose, ABO-24 (PADRE+GM-CSF) immunized mice exhibited superior KRAS G12A and G12C specific immunogenicity to ABO-25 (PADRE+MITD) and ABO-26 (GM-CSF+MITD) in splenocytes, and also induced the strongest immunogenicity compared with other eleme nts of the vaccines when stimulated by PADRE peptide.

All references mentioned in the present invention are incorporated herein by reference as if each of those references has been incorporated by reference individually. Although the description referred to particular embodiments, it will be clear to a person skilled in the art that the present invention may be practiced with variation of these specific details. Hence this invention should not be construed as limited to the embodiments set forth herein.

SEQUENCE CHART

Claims

An immunomodulating nucleic acid system encoding a target antigen, wherein the system comprises a first coding sequence encoding a helper T cell epitope and a second coding sequence encoding an immunomodulator, wherein the helper T cell epitope and the immunomodulator are configured to enhance immunogenicity of the target antigen upon co-expression with the target antigen.
The immunomodulating nucleic acid system of claim 1, wherein the system further comprises a third coding sequence encoding a trafficking peptide configured for intracellularly trafficking the target antigen towards proteosome upon expression.
The immunomodulating nucleic acid system of claim 1 or 2, wherein the system further comprises a fourth coding sequence encoding a signal peptide.
The immunomodulating nucleic acid system of any one of claims 1 to 3, wherein the system further comprises a fifth coding sequence encoding a ubiquitin peptide.
The immunomodulating nucleic acid system of any one of claims 1 to 4, wherein the first coding sequence encodes a fusion protein comprising the helper T cell epitope and the target antigen.
The immunomodulating nucleic acid system of any one of claims 1 to 4, wherein the second coding sequence encodes a fusion protein comprising the immunomodulator and the target antigen; optionally wherein the immunomodulator is positioned N-terminal to the target antigen or is positioned C-terminal to the target antigen in the fusion protein.
The immunomodulating nucleic acid system of any one of claims 1 to 4, wherein the third coding sequence encodes a fusion protein comprising the trafficking peptide and the target antigen; optionally wherein the trafficking peptide is positioned C-terminal to the target antigen in the fusion protein.
The immunomodulating nucleic acid system of any one of claims 1 to 4, wherein the fourth coding sequence encodes a fusion protein comprising the signal peptide and the target antigen; optionally wherein the signal peptide is positioned N-terminal to the target antigen in the fusion protein.
The immunomodulating nucleic acid system of any one of claims 1 to 4, wherein the fifth coding sequence encodes a fusion protein comprising the ubiquitin peptide and the target antigen; optionally wherein the ubiquitin peptide is positioned N-terminal to the target antigen in the fusion protein; optionally wherein fusion protein further comprises a spacer peptide positioned in between the ubiquitin peptide and the target antigen in the fusion protein; optionally the spacer peptide is at least about 25 amino acids in length.
The immunomodulating nucleic acid system of any one of claims 1 to 9, wherein at least one of the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences are in a first nucleic acid molecule.
The immunomodulating nucleic acid system of claim 9, wherein:

(a) the second coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator;

(b) the first coding sequence and the second coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator and the target antigen;

(c) the first coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the trafficking peptide and the target antigen;

(d) the first coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the signal peptide and the target antigen;

(e) the first coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the ubiquitin peptide and the target antigen;

(f) the second coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the trafficking peptide and the target antigen;

(g) the second coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the signal peptide, and the target antigen;

(h) the second coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the ubiquitin peptide and the target antigen;

(i) the third coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the trafficking peptide, the signal peptide and the target antigen;

(j) the third coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the trafficking peptide, the ubiquitin peptide and the target antigen;

(k) the fourth coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the signal peptide, the ubiquitin peptide and the target antigen;

(l) the first coding sequence, the second coding sequence, and the third coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, and the target antigen;

(m) the first coding sequence, the second coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the signal peptide, and the target antigen;

(n) the first coding sequence, the second coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the ubiquitin peptide, and the target antigen;

(o) the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the trafficking peptide, the signal peptide, and the target antigen;

(p) the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the target antigen;

(q) the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the trafficking peptide, the signal peptide, the ubiquitin peptide, and the target antigen;

(r) the first coding sequence, the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the target antigen;

(s) the first coding sequence, the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the target antigen; or

(t) the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the fusion protein comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the ubiquitin peptide, and the target antigen.
The immunomodulating nucleic acid system of claim 10, wherein at least one of the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences is in a second nucleic acid molecule, and wherein the first nucleic acid molecule and the second nucleic acid molecule are different molecules.
The immunomodulating nucleic acid system of claim 12, wherein the second nucleic acid molecule does not encode the target antigen.
The immunomodulating nucleic acid system of claim 12 or 13, wherein the first nucleic acid molecule encodes the fusion protein comprising the target antigen; wherein the fusion protein does not comprise the immunomodulator; and wherein the second nucleic acid molecule comprises the second coding sequence encoding the immunomodulator.
The immunomodulating nucleic acid system of any one of claims 10 to 13, wherein at least the second coding sequence is in the first nucleic acid molecule encoding the fusion protein comprising at least the immunomodulator and the target antigen; and wherein the first nucleic acid molecule further comprises a means for producing the immunomodulator and the target antigen as separate proteins or peptides.
The immunomodulating nucleic acid system of claim 15, wherein the means for producing the immunomodulator and the target antigen as separate proteins or peptides comprises a sixth coding sequence encoding a cleavable linker in between the second coding sequence and a coding sequence for the target antigen in the first nucleic acid.
The immunomodulating nucleic acid system of claim 16, wherein the cleavable linker is a 2A peptide, selected from the group consisting of P2A, F2A, T2A, and E2A.
The immunomodulating nucleic acid system of any one of claims 1 to 17, wherein the helper T cell epitope is a universal CD4 epitope.
The immunomodulating nucleic acid system of any one of claims 1 to 17, wherein the helper T cell epitope is an epitope of tetanus and diphtheria toxoids.
The immunomodulating nucleic acid system of any one of claims 1 to 17, wherein the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE.
The immunomodulating nucleic acid system of any one of claims 1 to 17, wherein the first coding sequence encoding the helper T cell epitope comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 72-75 and 171.
The immunomodulating nucleic acid system of any one of claims 1 to 17, wherein the first coding sequence encodes the helper T cell epitope that is a pan DR-binding epitope (PADRE) , and wherein the first coding sequence comprises the nucleic acid sequence of SEQ ID NO: 75 or 171.
The immunomodulating nucleic acid system of any one of claims 1 to 22, wherein the immunomodulator is selected from the group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, and any combination thereof.
The immunomodulating nucleic acid system of any one of claims 1 to 22, wherein the immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING.
The immunomodulating nucleic acid system of claim 1, wherein

(a) the first coding sequence encodes a fusion protein comprising the target antigen and a helper T cell epitope, and

(b) the second coding sequence encodes an immunomodulator, wherein the immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING.
The isolated nucleic acid of any one of claims 23 to 25, wherein the GM-CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) .
The isolated nucleic acid of any one of claims 23 to 25, wherein the GM-CSF is a full-length GM-CSF polypeptide.
The isolated nucleic acid of claim 27, wherein the full-length GM-CSF polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 108 or 181.
The isolated nucleic acid of any one of claims 23 to 25, wherein the GM-CSF comprises a truncated GM-CSF or a mutant GM-CSF comprising an amino acid sequence comprising one or more variations compared to the amino acid sequence set forth in SEQ ID NO: 108 or 181, and wherein the truncated GM-CSF or mutant GM-CSF is capable of stimulating macrophage differentiation and proliferation, and/or activating antigen presenting cells (APCs) .
The isolated nucleic acid of any one of claims 23 to 25, wherein the STING is human STING (hSTING) (V155M) .
The isolated nucleic acid of claim 30, wherein the STING comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 107.
The immunomodulating nucleic acid system of any one of claims 2 to 31, wherein the trafficking peptide is derived from one or more polypeptides selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.
The immunomodulating nucleic acid system of any one of claims 2 to 31, wherein the trafficking peptide is MHC class I trafficking domain.
The immunomodulating nucleic acid system of any one of claims 2 to 31, the trafficking peptide is MHC class I trafficking domain or LAMP3 transmembrane domain (LAMP3 TM) .
The immunomodulating nucleic acid system of any one of claims 2 to 31, wherein the third coding sequence encoding the trafficking peptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 76-80, 83-86, 172, and 201.
The immunomodulating nucleic acid system of any one of claims 3 to 35, wherein the fourth coding sequence encoding the signal peptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 64-71, 163-164, and 199.
The immunomodulating nucleic acid system of any one of claims 4 to 36, wherein the ubiquitin peptide comprises a naturally-existing ubiquitin peptide or a functional derivative thereof.
The immunomodulating nucleic acid system of claim 37, wherein the fifth coding sequence encoding the ubiquitin peptide comprises the nucleic acid sequence set forth in SEQ ID NO: 81.
The immunomodulating nucleic acid system of any one of claims 1 to 38, wherein the target antigen is selected from the group consisting of an HPV antigen, EGFR, KRAS, an HCC antigen, a portion thereof, and any combination thereof.
The immunomodulating nucleic acid system of any one of claims 10 to 39, wherein the first nucleic acid molecule is DNA or RNA.
The immunomodulating nucleic acid system of any one of claims 12 to 40, wherein the second nucleic acid molecule is DNA or RNA.
The immunomodulating nucleic acid system of claim 41, wherein the RNA is mRNA, self-amplifying RNA, or circular RNA.
The immunomodulating nucleic acid system of any one of claims 1 to 42, wherein the one or more coding sequences are codon optimized.
The immunomodulating nucleic acid system of any one of claims 1 to 43, wherein the one or more coding sequences are in one or more mRNA molecule.
The immunomodulating nucleic acid system of claim 44, wherein the mRNA molecule further comprises a 5’ untranslated region (UTR) , a 3’ UTR, or both a 5’ UTR and a 3’ UTR.
The immunomodulating nucleic acid system of claim 45, wherein the 5’ UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 198.
The immunomodulating nucleic acid system of claim 45 or 46, wherein the 3’ UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 16.
The immunomodulating nucleic acid system of any one of claims 44 to 47, wherein the mRNA molecule further comprises a 5’ Cap.
The immunomodulating nucleic acid system of any one of claims 44 to 48, wherein the mRNA molecule further comprises a poly (A) sequence.
The immunomodulating nucleic acid system of claim 49, wherein the poly (A) sequence has a length of about 50 nucleotides or longer.
The immunomodulating nucleic acid system of any one of claims 10 to 50, wherein the first nucleic acid molecule comprises a chemical modification.
The immunomodulating nucleic acid system of any one of claims 12 to 51, wherein the second nucleic acid molecule comprises a chemical modification.
The immunomodulating nucleic acid system of claim 51 or 52, wherein the chemical modification comprises pseudouridine; optionally wherein the pseudouridine is 1-methylpseudouridine.
The immunomodulating nucleic acid system of any one of claims 5 to 53, wherein the coding sequence encoding the fusion protein comprises a nucleic acid sequence having a least 80%identity to:

nucleotides 72-1, 436 of SEQ ID NO: 2;

nucleotides 72-1, 523 of SEQ ID NO: 3;

nucleotides 72-2, 423 of SEQ ID NO: 4;

nucleotides 72-1, 709 of SEQ ID NO: 5;

nucleotides 72-1, 304 of SEQ ID NO: 6;

nucleotides 72-1, 295 of SEQ ID NO: 7;

nucleotides 72-2, 396 of SEQ ID NO: 8;

nucleotides 72-1, 220 of SEQ ID NO: 10;

nucleotides 72-1, 208 of SEQ ID NO: 11;

nucleotides 72-1, 178 of SEQ ID NO: 12;

nucleotides 72-1, 175 of SEQ ID NO: 13;

nucleotides 72-1, 178 of SEQ ID NO: 14;

nucleotides 72-1, 781 of SEQ ID NO: 156;

nucleotides 72-1, 781 of SEQ ID NO: 157;

nucleotides 72-1, 781 of SEQ ID NO: 158;

nucleotides 72-1, 790 of SEQ ID NO: 161; or

nucleotides 72-1, 790 of SEQ ID NO: 162.
The immunomodulating nucleic acid system of any one of claims 10 to 53, wherein the first nucleic acid molecule encoding the fusion protein comprises a nucleic acid sequence having at least 80%identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8, 10 to 14, 156 to 158, 161, and 162.
The immunomodulating nucleic acid system of claim 55, wherein the first nucleic acid molecule encoding the fusion protein comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8, 10 to14, 156 to 158, 161, and 162.
The immunomodulating nucleic acid system of any one of claims 10 to 67, wherein the first nucleic acid molecule is in a first vector.
The immunomodulating nucleic acid system of any one of claims 12 to 57, wherein the second nucleic acid molecule is a second vector.
A vector system comprising the first vector of claim 57, the second vector of claim 58, or both of the first vector and the second vector.
A polypeptide encoded by the first nucleic acid molecule of the immunomodulating nucleic acid system of any one of claims 1 to 58 or the vector system of claim 59.
A composition comprising the immunomodulating nucleic acid system of any one of claims 1 to 58, the vector system of claim 59, or the polypeptide of claim 60, and a pharmaceutically acceptable carrier.
The composition of claim 61, wherein the immunomodulating nucleic acid system or the vector system is formulated in a lipid nanoparticle (LNP) .
The composition of claim 62, wherein the LNP comprises a cationic lipid.
The composition of claim 62 or 63, wherein the LNP comprises a phospholipid.
The composition of any one of claims 62 to 64, wherein the LNP comprises a sterol.
The composition of any one of claims 62 to 65, wherein the LNP comprises a polymer conjugated lipid.
The composition of claim 66, wherein the LNP comprises:

about 30%to about 55%cationic lipid,

about 5%to about 40%phospholipid,

about 20%to about 50%sterol, and

a polymer conjugated lipid.
A method of stimulating an immune response against a heterologous antigen in an subject, comprising administering to the subject an effective amount of the composition of any one of claims 61 to 68.
The method of claim 68, wherein the amount of composition is effective to induce cytotoxic and/or helper T lymphocyte activity in the individual.
The method of claim 68 or 69, wherein the amount of the composition is effective to induce production of antibodies in the individual.
The method of any one of claims 68 to 70, wherein at least two doses of the composition are administered to the individual.
The method of claim 71, wherein the at least two doses are administered at least one week apart.
The method of any one of claims 68 to 72, wherein the subject is human.