WO2025131119A1

WO2025131119A1 - Cancer vaccines and uses thereof

Info

Publication number: WO2025131119A1
Application number: PCT/CN2024/141468
Authority: WO
Inventors: Gang Hu; Xinan LIU; Liang XIE; Yue FAN; Wei Jennifer Yang
Original assignee: Everest Medicines China Co Ltd; Everest Medicines Ii Hk Ltd
Current assignee: Everest Medicines China Co Ltd; Everest Medicines Ii Hk Ltd
Priority date: 2023-12-22
Filing date: 2024-12-23
Publication date: 2025-06-26
Anticipated expiration: 2026-06-22

Abstract

Provided herein are nucleic acids, pharmaceutical compositions and vaccines that express a cancer antigen (e.g., PRAME). Also provided herein are methods for treating a cancer.

Description

CANCER VACCINES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of International Application No. PCT/CN2023/140956 filed on December 22, 2023 and International Application No. PCT/CN2024/070583 filed on January 4, 2024, which are incorporated herein by reference in their entireties for all purposes.

TECHNICAL FIELD

The disclosure relates to compositions and methods for treating cancer and in particular, vaccines that treat and provide protection against tumor growth.

BACKGROUND

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide (890,000 new cases and 450,000 deaths annually) and occurs in the mucosal surfaces of four major anatomical sites: oral cavity, sinonasal cavity, pharynx and larynx. The major cause of HNSCC are tobacco, alcohol and human papillomavirus (HPV) infection.

HNSCC of the oral cavity is generally treated with surgical resection, followed by adjuvant chemotherapy plus radiotherapy. Molecularly targeted agents, such as the epidermal growth factor receptor (EGFR) inhibitor cetuximab, have shown modest success in locally advanced disease. Finally, two immune-checkpoint inhibitors (ICIs) targeting PD1 (nivolumab and pembrolizumab) are approved for the treatment of recurrent and/or metastatic (R/M) HNSCC. Unfortunately, only 15–20%of patients with HNSCC achieve a durable response to these agents despite a twofold to threefold higher expression of PD1 and PD-L1 within the tumor.

Lung cancer is the leading cause of cancer deaths worldwide and the third most common cancer in the U.S. Lung cancer typically doesn't cause signs and symptoms in its earliest stages. Signs and symptoms of lung cancer typically occur when the disease is advanced. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. It accounts for over 80%of lung cancer cases. Common types include adenocarcinoma and squamous cell carcinoma. Adenosquamous carcinoma and sarcomatoid carcinoma are two less common types of NSCLC.

Lung cancer treatments include surgery, radiofrequency ablation, radiation therapy, chemotherapy, targeted drug therapy and immunotherapy. Targeting single molecular abnormalities or cancer pathways has achieved good clinical responses that have modestly affected survival in some cancers. However, this approach to cancer treatment is still reductionist, and many challenges need to be met to improve treatment outcomes.

Thus, there is an unmet need to develop new modalities and therapies that can improve therapeutic outcomes and prolong survival for patients with cancer.

SUMMARY

In one aspect, provided herein are nucleic acids encoding a polypeptide comprising: (a) an Igκ light chain signal peptide (SP) sequence; (b) an antigen sequence; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence.

In some embodiments, the antigen is a cancer antigen. In some embodiments, the cancer antigen is preferentially expressed antigen in melanoma (PRAME) .

In one aspect, provided herein are nucleic acids encoding a polypeptide comprising: (a) an Igκ light chain signal peptide (SP) sequence; (b) a preferentially expressed antigen in melanoma (PRAME) antigen sequence; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence.

In some embodiments, the Igκ light chain signal peptide (SP) sequence is at least 80%identical to the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, the nucleic acid described herein comprises a nucleic acid encoding the Igκ light chain signal peptide (SP) sequence, wherein the nucleic acid encoding the Igκ light chain signal peptide (SP) sequence is at least 80%identical to the sequence set forth in SEQ ID NO: 8, 9, 18 or 19.

In some embodiments, the MITD sequence comprises a sequence that is at least 80%identical to the amino acid sequence set forth in SEQ ID NO: 4.

In some embodiments, the nucleic acid described herein comprises a nucleic acid encoding the MITD sequence, wherein the nucleic acid encoding the MITD sequence is at least 80%identical to the sequence set forth in SEQ ID NO: 14, 15, 24 or 25.

In some embodiments, the PRAME antigen sequence comprises a sequence that is at least 80%identical to the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the nucleic acid described herein comprises a nucleic acid encoding the PRAME antigen sequence, wherein the nucleic acid encoding the PRAME antigen sequence is at least 80%identical to the sequence set forth in SEQ ID NO: 10, 11, 20 or 21.

In some embodiments, the Igκ light chain signal peptide (SP) sequence, the antigen sequence and/or the MITD sequence are linked via a linker.

In some embodiments, the linker comprises a sequence set forth in SEQ ID NO: 6.

In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding the linker, wherein the nucleic acid sequence encoding the linker is at least 80%identical to the sequence set forth in SEQ ID NO: 12, 13, 22 or 23.

In some embodiments, the nucleic acid described herein comprises a nucleotide sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 1, 2, 16 or 17.

In some embodiments, the nucleic acid comprises a stop codon.

In some embodiments, the nucleic acid is an mRNA.

In some embodiments, the mRNA comprises at least one chemical modification.

In some embodiments, the mRNA comprises a 5’UTR and/or a 3’ UTR. In some embodiments, the 5’ UTR comprises a sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the 3’ UTR comprises a sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 31 or 33.

In some embodiments, the chemical modification is selected from pseudouridine, N1-Methyl-pseudouridine, m7G (5') ppp (5') (2'-OMeA) pG, uridine, N1-ethylpseudouridine, 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2’-O-methyl uridine.

In some embodiments, the nucleic acid comprises a capping enhancing sequence.

In some embodiments, the capping enhancing sequence comprises a sequence set forth in SEQ ID NO: 27.

In some embodiments, the nucleic acid comprises a poly (A) sequence.

In some embodiments, the poly (A) sequence comprises a sequence set forth in SEQ ID NO: 34.

In one aspect, provided herein are pharmaceutical compositions comprising any one of the nucleic acids described herein.

In one aspect, provided herein are vaccines comprising any one of the nucleic acids described herein. In some embodiments, the vaccine is formulated in a lipid nanoparticle (LNP) .

In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.

In some embodiments, the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.

In one aspect, provided herein are polypeptides comprising a) an Igκ light chain signal peptide (SP) sequence; (b) an antigen sequence; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence. In one aspect, provided herein are polypeptides comprising a sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 7.

In one aspect, provided herein are methods of eliciting an immune response in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition described herein, the vaccine described herein, or the polypeptide described herein.

In one aspect, provided herein are methods of preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition described herein, the vaccine described herein, or the polypeptide described herein.

In some embodiments, the disease or disorder is cancer.

In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC) or lung cancer.

In some embodiments, the method described herein further comprises administering to the subject one or more additional therapeutic agents.

In some embodiments, the one or more additional therapeutic agents are anti-cancer therapeutic agents.

In one aspect, provided herein are methods of making a vaccine comprising mixing a nucleic acid described herein with a lipid nanoparticle formulation, thereby producing a vaccine.

In one aspect, provided herein is a nucleic acid comprising a sequence that is at least 80%, 90%, 95%, 99%, or 100%identical to a sequence set forth in any one of SEQ ID NOs: 1, 2, and 8-33.

In some embodiments, the nucleic acid is an mRNA.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the Western Blot result of the PRAME protein expression level. PRAME-1 and PRAME-2 are different mRNA sequences encoding the PRAME antigen.

FIG. 2 shows the PRAME antigen-specific immune response under the single dose regimen.

FIG. 3 shows the PRAME antigen-specific immune response under the intensive dosing schedule.

FIG. 4 shows the tolerance of the PRAME mRNA-LNP immunization in mice.

FIG. 5 shows inhibition of tumor growth by the PRAME mRNA-LNP vaccine.

FIG. 6 shows the percentage of mCD8⁺ central memory T cells (T_CM cells) in splenocytes of mice after the PRAME mRNA-LNP vaccine treatments.

FIG. 7 shows the percentage of mCD8⁺ effector memory T cells (T_EM cells) in splenocytes of mice after the PRAME mRNA-LNP vaccine treatments.

FIG. 8 shows inhibition of tumor growth in mice treated with the PRAME mRNA-LNP vaccine.

FIG. 9 shows inhibition of tumor growth in mice treated with multiple dosing of the PRAME mRNA-LNP vaccine.

FIG. 10 shows the number mCD3⁺ T cells per gram tumor tissue after the PRAME mRNA-LNP vaccination.

FIG. 11 shows the number mCD4⁺ helper T cells per gram tumor tissue after the PRAME mRNA-LNP vaccination.

FIG. 12 shows the number mCD8⁺ cytotoxic T cells (CTLs) per gram tumor tissue after the PRAME mRNA-LNP vaccination.

FIG. 13 shows the number mGranzymeB⁺ CTLs per gram tumor tissue after the PRAME mRNA-LNP vaccination.

FIG. 14 shows the numberCTLs per gram tumor tissue after the PRAME mRNA-LNP vaccination.

FIG. 15 shows selected sequences in the present disclosure.

DETAILED DESCRIPTION

The use of mRNA technology allows for induced production of a broad array of secreted, membrane-bound, and intracellular proteins in humans. Antigen-encoded mRNA is an attractive technology platform for antigen vaccination as an mRNA vaccine can deliver multiple antigens in a single molecule, a vaccine unique to each particular subject can be rapidly manufactured, and the antigens are endogenously translated and enter into the natural cellular antigen processing and presentation pathway. Moreover, this mRNA-based vaccine technology overcomes the challenges commonly associated with DNA-based vaccines, such as risk of genome integration or the high doses and devices needed for administration (e.g., electroporation) .

The present disclosure is related to nucleic acids, and pharmaceutical compositions or vaccines including the nucleic acids (e.g., an mRNA vaccine) , wherein the nucleic acid encodes a polypeptide comprising (a) an Igκ light chain signal peptide (SP) sequence; (b) an antigen (e.g., a cancer antigen such as preferentially expressed antigen in melanoma (PRAME) ) ; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) .

The present disclosure is also related to polypeptides comprising (a) an Igκ light chain signal peptide (SP) sequence; (b) an antigen (e.g., a cancer antigen such as preferentially expressed antigen in melanoma (PRAME) ) ; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) .

The present disclosure is also related to methods of eliciting an immune response or treating a cancer by administering to a subject a vaccine described herein (e.g., an mRNA cancer vaccine) formulated as a lipid nanoparticle.

The present disclosure further relates to a method of treating cancer by combining anti-cancer immunotherapy with the administration of the aforementioned vaccine (e.g., mRNA cancer vaccine) .

Nucleic Acids

In one aspect, the present disclosure is related to nucleic acids encoding a polypeptide comprising: (a) an immunoglobulin kappa (Igκ) light chain signal peptide (SP) sequence; (b) an antigen; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) .

In one aspect, the present disclosure is related to nucleic acids encoding a polypeptide comprising: (a) an Igκ light chain signal peptide (SP) sequence; (b) a preferentially expressed antigen in melanoma (PRAME) antigen sequence; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) .

Signal peptides (SP) , also referred to as signal sequences, are short peptides that can influence the targeting pathway of the protein and promote protein secretion or specific post-translational modifications such as glycosylation. As a result, SP from highly secreted proteins can be used to improve protein secretion levels of recombinant proteins in cell lines, as well as for ectopic expression of endogenous genes.

In some embodiments, the nucleic acid described herein encodes a polypeptide including an immunoglobulin kappa (Igκ) light chain signal peptide (SP) sequence. “Igκ signal peptide (SP) ” as used herein refers to the sequence derived from an immunoglobulin kappa (Igκ) light chain. In some embodiments, the Igκ signal peptide (SP) is a human Igκ signal peptide (SP) . As used herein, an “Igκ signal peptide (SP) sequence” refers to a full-length sequence, or a sequence variant thereof, e.g., a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the full-length sequence of an Igκ signal peptide (SP) . An example full-length sequence of Igκ signal peptide (SP) is shown in SEQ ID NO: 3. In some embodiments, the Igκ SP has an amino acid sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the amino acid sequence set forth in SEQ ID NO: 3.

In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding the Igκ signal peptide (SP) sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) . In some embodiments, the nucleic acid sequence encoding the Igκsignal peptide (SP) sequence comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 8 or 9. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid sequence encoding the Igκ signal peptide (SP) sequence comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 18 or 19.

In one aspect, provided herein is a nucleic acid comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 8 or 9. In some embodiments, the nucleic acid encodes a Igκ signal peptide (SP) sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) .

In one aspect, provided herein is a nucleic acid comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 18 or 19. In some embodiments, the nucleic acid encodes a Igκ signal peptide (SP) sequence. In some embodiments, the nucleic acid is a DNA.

Genetic modification of vaccines by linking an antigen to lysosomal or endosomal targeting signals has been used to route antigens into MHC class II processing compartments for improvement of CD4⁺ T cell responses. Combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to an antigen (e.g., a cancer antigen) can improve the presentation of MHC class I and class II epitopes in human cells. Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature immune cells (e.g., dendritic cells (DCs) ) , mimicking the dynamic trafficking properties of MHC molecules. Linking antigens (e.g., cancer antigens) to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8⁺ and CD4⁺ T cells, resulting in distinct CD8⁺ T cell specificities and a broad and variable Ag-specific CD4⁺ repertoire.

In some embodiments, the nucleic acid described herein encodes a polypeptide including MHC class I trafficking signal (MITD) sequence. “MHC class I trafficking signal (MITD) ” as used herein refers to the sequence derived from an MHC class I trafficking signal (MITD) . In some embodiments, the MITD is a human MITD. As used herein, an “MHC class I trafficking signal (MITD) sequence” refers to a full-length sequence, or a sequence variant thereof, e.g., a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the full-length sequence of an MHC class I trafficking signal (MITD) . An example full-length sequence of MHC class I trafficking signal (MITD) is shown in SEQ ID NO: 4. In some embodiments, the MHC class I trafficking signal (MITD) has an amino acid sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the amino acid sequence set forth in SEQ ID NO: 4.

In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding the MITD sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) . In some embodiments, the nucleic acid sequence encoding the MITD sequence comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 14 or 15. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid sequence encoding the MITD sequence comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 24 or 25.

In one aspect, provided herein is a nucleic acid comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 14 or 15. In some embodiments, the nucleic acid encodes an MITD sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) .

In one aspect, provided herein is a nucleic acid comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 24 or 25. In some embodiments, the nucleic acid encodes an MITD sequence. In some embodiments, the nucleic acid is a DNA.

In some embodiments, the Igκ signal peptide (SP) is located at the N-terminus of the antigen sequence (e.g., a cancer antigen sequence such as PRAME) . In some embodiments, the Igκ signal peptide (SP) sequence is located at the N-terminus of the MITD sequence. In some embodiments, the antigen sequence (e.g., a cancer antigen sequence such as PRAME) is located at the N-terminus of the MITD sequence. In some embodiments, the nucleic acid described herein encodes a polypeptide comprising, from the N-terminus to the C-terminus, the Igκ signal peptide (SP) sequence, the antigen sequence (e.g., a cancer antigen sequence such as PRAME sequence) , and the MITD sequence.

In some embodiments, the Igκ signal peptide (SP) sequence and the antigen sequence (e.g., a cancer antigen sequence such as PRAME sequence) ; the antigen sequence (e.g., a cancer antigen sequence such as PRAME sequence) and the MITD sequence are linked via a linker; or the Igκ signal peptide (SP) sequence and the MITD sequence are linked via a linker. Any suitable linker known in the art can be used herein. In some embodiments, the linker comprises an amino acid sequence set forth in SEQ ID NO: 6.

In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding the linker sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) . In some embodiments, the nucleic acid sequence encoding the linker sequence comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 12 or 13. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid sequence encoding the linker sequence comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 22 or 23.

In one aspect, provided herein is a nucleic acid comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 12 or 13. In some embodiments, the nucleic acid encodes a linker sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) .

In one aspect, provided herein is a nucleic acid comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 22 or 23. In some embodiments, the nucleic acid encodes a linker sequence. In some embodiments, the nucleic acid is a DNA.

In some embodiments, the nucleic acid described herein comprises a nucleotide sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) .

In some embodiments, the nucleic acid described herein comprises a nucleotide sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the amino acid sequence set forth in SEQ ID NO: 16 or 17. In some embodiments, the nucleic acid is a DNA.

In some embodiments, the nucleic acid described herein comprises a stop codon (e.g., a TAA, TGA, or TAG) . In some embodiments, the nucleic acid described herein comprises a TAA (UAA in the context of RNA) stop codon. In some embodiments, the nucleic acid described herein comprises a TGA (UGA in the context of RNA) stop codon. In some embodiments, the nucleic acid described herein comprises a TAG (UAG in the context of RNA) stop codon.

In some embodiments, the stop codon is at the 3’ end of the nucleic acid described herein. In some embodiments, the stop codon is at the 3’ end of the nucleic acid encoding the antigen (e.g., cancer antigen such as PRAME) and/or MITD.

The stop codons were originally identified by mutations in bacteriophage T4. The first one identified was TAG (UAG in the context of RNA) , the amber codon. The second stop codon to be found TAA (UAA in the context of RNA) was called the “ochre codon. ” The third stop codon TGA (UGA in the context of RNA) is called the “opal” or “umber” codon.

Translation of an mRNA into a polypeptide is terminated when the release factor eRF1 interacts with a UAA, UAG, or UGA stop codon in the ribosomal A site and another release factor, eRF3, hydrolyzes GTP and stimulates the polypeptide release activity of eRF1. However, at low frequency, a near-cognate tRNA (nc-tRNA; a tRNA with one base pair mismatch in its anticodon) outcompetes eRF1 in decoding the stop codon, resulting in the continuation of translation elongation. When the stop codon is located at its normal site at the end of an open reading frame (ORF) elongation will continue into the mRNA 3’-untranslated region (3’-UTR) , producing a C-terminally extended polypeptide product. Such events are termed stop codon readthrough or nonsense suppression.

In some embodiments, nucleic acids of the present disclosure are messenger RNA (mRNA) . “Messenger RNA” (mRNA) refers to any nucleic acid that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.

The basic components of an mRNA molecule typically include at least one coding region, a 5′untranslated region (UTR) , a 3′UTR, a 5′cap (e.g., a capping enhancing sequence) and a poly-Atail. Nucleic acids of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.

In some embodiments, the nucleic acid described herein comprises a cap sequence. In some embodiments, the cap sequence is a 5’ cap sequence. The 5’ capping in mature mRNA is required for protection of mRNA from degradation, facilitating recruitment of the ribosomes, gene expression, and self-versus non-self-identification. Several variations of 5’ cap structures have been found to exist in nature. The 5' cap of the eukaryotic mRNA contains 7-methylguanosine (m7G) through a 5’-5’-triphosphate bridge (m7GpppN) via a series of enzymatic capping reactions involving RNA triphosphatase, guanosyltransferase, and S-adenosyl methionine. To further enhance the translation efficiency, additional methylation can be introduced at the first nucleotide (cap1: m7GpppNmpN) or both first and second nucleotides (cap2: m7GpppNmpNm) . The 5’ capping modifications improve the translation initiation by recruiting translation initiation factors, protect the synthetic mRNA against exonuclease degradation, and avoid an innate immunity overactivation response. mRNA capping can be performed during the IVT reaction by substituting a part of the guanosine triphosphate (GTP) substrate for a cap analog. Alternatively, mRNA can be capped in a second enzymatic reaction using the vaccinia capping enzyme (VCC) and a methyl donor as a substrate.

In some embodiments, the nucleic acid described herein comprises a capping enhancing sequence. As used herein, a “capping enhancing sequence” is a sequence that enhances the functions of the 5’ cap of an RNA (e.g., mRNA) . In some embodiments, the capping enhancing sequence is an RNA (e.g., mRNA) sequence. In some embodiments, the capping enhancing sequence comprises a sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 27. In some embodiments, a DNA sequence encodes the capping enhancing sequence. In some embodiments, the sequence comprises a sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 26.

Also provided herein are nucleic acid sequences that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 26. In some embodiments, the nucleic acid encodes a capping enhancing sequence. In some embodiments, the nucleic acid is a DNA sequence.

Also provided herein are nucleic acid sequences that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 27. In some embodiments, the nucleic acid encodes a capping enhancing sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) sequence.

In some embodiments, the capping enhancing sequence is located 3’ to the 5’ cap of the nucleic acid described herein. In some embodiments, the capping enhancing sequence is located 5’ to the 5’ cap of the nucleic acid described herein.

In some embodiments, the nucleic acid described herein comprises a 3’ untranslated region (3’ UTR) and/or a 5’ UTR. The untranslated regions (UTRs) are responsible for the transcription regulation and mRNA stability. These regions strongly affect translation efficiency as the sequences used are involved in the translation machinery recognition, recruitment, and mRNA trafficking.

In some embodiments, the nucleic acid comprises a DNA sequence encoding a 3’ UTR. In some embodiments, the DNA sequence encoding the 3’ UTR comprises a sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 30 or 32. In some embodiments, the 3’ UTR sequence is an RNA (e.g., mRNA) sequence. In some embodiments, the 3’ UTR comprises a sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 31 or 33.

Also provided herein are nucleic acid sequences that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 30 or 32. In some embodiments, the nucleic acid encodes a 3’ UTR. In some embodiments, the nucleic acid is a DNA sequence.

Also provided herein are nucleic acid sequences that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 31 or 33. In some embodiments, the nucleic acid is a 3’ UTR. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) sequence.

In some embodiments, the nucleic acid comprises a DNA sequence encoding a 5’ UTR. In some embodiments, the DNA sequence encoding the 5’ UTR comprises a sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 28. In some embodiments, the 5’ UTR sequence is an RNA (e.g., mRNA) sequence. In some embodiments, the 5’ UTR comprises a sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 29.

Also provided herein are nucleic acid sequences that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 28. In some embodiments, the nucleic acid encodes a 5’ UTR. In some embodiments, the nucleic acid is a DNA sequence.

Also provided herein are nucleic acid sequences that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 29. In some embodiments, the nucleic acid is a 5’ UTR. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) sequence.

In some embodiments, the nucleic acid described herein comprises a poly (A) sequence. Addition of a poly (A) sequence (e.g., a 3′poly (A) tail) improves mRNA stability and translational activities, as it protects mRNA from nuclease degradation by the poly (A) -binding protein (PABP) . This tail can be added to the transcript by inserting a poly (A) sequence in the DNA template or by an enzymatic reaction to the RNA sequence. Tail size optimization is an important factor for the stabilization and expression of mRNA. Longer poly-Atails can improve mRNA stability and translation. However, this effect is not linear, and the best tail size is dependent on cell type.

In some embodiments, the poly (A) sequence can be in a DNA sequence described herein and be transcribed into the RNA (e.g., mRNA) sequence described herein. In some embodiments, the poly (A) sequence is added after the transcription of the RNA (e.g., mRNA) sequence. In some embodiments, the poly (A) sequence comprises a sequence that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 34.

Also provided herein are nucleic acid sequences that is about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99%or 100%identical to the sequence set forth in SEQ ID NO: 34. In some embodiments, the nucleic acid is a poly (A) sequence.

In some embodiments, the poly (A) sequence is located at the 3’ end of the nucleic acid described herein.

In one aspect, the nucleic acid described herein comprises from 5’ to 3’:

(a) a capping enhancing sequence;

(b) a 5’ UTR;

(c) an Igκ light chain signal peptide (SP) sequence;

(d) an antigen sequence (e.g., preferentially expressed antigen in melanoma (PRAME) ) ;

(e) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence;

(f) a 3’ UTR; and

(g) optionally a poly (A) sequence.

In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an RNA. In some embodiments, the nucleic acids described herein are codon optimized.

Cancer Antigens

The nucleic acid described herein encodes a polypeptide comprising an antigen. In some embodiments, the antigen is cancer antigen. In some embodiments, the antigen is the full-length or a portion of a cancer antigen.

Many cancer antigens are known in the art. Cancer or tumor antigens (e.g., traditional cancer antigens) in the compositions (e.g., nucleic acids, pharmaceutical compositions or vaccines) and methods described herein may be any such cancer or tumor antigens known in the field. In some embodiments, the cancer antigen is PRAME.

In some embodiments, the cancer antigen is a PRAME antigen sequence. PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen with restricted expression in somatic tissues and re‐expression in poor prognostic solid tumors. PRAME is a target for immunotherapy and its expression is associated with worse survival in the TCGA breast cancer cohort, particularly in immune‐unfavorable tumors. PRAME overexpressing breast cancer cells inhibit T cell activation and cytolytic potential, which could be partly restored by silencing of PRAME. Furthermore, silencing of PRAME reduces expression of several immune checkpoints and their ligands, including PD‐1, LAG3, PD‐L1, CD86, Gal‐9 and VISTA. PRAME tumor expression can suppress the expression and secretion of multiple pro‐inflammatory cytokines, and mediators of T cell activation, differentiation and cytolysis. A detailed review of the PRAME antigen can be found, for example, at Naik et al., J Cell Mol Med. 2021 Nov; 25 (22) : 10376–10388, which is incorporated by reference in its entirety herein. PRAME is expressed primarily in the cytoplasm in seminiferous ducts in testis. It is localized to the nucleoplasm in addition to the plasma membrane. It is enriched in testis tissue. It can serve as a prognostic marker for cancer (e.g., renal cancer (unfavorable) , lung cancer (unfavorable) and head and neck cancer (unfavorable) ) . The expression profile of PRAME can be found, e.g., at The Human Protein Atlas website.

As used herein, an “antigen sequence” refers to a full-length sequence, or a sequence variant thereof, e.g., a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the full-length sequence of a cancer antigen. An example full-length sequence of PRAME is shown in SEQ ID NO: 5. In some embodiments, the PRMAE has a Uniprot ID No. of P78395. In some embodiments, the PRAME comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the amino acid sequence set forth in SEQ ID NO: 5.

In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding the PRAME antigen sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) . In some embodiments, the nucleic acid sequence encoding the PRAME antigen sequence comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 10 or 11. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid sequence encoding the PRAME antigen sequence comprises a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 20 or 21.

In one aspect, provided herein is a nucleic acid comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 10 or 11. In some embodiments, the nucleic acid encodes a PRAME antigen sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) .

In one aspect, provided herein is a nucleic acid comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the sequence set forth in SEQ ID NO: 20 or 21. In some embodiments, the nucleic acid encodes a PRAME antigen sequence. In some embodiments, the nucleic acid is a DNA.

In some embodiments, the antigen is a personalized cancer antigen or a portion thereof.

In some embodiments, the antigen sequence (e.g., cancer antigen sequence) is a sequence of an antigen epitope. An epitope, also known as an antigenic determinant, as used herein is a portion of an antigen that is recognized by the immune system in the appropriate context, specifically by antibodies, B cells, or T cells. Epitopes may include B cell epitopes (e.g., predicted B cell reactive epitopes) and T cell epitopes (e.g., predicted T cell reactive epitopes) . B-cell epitopes (e.g., predicted B cell reactive epitopes) are peptide sequences which are required for recognition by specific antibody producing B-cells. B cell epitopes (e.g., predicted B cell reactive epitopes) refer to a specific region of the antigen that is recognized by an antibody. T-cell epitopes (e.g., predicted T cell reactive epitopes) are peptide sequences which, in association with proteins on APC, are required for recognition by specific T-cells. T cell epitopes (e.g., predicted T cell reactive epitopes) are processed intracellularly and presented on the surface of APCs, where they are bound to MHC molecules including e.g., MHC class II and/or MHC class I molecules. The portion of an antibody that binds to the epitope is called a paratope. An epitope may be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope. A linear, or continuous, epitope is defined by the primary amino acid sequence of a particular region of a protein. The sequences that interact with the antibody are situated next to each other sequentially on the protein, and the epitope can usually be mimicked by a single peptide. Conformational epitopes are epitopes that are defined by the conformational structure of the native protein. These epitopes may be continuous or discontinuous (i.e., may be components of the epitope which can be situated on disparate parts of the protein, and are brought close to each other in the folded native protein structure) .

In one aspect, provided herein are polypeptides comprising a) an Igκ light chain signal peptide (SP) sequence described herein; (b) an antigen sequence described herein; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence described herein.

In some embodiments, the antigen sequence is a PRAME sequence.

In one aspect, provided herein are polypeptides comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the amino acid sequence of the protein translation product of the nucleic acid described herein. The translation product can be the translation of the full-length or a portion of the nucleic acid described herein. In one aspect, provided herein are polypeptides comprising a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identical to the amino acid sequence set forth in SEQ ID NO: 7.

Chemical Modifications

In some embodiments, the nucleic acid described herein comprises one or more chemically modified nucleobases. The modified polynucleotides comprise a nucleic acid (or “polynucleotide” used exchangeable herein) described herein. The modified nucleic acids can be chemically modified and/or structurally modified. When the nucleic acids of the present disclosure are chemically and/or structurally modified the polynucleotides can be referred to as “modified nucleic acids” .

The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA polynucleotides, such as mRNA polynucleotides) encoding a polypeptide comprising (a) an Igκ light chain signal peptide (SP) sequence; (b) an antigen sequence (e.g., a cancer antigen sequence such as PRAME sequence) ; and (c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence. A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase” ) . A “nucleotide” refers to a nucleoside including a phosphate group. Modified nucleotides can by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.

The modified nucleic acids disclosed herein can comprise various distinct modifications. In some embodiments, the modified polynucleotides contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide introduced to a cell can exhibit one or more desirable properties such as, e.g., improved protein expression, reduced innate immune response, or reduced degradation in the cell, as compared to an unmodified polynucleotide.

In some embodiments, a nucleic acid disclosed herein (e.g., a nucleic acid encoding one or more peptide epitopes) is structurally modified. As used herein, a “structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted, or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” can be chemically modified to “AT-5meC-G. ” In some embodiments, the nucleic acids of the present disclosure are chemically modified. As used herein in reference to a nucleic acid, the terms “chemical modification” or, as appropriate, “chemically modified” refer to modification with respect to adenosine (A) , guanosine (G) , uridine (U) , or cytidine (C) ribo-or deoxyribonucleosides in one or more of their position, pattern, percentage, or population. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties.

In some embodiments, the nucleic acids of the present disclosure can have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by mere downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine or 5-methoxyuridine. In another embodiment, the polynucleotides can have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modified in the same way) .

Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base/sugar or linker can be incorporated into polynucleotides of the present disclosure.

The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the present disclosure will recite “T” s in a representative DNA sequence but where the sequence represents RNA, the “T” s would be substituted for “U” s.

In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.

In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; or PCT/IB2017/051367 all of which are incorporated by reference herein for this purpose.

Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.

Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) , in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid) , introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid) , introduced into a cell or organism, may exhibit reduced innate immune response relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) , in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., DNA nucleic acids or RNA nucleic acids, such as mRNA nucleic acids) .

In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1-methyl-pseudouridine (m1ψ) , 1-ethyl-pseudouridine (e1ψ) , 5-methoxy-uridine (mo5U) , 5-methyl-cytidine (m5C) , and/or pseudouridine (ψ) . In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

In some embodiments, the modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise m¹A (1-methyladenosine) ; m²A (2-methyladenosine) ; Am (2′-O-methyladenosine) ; ms² m⁶A (2-methylthio-N⁶-methyladenosine) ; i⁶A (N⁶-isopentenyladenosine) ; ms²i6A (2-methylthio-N⁶ isopentenyladenosine) ; io⁶A (N⁶- (cis-hydroxyisopentenyl) adenosine) ; ms²i⁶A (2-methylthio-N⁶- (cis-hydroxyisopentenyl) adenosine) ; g⁶A (N⁶-glycinylcarbamoyladenosine) ; t⁶A (N⁶-threonylcarbamoyladenosine) ; ms²t⁶A (2-methylthio-N⁶-threonyl carbamoyladenosine) ; m⁶t⁶A (N⁶-methyl-N⁶-threonylcarbamoyladenosine) ; hn⁶A (N⁶-hydroxynorvalylcarbamoyladenosine) ; ms²hn⁶A (2-methylthio-N⁶-hydroxynorvalyl carbamoyladenosine) ; Ar (p) (2′-O-ribosyladenosine (phosphate) ) ; I (inosine) ; m¹I (1-methylinosine) ; m¹Im (1, 2′-O-dimethylinosine) ; m³C (3-methylcytidine) ; Cm (2′-O-methylcytidine) ; s²C (2-thiocytidine) ; ac⁴C (N⁴-acetylcytidine) ; f⁵C (5-formylcytidine) ; m⁵ Cm (5, 2′-O-dimethylcytidine) ; ac⁴Cm (N⁴-acetyl-2′-O-methylcytidine) ; k²C (lysidine) ; m¹G (1-methylguanosine) ; m²G (N²-methylguanosine) ; m⁷G (7-methylguanosine) ; Gm (2′-O-methylguanosine) ; m² ₂G (N², N²-dimethylguanosine) ; m²Gm (N², 2′-O-dimethylguanosine) ; m² ₂Gm (N², N², 2′-O-trimethylguanosine) ; Gr (p) (2′-O-ribosylguanosine (phosphate) ) ; yW (wybutosine) ; o₂yW (peroxywybutosine) ; OHyW (hydroxywybutosine) ; OHyW* (undermodified hydroxywybutosine) ; imG (wyosine) ; mimG (methylwyosine) ; Q (queuosine) ; oQ (epoxyqueuosine) ; galQ (galactosyl-queuosine) ; manQ (mannosyl-queuosine) ; preQ₀ (7-cyano-7-deazaguanosine) ; preQ₁ (7-aminomethyl-7-deazaguanosine) ; G⁺ (archaeosine) ; D (dihydrouridine) ; m⁵Um (5, 2′-O-dimethyluridine) ; s⁴U (4-thiouridine) ; m⁵s²U (5-methyl-2-thiouridine) ; s²Um (2-thio-2′-O-methyluridine) ; acp³U (3- (3-amino-3-carboxypropyl) uridine) ; ho⁵U (5-hydroxyuridine) ; mo⁵U (5-methoxyuridine) ; cmo⁵U (uridine 5-oxyacetic acid) ; mcmo⁵U (uridine 5-oxyacetic acid methyl ester) ; chm⁵U (5- (carboxyhydroxymethyl) uridine) ) ; mchm⁵U (5- (carboxyhydroxymethyl) uridine methyl ester) ; mcm⁵U (5-methoxycarbonylmethyluridine) ; mcm⁵Um (5-methoxycarbonylmethyl-2′-O-methyluridine) ; mcm⁵s²U (5-methoxycarbonylmethyl-2-thiouridine) ; nm⁵s²U (5-aminomethyl-2-thiouridine) ; mnm⁵U (5-methylaminomethyluridine) ; mnm⁵s²U (5-methylaminomethyl-2-thiouridine) ; mnm⁵se²U (5-methylaminomethyl-2-selenouridine) ; ncm⁵U (5-carbamoylmethyluridine) ; ncm⁵Um (5-carbamoylmethyl-2′-O-methyluridine) ; cmnm⁵U (5-carboxymethylaminomethyluridine) ; cmnm⁵Um (5-carboxymethylaminomethyl-2′-O-methyluridine) ; cmnm⁵s²U (5-carboxymethylaminomethyl-2-thiouridine) ; m⁶ ₂A (N⁶, N⁶-dimethyladenosine) ; Im (2′-O-methylinosine) ; m⁴C (N⁴-methylcytidine) ; m⁴ Cm (N⁴, 2′-O-dimethylcytidine) ; hm⁵C (5-hydroxymethylcytidine) ; m³U (3-methyluridine) ; cm⁵U (5-carboxymethyluridine) ; m⁶Am (N⁶, 2′-O-dimethyladenosine) ; m⁶ ₂Am (N⁶, N⁶, O-2′-trimethyladenosine) ; m^2, 7G (N², 7-dimethylguanosine) ; m^2, 2, 7G (N², N², 7-trimethylguanosine) ; m³Um (3, 2′-O-dimethyluridine) ; m⁵D (5-methyldihydrouridine) ; f⁵Cm (5-formyl-2′-O-methylcytidine) ; m¹Gm (1, 2′-O-dimethylguanosine) ; m¹Am (1, 2′-O-dimethyladenosine) ; τm⁵U (5-taurinomethyluridine) ; τm⁵s²U (5-taurinomethyl-2-thiouridine) ) ; imG-14 (4-demethylwyosine) ; imG2 (isowyosine) ; or ac⁶A (N⁶-acetyladenosine) .

In some embodiments, a RNA nucleic acid of the disclosure comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid. In some embodiments, the nucleic acid described herein (e.g., mRNA) includes N1-Methyl-pseudouridine substitutions at one or more positions and m7G (5') ppp (5') (2'-OMeA) pG substitutions at one or more positions.

In some embodiments, an RNA nucleic acid of the disclosure comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid.

In some embodiments, an RNA nucleic acid of the disclosure comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

In some embodiments, nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail) . In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

The nucleic acid may contain from about 1%to about 100%modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U, or C) or any intervening percentage (e.g., from 1%to 20%, from 1%to 25%, from 1%to 50%, from 1%to 60%, from 1%to 70%, from 1%to 80%, from 1%to 90%, from 1%to 95%, from 10%to 20%, from 10%to 25%, from 10%to 50%, from 10%to 60%, from 10%to 70%, from 10%to 80%, from 10%to 90%, from 10%to 95%, from 10%to 100%, from 20%to 25%, from 20%to 50%, from 20%to 60%, from 20%to 70%, from 20%to 80%, from 20%to 90%, from 20%to 95%, from 20%to 100%, from 50%to 60%, from 50%to 70%, from 50%to 80%, from 50%to 90%, from 50%to 95%, from 50%to 100%, from 70%to 80%, from 70%to 90%, from 70%to 95%, from 70%to 100%, from 80%to 90%, from 80%to 95%, from 80%to 100%, from 90%to 95%, from 90%to 100%, and from 95%to 100%) . It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

The nucleic acids may contain at a minimum 1%and at maximum 100%modified nucleotides, or any intervening percentage, such as at least 5%modified nucleotides, at least 10%modified nucleotides, at least 25%modified nucleotides, at least 50%modified nucleotides, at least 80%modified nucleotides, or at least 90%modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%or 100%of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil) . The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures) . In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100%of the cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine) . The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures) .

In some embodiments, the nucleic acid can include any useful linker between the nucleosides. Such linkers, including backbone modifications, that are useful in the composition of the present disclosure include, but are not limited to the following: 3′-alkylene phosphonates, 3′-amino phosphoramidate, alkene containing backbones, aminoalkylphosphoramidates, aminoalkylphosphotriesters, boranophosphates, -CH₂-O-N (CH₃) -CH₂-, -CH₂-N (CH₃) -N (CH₃) -CH₂-, -CH₂-NH-CH₂-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene (methylimino) , methylene formacetyl and thioformacetyl backbones, methyleneimino and methylenehydrazino backbones, morpholino linkages, -N (CH₃) -CH₂-CH₂-, oligonucleosides with heteroatom internucleoside linkage, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphotriesters, PNA, siloxane backbones, sulfamate backbones, sulfide sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates.

The modified nucleosides and nucleotides (e.g., building block molecules) , which can be incorporated into a nucleic acid (e.g., RNA or mRNA, as described herein) , can be modified on the sugar of the ribonucleic acid. For example, the 2′hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C_1-6 alkyl; optionally substituted C_1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C_3-8 cycloalkyl; optionally substituted C_3-8 cycloalkoxy; optionally substituted C_6-10 aryloxy; optionally substituted C_6-10 aryl-C1-6 alkoxy, optionally substituted C_1-12 (heterocyclyl) oxy; a sugar (e.g., ribose, pentose, or any described herein) ; a polyethyleneglycol (PEG) , -O (CH₂CH₂O) _nCH₂CH₂OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20) ; “locked” nucleic acids (LNA) in which the 2′-hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to the 4′-carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.

Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene) ; addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl) ; ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane) ; ring expansion of ribose (e.g., to form a 6-or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone) ; multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds) , threose nucleic acid (TNA, where ribose is replace with α-L-threofuranosyl- (3′→2′) ) , and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone) . The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar. Such sugar modifications are described in, for example, International Patent Publication Nos. WO2013052523 and WO2014093924, the contents of each of which are incorporated herein by reference in their entireties for this purpose.

The nucleic acids of the disclosure can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein.

Pharmaceutical Compositions and Vaccine Formulations

Provided herein are compositions (e.g., pharmaceutical compositions) , methods, kits, and reagents for prevention and/or treatment of cancer in humans (e.g., subjects or patients) and other mammals.

In one aspect, provided herein are pharmaceutical compositions and vaccines comprising the nucleic acid described herein.

Vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. As a non-limiting set of examples, cancer vaccines can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation) ; (4) alter the biodistribution (e.g., target to specific tissues or cell types) ; (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with cancer vaccines (e.g., for transplantation into a subject) , hyaluronidase, nanoparticle mimics and combinations thereof.

In some embodiments, vaccine compositions comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety for this purpose) .

In some embodiments, cancer vaccines are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the cancer vaccines or the nucleic acids contained therein, for example, RNA (e.g., mRNA) encoding antigenic polypeptides.

Formulations of the vaccine 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 bringing the active ingredient (e.g., nucleic acids such as mRNA) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single-or multi-dose unit.

The formulation of any of the compositions disclosed herein can include one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants) , or other components. For example, a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No. 2005/0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof) .

A polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form) . A polymer can be biodegradable and/or biocompatible. A polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

In some embodiments, the compositions disclosed herein may be formulated as lipid nanoparticles (LNP) . Accordingly, the present disclosure also provides vaccines comprising (i) a lipid composition comprising a delivery agent, and (ii) a nucleic acid described herein. In such vaccine, the lipid composition disclosed herein can encapsulate the nucleic acid described herein.

Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs) , liposomes (e.g., lipid vesicles) , and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

Nanoparticle compositions include, for example, lipid nanoparticles (LNPs) , liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and/or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels.

In one embodiment, a vaccine comprises an ionizable lipid, a structural lipid, a phospholipid, and the nucleic acid described herein (e.g., mRNA) . In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid and a structural lipid.

The ratio between the lipid composition and the vaccine may be from about 10: 1 to about 60: 1 (wt/wt) . In some embodiments, the ratio between the lipid composition and the nucleic acid may be about 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 21: 1, 22: 1, 23: 1, 24: 1, 25: 1, 26: 1, 27: 1, 28: 1, 29: 1, 30: 1, 31: 1, 32: 1, 33: 1, 34: 1, 35: 1, 36: 1, 37: 1, 38: 1, 39: 1, 40: 1, 41: 1, 42: 1, 43: 1, 44: 1, 45: 1, 46: 1, 47: 1, 48: 1, 49: 1, 50: 1, 51: 1, 52: 1, 53: 1, 54: 1, 55: 1, 56: 1, 57: 1, 58: 1, 59: 1 or 60: 1 (wt/wt) . In some embodiments, the wt/wt ratio of the lipid composition to the vaccine is about 20: 1 or about 15: 1.

In some embodiments, the vaccine (e.g., the nucleic acid cancer vaccine) may be comprised in lipid nanoparticles such that the lipid: polynucleotide weight ratio is 5: 1, 10: 1, 15: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, 55: 1, 60: 1 or 70: 1, or a range or any of these ratios such as, but not limited to, 5: 1 to about 10: 1, from about 5: 1 to about 15: 1, from about 5: 1 to about 20: 1, from about 5: 1 to about 25: 1, from about 5: 1 to about 30: 1, from about 5: 1 to about 35: 1, from about 5: 1 to about 40: 1, from about 5: 1 to about 45: 1, from about 5: 1 to about 50: 1, from about 5: 1 to about 55: 1, from about 5: 1 to about 60: 1, from about 5: 1 to about 70: 1, from about 10: 1 to about 15: 1, from about 10: 1 to about 20: 1, from about 10: 1 to about 25: 1, from about 10: 1 to about 30: 1, from about 10: 1 to about 35: 1, from about 10: 1 to about 40: 1, from about 10: 1 to about 45: 1, from about 10: 1 to about 50: 1, from about 10: 1 to about 55: 1, from about 10: 1 to about 60: 1, from about 10: 1 to about 70: 1, from about 15: 1 to about 20: 1, from about 15: 1 to about 25: 1, from about 15: 1 to about 30: 1, from about 15: 1 to about 35: 1, from about 15: 1 to about 40: 1, from about 15: 1 to about 45: 1, from about 15: 1 to about 50: 1, from about 15: 1 to about 55: 1, from about 15: 1 to about 60: 1 or from about 15: 1 to about 70: 1.

In some embodiments, the vaccine (e.g., the nucleic acid cancer vaccine) may be comprised in lipid nanoparticles in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

As generally defined herein, the term “lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids lead them to form liposomes, vesicles, or membranes in aqueous media.

In some embodiments, a lipid nanoparticle (LNP) may comprise an ionizable lipid. As used herein, the term “ionizable lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged or negatively charged. An ionizable lipid may be positively charged, in which case it can be referred to as “cationic lipid” . In certain embodiments, an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipids. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1) , divalent (+2, or -2) , trivalent (+3, or -3) , etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged) . Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines) , ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively-charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired. Ionizable lipids can also be the compounds disclosed in International Publication Nos.: WO2017075531, WO2015199952, WO2013086354, or WO2013116126, or selected from formulae CLI-CLXXXXII of U.S. Pat. No. 7,404,969; each of which is hereby incorporated by reference in its entirety for this purpose.

It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge” or “partial positive charge” on a molecule. The terms “partial negative charge” and “partial positive charge” are given its ordinary meaning in the art. A “partial negative charge” may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

In some embodiments, the ionizable lipid is an ionizable amino lipid, sometimes referred to in the art as an “ionizable cationic lipid” . In one embodiment, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure. In addition to these, an ionizable lipid may also be a lipid including a cyclic amine group.

Vaccines of the present disclosure are typically formulated into lipid nanoparticles. In some embodiments, the lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG) -modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60%ionizable amino lipid. For example, the lipid nanoparticle may comprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60%ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%, 40%, 50, or 60%ionizable amino lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25%non-cationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25%non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or 25%non-cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55%sterol. For example, the lipid nanoparticle may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55%sterol. In some embodiments, the lipid nanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or 55%sterol.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15%PEG-modified lipid. For example, the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%PEG-modified lipid.

In some embodiments, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety for this purpose. In one embodiment, the lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2013086354; the contents of which is herein incorporated by reference in its entirety for this purpose.

Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

The size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide. As used herein, “size” or “mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

Relative amounts of the active ingredient (e.g., the nucleic acid cancer vaccine) , the pharmaceutically acceptable excipient, and/or any additional ingredients in a vaccine composition may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1%and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1%and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

In some embodiments, provided herein are packages containing the vaccine containing about 0.1 mg to about 1 mg of the nucleic acid (e.g., mRNA) described herein.

Methods of Treatment

In one aspect, provided herein are methods of eliciting an immune response in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition described herein or the vaccine described herein.

In one aspect, provided herein are methods of preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition described herein or the vaccine described herein.

Vaccines described herein (e.g., nucleic acid cancer vaccines) may be used as therapeutic or prophylactic agents in medicine to prevent and/or treat cancer. In some embodiments, the cancer vaccines of the present disclosure are used to provide prophylactic protection from cancer. Prophylactic protection from cancer can be achieved following administration of a cancer vaccine of the present disclosure. Vaccines can be administered once, twice, three times, four times, or more but it may be sufficient to administer the vaccine once (optionally followed by a single booster) . It may also be desirable to administer the vaccine to an individual having cancer to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

In some embodiments, the disease or disorder is cancer. A non-limiting list of cancers that the cancer vaccines may treat is presented below. Peptide epitopes or antigens may be derived from any antigen of these cancers or tumors. Such epitopes may be referred to as cancer or tumor antigens. Cancer cells may differentially express cell surface molecules during different phases of tumor progression. For example, a cancer cell may express a cell surface antigen in a benign state, yet down-regulate that particular cell surface antigen upon metastasis. As such, it is envisioned that the tumor or cancer antigen may encompass antigens produced during any stage of cancer progression. The methods of the disclosure may be adjusted to accommodate for these changes. For instance, several different cancer vaccines may be generated for a particular patient. For instance, a first vaccine may be used at the start of the treatment. At a later time point, a new cancer vaccine may be generated and administered to the patient to account for different antigens being expressed.

Cancers or tumors include but are not limited to neoplasms, malignant tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth such that it would be considered cancerous. The cancer may be a primary or metastatic cancer. Specific cancers that can be treated according to the present disclosure include, but are not limited to, those listed below (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia) . Cancers for use with the instantly described methods and compositions may include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma, teratomas; tumor mutational burden high tumors; choriocarcinomas; stromal tumors and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms' tumor. In some embodiments that cancer is any one of melanoma, bladder carcinoma, HPV negative head and neck squamous cell carcinoma (HNSCC) , NSCLC, SCLC, MSI-High tumors, or TMB (tumor mutational burden) High cancers.

In some embodiments, the cancer is a cancer wherein the expression of PRMAE is increased. In some embodiments, the cancer is head and neck squamous cell carcinoma (HNSCC) , melanoma or lung cancer.

Once a vaccine (e.g., a nucleic acid cancer vaccine) is synthesized, it is administered to the patient. In some embodiments the vaccine is administered on a schedule for up to two months, up to three months, up to four month, up to five months, up to six months, up to seven months, up to eight months, up to nine months, up to ten months, up to eleven months, up to 1 year, up to 1 and 1/2 years, up to two years, up to three years, or up to four years. The schedule may be the same or varied. In some embodiments the schedule is weekly for the first 3 weeks and then monthly thereafter. The schedule may be determined or varied by one of skill in the art (e.g., a medical doctor) depending on the individual patient or subject's criteria (e.g., weight, age, type of cancer, etc. ) .

The vaccine may be administered by any route. In some embodiments the vaccine is administered by an intradermal, intramuscular, intravascular, intratumoral, and/or subcutaneous route.

In some embodiments, the nucleic acid cancer vaccine may also be administered with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are anti-cancer therapeutic agents. The nucleic acid cancer vaccine and other therapeutic agent may be administered simultaneously or sequentially. When the other therapeutic agents are administered simultaneously they can be administered in the same or separate formulations, but are administered at the same time. The other therapeutic agents are administered sequentially with one another and with the nucleic acid cancer vaccine, when the administration of the other therapeutic agents and the nucleic acid cancer vaccine is temporally separated. The separation in time between administrations of these compounds may be a matter of minutes or it may be longer, e.g., hours, days, weeks, months. Other therapeutic agents include but are not limited to anti-cancer therapeutics, adjuvants, cytokines, antibodies, antigens, etc.

At any point in the treatment the patient may be examined to determine whether the mutations in the vaccine are still appropriate. Based on that analysis the vaccine may be adjusted or reconfigured to include one or more different mutations or to remove one or more mutations.

In some embodiments, a cancer vaccine containing the nucleic acid (e.g., RNA polynucleotides) as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject) , and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide.

The vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) may be induced for translation of a polypeptide (e.g., antigen or immunogen) in a cell, tissue or organism. In exemplary embodiments, such translation occurs in vivo, although there can be envisioned embodiments where such translation occurs ex vivo, in culture or in vitro. In exemplary embodiments, the cell, tissue or organism is contacted with an effective amount of a composition containing a cancer vaccine that contains a polynucleotide that has at least one a translatable region encoding an antigenic polypeptide.

An “effective amount” of a cancer RNA vaccine may be provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the cancer vaccine, and other determinants. In general, an effective amount of the cancer vaccine composition provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the cancer vaccine) , increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide) , or altered antigen specific immune response of the host cell.

The vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in cancer or during active cancer after onset of symptoms. In some embodiments, the amount of RNA vaccines of the present disclosure provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

In some embodiments, the vaccine may be administered prophylactically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In some embodiments, the vaccine is administered every about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In some embodiments, vaccine is administered every about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. vaccine is administered every about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more months. In some embodiments, the subject has not been diagnosed of cancer or does not have detectable level (s) of cancer-related markers.

In some embodiments, the vaccine may be administered therapeutically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. In some embodiments, the vaccine is administered every about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In some embodiments, vaccine is administered every about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks. vaccine is administered every about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more months. In some embodiments, the subject has been diagnosed of cancer or has detectable level (s) of cancer-related markers.

In some embodiments, the vaccine is administered once to the subject. In some embodiments, the vaccine is administered three times to the subject.

The vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) may be administered with other prophylactic or therapeutic compounds in addition to checkpoint inhibitors. As a non-limiting example, a prophylactic or therapeutic compound may be an immune potentiator or a booster. As used herein, when referring to a composition, such as a vaccine, the term “booster” refers to an extra administration of the prophylactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years. In exemplary embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In some embodiments, the nucleic acid (e.g., mRNA) vaccine compositions may be administered at dosage levels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one or more times a day, per week, per month, etc. to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see e.g., the range of unit doses described in International Publication No. WO2013078199, herein incorporated by reference in its entirety) . In some embodiments, the nucleic acid (e.g., mRNA) vaccine is administered at a dosage level sufficient to deliver about 0.0100 mg, 0.025 mg, 0.040 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.130 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.390 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, 1.0 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg or more nucleic acid (e.g., mRNA) to the subject. In some embodiments, the nucleic acid (e.g., mRNA) vaccine is administered at a dosage level sufficient to deliver between 10 μg and 400 μg of the mRNA vaccine to the subject. In some embodiments, the nucleic acid (e.g., mRNA) vaccine is administered at a dosage level sufficient to deliver 10 mg nucleic acid (e.g., mRNA) to the subject.

The vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) may be utilized in various settings depending on the severity of the cancer or the degree or level of unmet medical need. As a non-limiting example, the cancer vaccines may be utilized to treat any stage of cancer.

In some embodiments, the vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) and/or checkpoint inhibitors may be used to treat PD-L1 positive tumors. In other embodiments the cancer vaccines and/or checkpoint inhibitors may be used to treat PD-L1 negative tumors. While emerging data support the use of PD-1 inhibitors such as pembrolizumab in tumors where PD-L1 expression can be demonstrated, the use of the combinations of the invention in treating PD-1 “negative” tumors is envisioned. Mechanistically, there is an adaptive component to PD-L1 expression by tumors, i.e., tumors may initially appear PD-L1 negative but upregulate PD-L1 expression in response to IFN-γ secretion by infiltrating tumor lymphocytes. This has translated clinically, such that the response rates of PD-L1 negative tumors to the combination of PD-1 and CTLA-4 blockade is higher than the response rate to single agent PD-1 inhibitors in both cutaneous melanoma and lung cancer. Aspects of the invention relate to the use of a personalized cancer vaccine to induce PD-L1 expression in PD-L1 low tumors, in combination with a PD-1 inhibitor.

In some embodiments that the vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) and/or checkpoint inhibitors may be used to treat tumors having a high tumor mutation burden. Thus in some embodiments a pool of subjects may be tested for TMB and the subjects having a TMB value over a threshold level may be treated with the combination therapy of the invention.

Provided herein are pharmaceutical compositions including cancer vaccines and RNA vaccine compositions and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients. The vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) may be formulated or administered alone or in conjunction with one or more other components as described herein.

In other embodiments, the vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) described herein may be combined with any other therapy useful for treating the patient. For instance a patient may be treated with the cancer vaccine and an anti-cancer agent. Thus, in one embodiment, the methods of the disclosure can be used in conjunction with one or more cancer therapeutics, for example, in conjunction with an anti-cancer agent, a traditional cancer vaccine, chemotherapy, radiotherapy, etc. (e.g., simultaneously, or as part of an overall treatment procedure) . Parameters of cancer treatment that may vary include, but are not limited to, dosages, timing of administration or duration or therapy; and the cancer treatment can vary in dosage, timing, or duration. Another treatment for cancer is surgery, which can be utilized either alone or in combination with any of the previous treatment methods. Any agent or therapy (e.g., traditional cancer vaccines, chemotherapies, radiation therapies, surgery, hormonal therapies, and/or biological therapies/immunotherapies) which is known to be useful, or which has been used or is currently being used for the prevention or treatment of cancer can be used in combination with a composition of the disclosure in accordance with the disclosure described herein. One of ordinary skill in the medical arts can determine an appropriate treatment for a subject.

Examples of such agents (i.e., anti-cancer agents) include, but are not limited to, DNA-interactive agents including, but not limited to, the alkylating agents (e.g., nitrogen mustards, e.g., Chlorambucil, Cyclophosphamide, Isofamide, Mechlorethamine, Melphalan, Uracil mustard; Aziridine such as Thiotepa; methanesulphonate esters such as Busulfan; nitroso ureas, such as Carmustine, Lomustine, Streptozocin; platinum complexes, such as Cisplatin, Carboplatin; bioreductive alkylator, such as Mitomycin, and Procarbazine, Dacarbazine and Altretamine) ; the DNA strand-breakage agents, e.g., Bleomycin; the intercalating topoisomerase II inhibitors, e.g., Intercalators, such as Amsacrine, Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin, Mitoxantrone, and nonintercalators, such as Etoposide and Teniposide; the nonintercalating topoisomerase II inhibitors, e.g., Etoposide and Teniposde; and the DNA minor groove binder, e.g., Plicamydin; the antimetabolites including, but not limited to, folate antagonists such as Methotrexate and trimetrexate; pyrimidine antagonists, such as Fluorouracil, Fluorodeoxyuridine, CB3717, Azacitidine and Floxuridine; purine antagonists such as Mercaptopurine, 6-Thioguanine, Pentostatin; sugar modified analogs such as Cytarabine and Fludarabine; and ribonucleotide reductase inhibitors such as hydroxyurea; tubulin Interactive agents including, but not limited to, colcbicine, Vincristine and Vinblastine, both alkaloids and Paclitaxel and cytoxan; hormonal agents including, but not limited to, estrogens, conjugated estrogens and Ethinyl Estradiol and Diethylstilbesterol, Chlortrianisen and Idenestrol; progestins such as Hydroxyprogesterone caproate, Medroxyprogesterone, and Megestrol; and androgens such as testosterone, testosterone propionate; fluoxymesterone, methyltestosterone; adrenal corticosteroid, e.g., Prednisone, Dexamethasone, Methylprednisolone, and Prednisolone; leutinizing hormone releasing hormone agents or gonadotropin-releasing hormone antagonists, e.g., leuprolide acetate and goserelin acetate; antihormonal antigens including, but not limited to, antiestrogenic agents such as Tamoxifen, antiandrogen agents such as Flutamide; and antiadrenal agents such as Mitotane and Aminoglutethimide; cytokines including, but not limited to, IL-1. alpha., IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-18, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-. γ, and Uteroglobins (U.S. Pat. No. 5,696,092) ; anti-angiogenics including, but not limited to, agents that inhibit VEGF (e.g., other neutralizing antibodies) , soluble receptor constructs, tyrosine kinase inhibitors, antisense strategies, RNA aptamers and ribozymes against VEGF or VEGF receptors, immunotoxins and coaguligands, tumor vaccines, and antibodies.

Specific examples of anti-cancer agents which can be used in accordance with the methods of the disclosure include, but not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interieukin II, or rIL2) , interferon alpha-2a; interferon alpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicin hydrochloride.

Other anti-cancer drugs which may be used with the instant compositions and methods include, but are not limited to: 20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; angiogenesis inhibitors; anti-dorsalizing morphogenetic protein-1; ara-CDP-DL-PTBA; BCR/ABL antagonists; CaRest M3; CARN 700; casein kinase inhibitors (ICOS) ; clotrimazole; collismycin A; collismycin B; combretastatin A4; crambescidin 816; cryptophycin 8; curacin A; dehydrodidemnin B; didemnin B; dihydro-5-azacytidine; dihydrotaxol, duocarmycin SA; kahalalide F; lamellarin-N triacetate; leuprolide+estrogen+progesterone; lissoclinamide 7; monophosphoryl lipid A+myobacterium cell wall sk; N-acetyldinaline; N-substituted benzamides; 06-benzylguanine; placetin A; placetin B; platinum complex; platinum compounds; platinum-triamine complex; rhenium Re 186 etidronate; RH retinamide; rubiginone B 1; SarCNU; sarcophytol A; sargramostim; senescence derived inhibitor 1; spicamycin D; tallimustine; 5-fluorouracil; thrombopoietin; thymotrinan; thyroid stimulating hormone; variolin B; thalidomide; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; zanoterone; zeniplatin; and zilascorb.

The disclosure also encompasses administration of a composition comprising a vaccine (e.g., nucleic acid cancer vaccine such as mRNA cancer vaccine) in combination with radiation therapy comprising the use of X-rays, gamma rays and other sources of radiation to destroy the cancer cells. In certain embodiments, the radiation treatment is administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. In other embodiments, the radiation treatment is administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass.

In some embodiments, an appropriate anti-cancer regimen is selected depending on the type of cancer (e.g., by a physician) . For instance, a patient with ovarian cancer may be administered a prophylactically or therapeutically effective amount of a composition comprising a cancer vaccine in combination with a prophylactically or therapeutically effective amount of one or more other agents useful for ovarian cancer therapy, including but not limited to, intraperitoneal radiation therapy, such as P32 therapy, total abdominal and pelvic radiation therapy, cisplatin, the combination of paclitaxel (Taxol) or docetaxel (Taxotere) and cisplatin or carboplatin, the combination of cyclophosphamide and cisplatin, the combination of cyclophosphamide and carboplatin, the combination of 5-FU and leucovorin, etoposide, liposomal doxorubicin, gemcitabine or topotecan. Cancer therapies and their dosages, routes of administration and recommended usage are known in the art and have been described in such literature as the Physician's Desk Reference (56th ed., 2002) .

In some embodiments, the cancer therapeutic agent is a cytokine. In yet other embodiments the cancer therapeutic agent is a vaccine comprising a population based tumor specific antigen.

In some embodiments, the vaccine described herein elicits antigen-specific immune response (s) . In some embodiments, the immune response is a T cell-mediated immune response. In some embodiments, the T cells can be isolated or detected from spleen, tumors, lymph nodes, thymus, peripheral blood, bone marrow, liver, lung, and/or lymphoid tissues. In some embodiments, the immune responses can be mediated by cytotoxic CD8⁺ T cells, CD4⁺ helper T cells, effector T cells, memory T cells, and/or tumor infiltrating lymphocytes (TILs) . In some embodiments, the immune response can be mediated by cytotoxic CD8⁺ T cells, e.g., central memory CD8⁺ T cells (T_CM) , effector memory CD8⁺ T cells (T_EM) , CD8⁺ GranzymeB⁺ T cells, and/or CD8⁺ IFN-γ⁺ T cells. In some embodiments, the immune response is featured with increased absolute number/percentage of CD8⁺ T_CM cells, CD8⁺ T_EM cells, CD8⁺ GranzymeB⁺ T cells, and/or CD8⁺ GranzymeB⁺ T cells. In some embodiments, the absolute number/percentage of CD8⁺ T_CM cells and CD8⁺ T_EM cells can be detected from spleen or splenocytes. In some embodiments, the absolute number/percentage of CD8⁺ GranzymeB⁺ T cells and CD8⁺ IFN-γ ⁺ T cells can be detected from tumors or TILs.

In some embodiments, the antigen-specific immune response is measured by the level of IFN-γ in the subject (e.g., in the splenocytes or in the tumor tissue) . In some embodiments, the level of IFN-γ is increased by about 2, 3, 4, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more folds. In some embodiments, the increased level of IFN-γ is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21 days or more. In some embodiments, the increased level of IFN-γ is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more.

In some embodiments, the antigen-specific immune response is measured by the percentage of CD8⁺ T_CM cells in the subject (e.g., in the splenocytes or in the tumor tissue) . In some embodiments, the percentage of CD8⁺ T_CM cells is increased by at least 1.2 folds (e.g., about 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, 5.5, 6, or more folds) . In some embodiments, the percentage of CD8⁺ T_CM cells is increased by up to 2, 3, 4, 5, or 6 folds. In some embodiments, the percentage of CD8⁺ T_CM cells in the tissue (e.g., in the splenocytes or in the tumor tissue) is at least 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. In some embodiments, the percentage of CD8⁺T_CM cells in the tissue (e.g., in the splenocytes or in the tumor tissue) is no more than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. In some embodiments, the antigen-specific immune response is measured by the percentage of CD8⁺ T_EM cells in the subject (e.g., in the splenocytes or in the tumor tissue) . In some embodiments, the percentage of CD8⁺ T_EM cells is increased by at least 1.2 folds (e.g., about 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, 5.5, 6, or more folds) . In some embodiments, the percentage of CD8⁺ T_EM cells is increased by up to 2, 3, 4, 5, or 6 folds. In some embodiments, the percentage of CD8⁺ T_EM cells in the tissue (e.g., in the splenocytes or in the tumor tissue) is at least 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. In some embodiments, the percentage of CD8⁺ T_EM cells in the tissue (e.g., in the splenocytes or in the tumor tissue) is no more than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

In some embodiments, the antigen-specific immune response is measured by the cell count/gram tissue of CD3⁺ T cells in the subject (e.g., in the splenocytes or in the tumor tissue) . In some embodiments, the cell count/gram tissue of CD3⁺ T cells is increased by at least 1.2 folds (e.g., about 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, or more folds) . In some embodiments, the increased cell count/gram tissue of CD3⁺ T cells is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21 days or more. In some embodiments, the increased cell count/gram tissue of CD3⁺ T cells is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more.

In some embodiments, the antigen-specific immune response is measured by the cell count/gram tissue of CD4⁺ helper T cells in the subject (e.g., in the splenocytes or in the tumor tissue) . In some embodiments, the cell count/gram tissue of CD4⁺ helper T cells is increased by at least 1.2 folds (e.g., about 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, or more folds) . In some embodiments, the increased cell count/gram tissue of CD4⁺ helper T cells is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21 days or more. In some embodiments, the increased cell count/gram tissue of CD4⁺helper T cells is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more.

In some embodiments, the antigen-specific immune response is measured by the cell count/gram tissue of CD8⁺ cytotoxic T cells (CTLs) in the subject (e.g., in the splenocytes or in the tumor tissue) . In some embodiments, the cell count/gram tissue of CD8⁺ CTLs is increased by at least 1.2 folds (e.g., about 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, or more folds) . In some embodiments, the increased cell count/gram tissue of CD8⁺ CTLs is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21 days or more. In some embodiments, the increased cell count/gram tissue of CD8⁺CTLs is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more.

In some embodiments, the antigen-specific immune response is measured by the cell count/gram tissue of CD8⁺ GranzymeB⁺ T cells in the subject (e.g., in the splenocytes or in the tumor tissue) . In some embodiments, the cell count/gram tissue of CD8⁺ GranzymeB⁺ T cells is increased by at least 1.2 folds (e.g., about 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, or more folds) . In some embodiments, the increased cell count/gram tissue of CD8⁺ GranzymeB⁺ T cells is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21 days or more. In some embodiments, the increased cell count/gram tissue of CD8⁺ GranzymeB⁺ T cells is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more.

In some embodiments, the antigen-specific immune response is measured by the cell count/gram tissue of CD8⁺ IFN-γ ⁺ T cells in the subject (e.g., in the splenocytes or in the tumor tissue) . In some embodiments, the cell count/gram tissue of CD8⁺ IFN-γ ⁺ T cells is increased by at least 1.2 folds (e.g., about 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, or more folds) . In some embodiments, the increased cell count/gram tissue of CD8⁺ IFN-γ ⁺ T cells is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21 days or more. In some embodiments, the increased cell count/gram tissue of CD8⁺IFN-γ ⁺ T cells is maintained for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more. In some embodiments, the administration of the vaccine described herein does not cause significant body weight loss of the subject.

In some embodiments, the administration of the vaccine described herein inhibits tumor growth in the subject. In some embodiments, the administration of the vaccine described herein reduces tumor volume in the subject. In some embodiments, the tumor volume is reduced by about 2, 3, 4, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more folds.

In some embodiments, the antibody has a tumor growth inhibition rate (TGI%) that is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibody has a tumor growth inhibition percentage that is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI%can be determined, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the treatment starts, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the treatment starts. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:
TGI (%) = [1- (Ti-T0) / (Vi-V0) ] ×100

Ti is the average tumor volume in the treatment group on day i. T0 is the average tumor volume in the treatment group on day zero. Vi is the average tumor volume in the control group on day i. V0 is the average tumor volume in the control group on day zero.

In some embodiments, the administration of the vaccine described herein can prevent tumor development in the subject. For example, the vaccine may stimulate an immune response that targets cancer cells or precancerous cells, thereby inhibiting the formation of new tumors.

In some embodiments, the subject can remain tumor free for a period of time, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more weeks following the administration of the vaccine described herein. This prolonged tumor-free state may be indicative of the vaccine's effectiveness in maintaining an anti-tumor immune response.

In some embodiments, the vaccine may also prevent or mitigate metastasis, reducing the likelihood of cancer spreading to secondary sites such as the lymph nodes, liver, lungs, or bones. This could result in improved survival outcomes and better quality of life for the subject.

In some embodiments, the administration of the vaccine described herein can slow or inhibit tumor growth in the subject with recurrent or metastatic tumors. Specifically, the vaccine may enhance the activation of cytotoxic T cells, natural killer cells, or other components of the immune system that mediate the suppression of tumor proliferation.

In some embodiments, the administration of the vaccine described herein reduces tumor volume in the subject with recurrent or metastatic tumors. For example, reductions in tumor size may be observed in imaging studies, such as MRI or CT scans, or in physical measurements of accessible tumors. Such reductions may occur within weeks or months after initiating vaccine treatment and could reflect both direct cytotoxic effects and enhanced anti-tumor immunity.

Methods of Producing Vaccines

Vaccines of the present disclosure may comprise at least one nucleic acid (e.g., an RNA polynucleotide, such as an mRNA (message RNA) or an mmRNA (modified mRNA) ) . mRNA, for example, is transcribed in vitro from template DNA, referred to as an “in vitro transcription template. ” In some embodiments, an in vitro transcription template encodes a 5′untranslated (UTR) region, contains an open reading frame, and encodes a 3′UTR and a polyA tail. In some embodiments, an in vitro transcription template encodes a capping enhancing sequence. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

In some embodiments, a nucleic acid includes about 15 to about 3,000 nucleotides. For example, a polynucleotide may include about 15 to 50, about 15 to 100, about 15 to 200, about 15 to 300, about 15 to 400, about 15 to 500, about 15 to 600, about 15 to 700, about 15 to 800, about 15 to 900, about 15 to 1000, about 15 to 1200, about 15 to 1400, about 15 to 1500, about 15 to 1800, about 15 to 2000, about 15 to 2500, about 15 to 3000, about 50 to 100, about 50 to 200, about 50 to 300, about 50 to 400, about 50 to 500, about 50 to 600, about 50 to 700, about 50 to 800, about 50 to 900, about 50 to 1000, about 50 to 1200, about 50 to 1400, about 50 to 1500, about 50 to 1800, about 50 to 2000, about 50 to 2500, about 50 to 3000, about 100 to 200, about 100 to 300, about 100 to 400, about 100 to 500, about 100 to 600, about 100 to 700, about 100 to 800, about 100 to 900, about 100 to 1000, about 100 to 1200, about 100 to 1400, about 100 to 1500, about 100 to 1800, about 100 to 2000, about 100 to 2500, about 100 to 3000, about 200 to 300, about 200 to 400, about 200 to 500, about 200 to 600, about 200 to 700, about 200 to 800, about 200 to 900, about 200 to 1000, about 200 to 1500, about 200 to 3000, about 500 to 1000, about 500 to 1500, about 500 to 2000, about 500 to 2500, about 500 to 3000, about 1000 to 1500, about 1000 to 2000, about 1000 to 2500, about 1000 to 3000, about 1500 to 3000, about 2500 to 3000, or about 2000 to 3000 nucleotides) .

In other aspects, the disclosure relates to a method of making or producing a nucleic acid vaccine (e.g., an mRNA cancer vaccine) by in vitro transcription (IVT) methods.

In one aspect, the disclosure relates to a method of making a vaccine comprising mixing a nucleic acid described herein with a lipid nanoparticle formulation, thereby producing a vaccine.

In vitro transcription (IVT) methods permit template-directed synthesis of RNA molecules of almost any sequence. In some embodiments, the RNA (e.g., mRNA) molecule described herein can be transcribed from a corresponding DNA molecule (e.g., a DNA molecule encoding the same amino acid sequence) described herein. In some embodiments, the DNA molecule described herein is in a vector. In some embodiments, the vector is a plasmid. The size of the RNA molecules that can be synthesized using IVT methods range from short oligonucleotides to long nucleic acid polymers of several thousand bases. IVT methods permit synthesis of large quantities of RNA transcript (e.g., from microgram to milligram quantities) . See Beckert et al., Synthesis of RNA by in vitro transcription, Methods Mol Biol. 703: 29-41 (2011) ; Rio et al. RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220; Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington D. C.: ASM Press, 2007.262-299, each of which is herein incorporated by reference for this purpose. Generally, IVT utilizes a DNA template featuring a promoter sequence upstream of a sequence of interest. The promoter sequence is most commonly of bacteriophage origin (e.g., the T7, T3 or SP6 promoter sequence) but many other promotor sequences can be tolerated including those designed de novo. Transcription of the DNA template is typically best achieved by using the RNA polymerase corresponding to the specific bacteriophage promoter sequence. Exemplary RNA polymerases include, but are not limited to T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase, among others. IVT is generally initiated at a dsDNA but can proceed on a single strand.

It will be appreciated that nucleic acid vaccines (e.g., mRNA cancer vaccines) of the present disclosure, e.g., mRNAs encoding the cancer antigen, may be made using any appropriate synthesis method. For example, in some embodiments, mRNA vaccines of the present disclosure are made using IVT from a single bottom strand DNA as a template and complementary oligonucleotide that serves as promotor. The single bottom strand DNA may act as a DNA template for in vitro transcription of RNA, and may be obtained from, for example, a plasmid, a PCR product, or chemical synthesis. In some embodiments, the single bottom strand DNA is linearized from a circular template. The single bottom strand DNA template generally includes a promoter sequence, e.g., a bacteriophage promoter sequence, to facilitate IVT. Methods of making RNA using a single bottom strand DNA and a top strand promoter complementary oligonucleotide are known in the art. An exemplary method includes, but is not limited to, annealing the DNA bottom strand template with the top strand promoter complementary oligonucleotide (e.g., T7 promoter complementary oligonucleotide, T3 promoter complementary oligonucleotide, or SP6 promoter complementary oligonucleotide) , followed by IVT using an RNA polymerase corresponding to the promoter sequence, e.g., aT7 RNA polymerase, a T3 RNA polymerase, or an SP6 RNA polymerase.

IVT methods can also be performed using a double-stranded DNA template. For example, in some embodiments, the double-stranded DNA template is made by extending a complementary oligonucleotide to generate a complementary DNA strand using strand extension techniques available in the art. In some embodiments, a single bottom strand DNA template containing a promoter sequence and sequence encoding one or more peptide epitopes of interest is annealed to a top strand promoter complementary oligonucleotide and subjected to a PCR-like process to extend the top strand to generate a double-stranded DNA template. Alternatively or additionally, a top strand DNA containing a sequence complementary to the bottom strand promoter sequence and complementary to the sequence encoding one or more peptide epitopes of interest is annealed to a bottom strand promoter oligonucleotide and subjected to a PCR-like process to extend the bottom strand to generate a double-stranded DNA template. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. In some embodiments, a double-stranded DNA template is synthesized wholly or in part by chemical synthesis methods. The double-stranded DNA template can be subjected to in vitro transcription as described herein.

In another aspect, nucleic acid cancer vaccines of the present disclosure comprising, e.g., mRNAs encoding the cancer antigen (e.g., PRAME) , may be made using two DNA strands that are complementary across an overlapping portion of their sequence, leaving single-stranded overhangs (i.e., sticky ends) when the complementary portions are annealed. These single-stranded overhangs can be made double-stranded by extending using the other strand as a template, thereby generating double-stranded DNA. In some cases, this primer extension method can permit larger ORFs to be incorporated into the template DNA sequence, e.g., as compared to sizes incorporated into the template DNA sequences obtained by top strand DNA synthesis methods. In the primer extension method, a portion of the 3F-end of a first strand (in the 5′-3′direction) is complementary to a portion the 3′-end of a second strand (in the 3′-5′direction) . In some such embodiments, the single first strand DNA may include a sequence of a promoter (e.g., T7, T3, or SP6) , optionally a 5′-UTR, and some or all of an ORF (e.g., a portion of the 5′-end of the ORF) . In some embodiments, the single second strand DNA may include complementary sequences for some or all of an ORF (e.g., a portion complementary to the 3′-end of the ORF) , and optionally a 3′-UTR, a stop sequence, and/or a poly (A) tail. Methods of making RNA using two synthetic DNA strands may include annealing the two strands with overlapping complementary portions, followed by primer extension using one or more PCR-like cycles to extend the strands to generate a double-stranded DNA template. In some embodiments, the number of PCR-like cycles ranges from 1 to 20 cycles, e.g., 3 to 10 cycles. Such double-stranded DNA can be subjected to in vitro transcription as described herein.

In another aspect, nucleic acid vaccines of the present disclosure comprising, e.g., mRNAs encoding the cancer antigen (e.g., PRAME) , may be made using synthetic double-stranded linear DNA molecules, such as (Integrated DNA Technologies, Coralville, Iowa) , as the double-stranded DNA template. An advantage to such synthetic double-stranded linear DNA molecules is that they provide a longer template from which to generate mRNAs. For example, can range in size from 45-1000 (e.g., 125-750 nucleotides) . In some embodiments, a synthetic double-stranded linear DNA template includes a full length 5′-UTR, a full length 3′-UTR, or both. A full length 5′-UTR may be up to 100 nucleotides in length, e.g., about 40-60 nucleotides. A full length 3′-UTR may be up to 300 nucleotides in length, e.g., about 100-150 nucleotides.

To facilitate generation of longer constructs, two or more double-stranded linear DNA molecules and/or gene fragments that are designed with overlapping sequences on the 3′strands may be assembled together using methods known in art. For example, the Gibson Assembly^TM Method (Synthetic Genomics, Inc., La Jolla, Calif. ) may be performed with the use of a mesophilic exonuclease that cleaves bases from the 5′-end of the double-stranded DNA fragments, followed by annealing of the newly formed complementary single-stranded 3′-ends, polymerase-dependent extension to fill in any single-stranded gaps, and finally, covalent joining of the DNA segments by a DNA ligase.

In another aspect, nucleic acid cancer vaccines of the present disclosure comprising, e.g., mRNAs encoding the cancer antigen (e.g., PRAME) , may be made using chemical synthesis of the RNA. Methods, for instance, involve annealing a first polynucleotide comprising an open reading frame encoding the polypeptide and a second polynucleotide comprising a 5′-UTR to a complementary polynucleotide conjugated to a solid support. The 3′-terminus of the second polynucleotide is then ligated to the 5′-terminus of the first polynucleotide under suitable conditions. Suitable conditions include the use of a DNA Ligase. The ligation reaction produces a first ligation product. The 5′terminus of a third polynucleotide comprising a 3′-UTR is then ligated to the 3′-terminus of the first ligation product under suitable conditions. Suitable conditions for the second ligation reaction include an RNA Ligase. A second ligation product is produced in the second ligation reaction. The second ligation product is released from the solid support to produce an mRNA encoding a polypeptide of interest. In some embodiments the mRNA is between 30 and 1000 nucleotides.

In some embodiments, template DNA encoding the nucleic acid (e.g., mRNA) cancer vaccines of the present disclosure includes an open reading frame (ORF) encoding one or more peptide epitopes. In some embodiments, the template DNA includes an ORF of up to 1000 nucleotides, e.g., about 10-350, 30-300 nucleotides or about 50-250 nucleotides. In some embodiments, the template DNA includes an ORF of about 150 nucleotides. In some embodiments, the template DNA includes an ORF of about 200 nucleotides.

In some embodiments, IVT transcripts are purified from the components of the IVT reaction mixture after the reaction takes place. For example, the crude IVT mix may be treated with RNase-free DNase to digest the original template. The nucleic acid (e.g., mRNA) can be purified using methods known in the art, including but not limited to, precipitation using an organic solvent or column based purification method. Commercial kits are available to purify RNA, e.g., MEGACLEAR^TM Kit (Ambion, Austin, Tex. ) . The nucleic acid (e.g., mRNA) can be quantified using methods known in the art, including but not limited to, commercially available instruments, e.g., NanoDrop. Purified nucleic acids (e.g., mRNAs) can be analyzed, for example, by agarose gel electrophoresis to confirm the nucleic acid is the proper size and/or to confirm that no degradation of the nucleic acid has occurred.

Methods of in vitro transcription of nucleic acid to make vaccines (e.g., mRNA vaccines) are described in, for example, US Patent Publication Nos. US20220125899A1, US20190351040A1 and US20180318409A1, the entire contents are hereby incorporated by reference.

In some embodiments, the in vitro transcribed nucleic acid (e.g., mRNA) is mixed with a mixture of lipids to produce the vaccine described herein. In some embodiments, the mixture of lipids comprises ionizable cationic lipid, phospholipid, cholesterol and PEG-conjugated lipid. In some embodiments, the lipid mixture is dissolved in ethanol. In some embodiments, the nucleic acid (e.g., mRNA) is dissolved in a sterile and RNase-free buffer (e.g., sodium acetate buffer) prior to preparation.

Lipid nanoparticles can be produced using any of the methods known in the art and described herein. In some embodiments, LNPs are prepared by a rapid mixing of lipids mixture containing ethanol phase with an aqueous phase containing the RNA using a microfluidic mixing device in a ratio of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6 1: 7, 1: 8, 1: 9, or 1: 10 (v/v) . In some embodiments, LNPs are prepared by a rapid mixing of lipids mixture containing ethanol phase with an aqueous phase containing the RNA using a microfluidic mixing device in a ratio of one volume of lipid mixture in ethanol and three volumes of RNA. In some embodiments, the lipid nanoparticles containing RNA are subjected to diafiltration and/or ultrafiltration. Any suitable methods of preparing LNPs known in the art can be used in the methods described herein.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLE 1: PRAME mRNA-LNP Vaccine Design and Anti-tumor Efficacy

Methods

Antigen design:

The Igκ light chain signal peptide (SP) sequence was fused at the N-terminal of full-length PRAME antigens, and the human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) was fused to the C-terminal of the target antigens.

Different nucleotide sequences which encoding the target antigen (with SP and MITD) were designed with proprietary codon optimization algorithms, which can improve mRNA stability and translational efficiency, and further protein expression.

Plasmid design

The nucleotide sequences of full-length antigen were cloned into the pUC57-kan plasmid backbone (Genscript) , in which contains the optimized 5’-Untranslated regions (UTR) , 3-UTR and polyA tail. The plasmid was extracted after amplification in E. coli.

mRNA preparation

The plasmid was linearized with the BspQI restriction enzyme (New England Biolabs, R0712L) . After being purified using QIAquick Gel Extraction Kit (QIAGEN, Cat.: 278704) , the linearized plasmid DNA was in vitro transcribed into mRNA by High yield T7 IVT kit (Hongene, Cat.: ON-040) following the manufacturer’s instructions, which containing the modified mRNA molecules (N1-Methyl-pseudouridine, Hongene, Cat.: R5-064) and m7G (5') ppp (5') (2'-OMeA) pG (Hongene, Cat.: ON-134) . Then the transcribed mRNAs were purified using RNA Cleanup Kit (NEW ENGLAND BioLabs, Cat.: T2050L) following the manufacturer’s instructions. The purified mRNA then was stored at -80 ℃.

mRNA-LNP preparation

A mix of lipids containing the ionizable cationic lipid, phospholipid, cholesterol and PEG-conjugated lipid was dissolved in ethanol. And mRNA was dissolved in a sterile and RNase-free 10 mM sodium acetate buffer (pH 4.0) prior to preparation. Lipid nanoparticles were prepared by a rapid mixing of lipids mixture containing ethanol phase with an aqueous phase containing the RNA using a microfluidic mixing device (one volume of lipid mixture in ethanol and three volumes of RNA in 10 mM sodium acetate buffer) at a total flow rate of 20 mL/min, and directly mixed and diluted with 10 volumes of 1x phosphate buffered saline (PBS) . The lipid nanoparticles containing RNA were subjected to diafiltration and ultrafiltration using Amicon Ultra-15 Centrifugal filters (Millipore) or Tangential Flow Filtration. The final lipid nanoparticle products were stored in 10%sucrose (w/v) , 5 mM Tris buffer (pH 8.0) and filtered through 0.22 μm filter in sterile condition. The mRNA-LNP was stored at -80℃ until use.

In vitro protein expression

The target mRNA was mixed with lipofectamine to transfect HEK293T cell lines. The cell lysate was collected and the target antigen expression was measured with Western Blot.

For in vitro expression assay, HEK293T cells were harvested by Trypsin-EDTA digestion and inoculated into 24-well plate (300000 cells/well) . 1 μg mRNA was mixed with 1 μL of lipofectamine (Invitrogen, LMRNA015) and incubated for 15 minutes at room temperature. Then the mixture was transfected to HEK293T cells. After 24 hours’ incubation, the cell was washed and cell lysate was prepared using RIPA (Pierce, Cat. # 89901) with protease and phosphatase Inhibitor Cocktail (Thermo Scientific, Cat. # 78441) . After incubation on ice, the tubes were spun at a speed > 12000 rpm and the supernatant was collected. The total protein concentration was quantitated with BCA protein assay kit (Pierce, Cat. # 23225) . 10 μg protein was loaded on SDS-PAGE and the PRAME protein expression level was measured by Western Blot.

In vivo immunogenicity in mice

The target encapsulated mRNA-LNP was injected into female C57BL/6 mice (Shanghai BK) via intramuscular (IM) injection at both sites of gastrocnemius muscle (0.1 mL/mouse) . The dose of mRNA is 10 μg/mouse. The dosing schedule was indicated in each experiment.

At different time points after the immunization, mice were euthanized, and the spleen and inguinal and popliteal lymph nodes were collected for the IFN-γ Enzyme-linked immunospot (ELISPOT) assay.

ELISPOT assay

The antigen-specific immunogenicity was measured by ELISPOT assay with the ex vivo stimulation of target peptide pools. Briefly, the mouse spleen or lymph nodes were grinded with a sterile syringe pushrod and filtered with a 70 μm cell-strainer to isolate single cell suspension. The single splenocytes or lymphocytes were inoculated into IFN-γ ELISPOT plates (Mabtech, Cat. # 3321-4AST-10) and incubated with the target peptide pools for 40-44 hours, then the IFN-γ spots were determined following the manufacture’s instructions.

Transgenic cell line generation and anti-tumor efficacy study

The full-length wild-type antigen was cloned into the genomic DNA of MC38, a murine colorectal cancer cell line, through CRISPR-Cas9 methods. In brief, the full-length wild-type human PRAME gene was inserted into the cloning vectors, which contained puromycin resistance gene. Then the ROSA26-sgRNA plasmid which expressed ROSA26 genome site targeting sgRNA and Cas9 protein, were co-electroporated with the PRAME cloning vector into MC38 cells. The transgenic PRAME-MC38 cells were enriched with puromycin treatment and then single cell clones were selected with limited dilution method. The transgenic PRAME-MC38 single cell clones were confirmed through q-PCR sequencing and protein expression and passed the mycoplasma test before used for in vivo study.

For in vivo efficacy study, 5x10⁵ transgenic PRAME-MC38 cancer cells were inoculated into the right flank of female C57BL/6 mice (Beijing VitalRiver) on Day 0. The dose level of PRAME mRNA-LNP was 10 μg/mouse via intramuscular injection and the dosing volume was 0.1 mL/mouse. Two dosing schedules were used for anti-tumor efficacy evaluation. The prophylactic dosing schedule was started before cancer cell inoculation and were dosed on Day -10, Day -7, Day -3, Day 4, Day 11, Day 18, and Day 25. The therapeutic dosing schedule was started after tumor cell inoculation and were dosed on Day 1, Day 4, Day 7, Day 11, Day 18, and Day 25. The mouse body weight and tumor volume were measure three times per week.

Memory T cell phenotype analysis

At the end of the efficacy study, mouse spleens were collected from the PBS control group and the PRAME mRNA-LNP treatment groups with different dosing schedules. The isolated splenocytes were stained with fluorescence-labeled antibodies (anti-mCD45, anti-mCD3, anti-mCD4, anti-CD8, anti-mCD44 and anti-mCD62L) . The cell suspension was loaded and analyzed with a flow cytometry. Central memory T cell (T_CM) was defined as mCD44^highmCD62L^high, and effector memory T cell (T_EM) was defined as mCD44^highmCD62L^- ^/low.

In vivo rechallenge study

The tumor-free mice, which achieved complete tumor regression in the prophylactic vaccination group after multiple PRAME mRNA-LNP doses, were rechallenged with 5×10⁵ PRAME-MC38 cancer cells on Day 46 (21 days post last PRAME mRNA-LNP vaccination) . female C57BL/6 mice (Beijing VitalRiver) were inoculated with PRAME-MC38 cancer cells at same time as control. On Day 90, 5×10⁵ wildtype MC38 cancer cells were inoculated into these tumor-free mice again. female C57BL/6 mice (Beijing VitalRiver) inoculated with 5×10⁵ wildtype MC38 cancer cells were used as a control. There was no PRAME mRNA-LNP treatment for mice during the rechallenge study. The mouse body weight and tumor volume were measured three times per week.

Tumor infiltrating lymphocyte (TIL) analysis

For in vivo pharmacodynamics analysis of PRAME mRNA-LNP treatment, 5×10⁵ transgenic PRAME-MC38 cancer cells were inoculated into the right flank of female C57BL/6 mice (Beijing VitalRiver) on Day 0. The mice were randomized into two groups on Day 11 when the tumor volume reached ～200 mm³. Mice in Group 1 were treated with PBS as controls, and mice in Group 2 were treated with PRAME mRNA-LNP (10 μg/mouse) via intramuscular route on Day 11, Day 14, and Day 18. The tumor tissue from each mouse was collected for TIL analysis on Day 27. Single cells were isolated from the tumor tissue of individual mice through enzyme digestion. The cell suspension was counted and stained with fluorescence-labeled antibody (anti-mCD45, anti-mCD3, anti-mCD4, anti-CD8, anti-mGranzymeB, and anti-IFN-γ) and analyzed with a flow cytometer.

Results

Two mRNA of different nucleotide sequences (SEQ ID NOs: 1 and 2) which encoding the full-length PRAME antigen, can translate into the protein product with expected molecular weight. The nucleotide sequence of SEQ ID NOs: 1 and 2 are shown below:

>PRAME-1

>PRAME-2

Both mRNAs showed similar protein expression level (FIG. 1) .

In a single dose kinetics assay, PRAME mRNA-LNP single dose via IM injection in C57BL/6 mice induced antigen-specific immune response in mouse spleens. After single dose on D0, The PRAME antigen-specific immune response reached to peak on D7 and maintained the plateau to D11 and decreased on D21 (FIG. 2) .

In an intensive dosing assay, PRAME mRNA-LNP were injected into C57BL/6 mice via IM on D0, D3 and D7, and the T cell response in spleen were measured on D7 and D14. The intensive dosing schedule can generate prolonged PRAME antigen-specific immune response (FIG. 3) .

In an anti-tumor efficacy study, the PRAME-MC38 transgenic tumor cells were inoculated into C57BL/6 mice (D0) , and 10 μg/mouse PRAME mRNA-LNP were vaccinated with two different dosing schedules (prophylactic: Day -10, Day -7, Day -3, Day 4, Day 11, Day 18, and Day 25; therapeutic: Day 1, Day 4, Day 7, Day 11, Day 18, and Day 25) . Each dosing schedule induced transient but recoverable body weight loss. The mice can tolerate the mRNA-LNP immunization (FIG. 4) . All mice developed tumors in PBS treated mice. In prophylactic schedule, PRAME mRNA-LNP completely inhibited the tumor growth, and 13/15 mice were tumor-free. In therapeutic schedule, PRAME mRNA-LNP effectively inhibited the tumor growth, and the tumor growth inhibition rate (TGI%) was 76.3% (FIG. 5) .

At the end of the efficacy study, mouse spleens were collected and splenocytes were isolated for memory T cell phenotype analysis. The central memory CD8⁺ T cells (T_CM) and effector memory CD8⁺ T cells (T_EM) percentage in splenocytes were significantly increased in both PRAME mRNA-LNP treatment groups with different schedules compared to the PBS treatment group (FIG. 6 and FIG. 7) .

For the tumor-rechallenge study, 5×10⁵ transgenic PRAME-MC38 cancer cells were inoculated into the tumor-free mice from the PRAME mRNA-LNP treatment group with prophylactic dosing schedule on Day 46 (21 days post the last PRAME mRNA-LNP vaccination) , and additionalC57BL/6 mice were inoculated with PRAME-MC38 cells as controls. No PRAME mRNA-LNP treatment was given to mice during rechallenge study. PRAME-MC38 cancer cells developed tumors inC57BL/6 mice as expected, while no tumor grew up in the tumor-free mice pre-vaccinated with PRAME mRNA-LNP for 45 days (FIG. 8) , demonstrating that the sustained immunogenicity induced by PRAME mRNA-LNP vaccination prevented tumor growth. Furthermore, the tumor-free mice were inoculated with wildtype MC38 cancer cells without human PRAME expression on Day 90, and there was still no tumor growth in one month after cell inoculation in these mice, while themice inoculated with wildtype MC38 cells developed the tumor rapidly (FIG. 8) , implying a memory immune response against the wildtype MC38 cancer cells.

In the in vivo pharmacodynamics study, C57BL/6 mice were inoculated with PRAME-MC38 cancer cells on Day 0, and treated with 10 μg/mouse PRAME mRNA-LNP on Day 11, Day 14, and Day 18. PRAME mRNA-LNP vaccination showed significant efficacy in tumor growth inhibition (FIG. 9) . Tumor tissue was collected for TIL analysis on Day 27. Significantly enhanced T cell infiltration into the PRAME-MC38 tumors were observed in mice post PRAME mRNA-LNP vaccination, including total T cells (mCD45⁺mCD3⁺, FIG. 10) , helper T cells (mCD45⁺mCD3⁺mCD4⁺, FIG. 11) , and cytotoxic T cells (CTLs, mCD45⁺mCD3⁺mCD8⁺, FIG. 12) compared to the PBS control group. In addition, the activated CD8⁺ CTLs (mCD8⁺mGranzymeB⁺ T cells shown in FIG. 13 and mCD8⁺mIFN-γ⁺ T cells shown in FIG. 14) were significantly increased in the tumors of PRAME mRNA-LNP treated group. These data indicated that PRAME mRNA-LNP vaccination can enhance T cell expansion, activation, and infiltration into tumor tissue, which correlates with its efficacy in tumor growth inhibition.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A nucleic acid encoding a polypeptide comprising:

(a) an Igκ light chain signal peptide (SP) sequence;

(b) an antigen sequence; and

(c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence.
The nucleic acid of claim 1, wherein the antigen is a cancer antigen.
The nucleic acid of claim 1 or 2, wherein the cancer antigen is preferentially expressed antigen in melanoma (PRAME) .
A nucleic acid encoding a polypeptide comprising:

(a) an Igκ light chain signal peptide (SP) sequence;

(b) a preferentially expressed antigen in melanoma (PRAME) antigen sequence; and

(c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence.
The nucleic acid of any one of claims 1-4, wherein the Igκ light chain signal peptide (SP) sequence is at least 80%identical to the amino acid sequence set forth in SEQ ID NO: 3.
The nucleic acid of any one of claims 1-5, comprising a nucleic acid encoding the Igκlight chain signal peptide (SP) sequence, wherein the nucleic acid encoding the Igκ light chain signal peptide (SP) sequence is at least 80%identical to the sequence set forth in SEQ ID NO: 8, 9, 18 or 19.
The nucleic acid sequence of any one of claims 1-6, wherein the MITD sequence comprises a sequence that is at least 80%identical to the amino acid sequence set forth in SEQ ID NO: 4.
The nucleic acid of any one of claims 1-7, comprising a nucleic acid encoding the MITD sequence, wherein the nucleic acid encoding the MITD sequence is at least 80%identical to the sequence set forth in SEQ ID NO: 14, 15, 24 or 25.
The nucleic acid of any one of claims 3-8, wherein the PRAME antigen sequence comprises a sequence that is at least 80%identical to the amino acid sequence set forth in SEQ ID NO: 5.
The nucleic acid of any one of claims 3-9, comprising a nucleic acid encoding the PRAME antigen sequence, wherein the nucleic acid encoding the PRAME antigen sequence is at least 80%identical to the sequence set forth in SEQ ID NO: 10, 11, 20 or 21.
The nucleic acid of any one of claims 1-10, wherein the Igκ light chain signal peptide (SP) sequence, the antigen sequence and/or the MITD sequence are linked via a linker.
The nucleic acid of claim 11, wherein the linker comprises a sequence set forth in SEQ ID NO: 6.
The nucleic acid of claim 11 or 12, comprising a nucleic acid sequence encoding the linker, wherein the nucleic acid sequence encoding the linker is at least 80%identical to the sequence set forth in SEQ ID NO: 12, 13, 22 or 23.
The nucleic acid of any one of claims 1-13, comprising a stop codon.
The nucleic acid of any one of claims 1-14, comprising a nucleotide sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 1, 2, 16 or 17.
The nucleic acid of any one of claims 1-15, wherein the nucleic acid is an mRNA.
The nucleic acid of claim 16, wherein the mRNA comprises at least one chemical modification.
The nucleic acid of claim 16 or 17, wherein the mRNA comprises a 5′UTR and/or a 3′ UTR.
The nucleic acid of claim 17 or 18, wherein the chemical modification is selected from pseudouridine, N1-Methyl-pseudouridine, m7G (5') ppp (5') (2'-OMeA) pG, uridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2′-O-methyl uridine.
The nucleic acid of claim 18, wherein the 5’ UTR comprises a sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 29.
The nucleic acid of claim 18 or 20, wherein the 3’ UTR comprises a sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 31 or 33.
The nucleic acid of any one of claims 16-21, wherein the nucleic acid comprises a capping enhancing sequence.
The nucleic acid of claim 22, wherein the capping enhancing sequence comprises a sequence set forth in SEQ ID NO: 27.
The nucleic acid of any one of claims 16-23, wherein the nucleic acid comprises a poly (A) sequence.
The nucleic acid of claim 24, wherein the poly (A) sequence comprises a sequence set forth in SEQ ID NO: 34.
A pharmaceutical composition comprising the nucleic acid of any one of claims 1-25.
A vaccine comprising the nucleic acid of any one of claims 1-25, formulated in a lipid nanoparticle (LNP) .
The vaccine of claim 27, wherein the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.
The vaccine of claim 28, wherein the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.
A polypeptide comprising:

(a) an Igκ light chain signal peptide (SP) sequence;

(b) an antigen sequence; and

(c) a human major histocompatibility complex (MHC) class I transmembrane and trafficking domain (MITD) sequence.
The polypeptide of claim 30, wherein the antigen is PRAME.
The polypeptide of claim 30 or 31, comprising a sequence that is at least 80%identical to the sequence set forth in SEQ ID NO: 7.
A method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 26, the vaccine of any one of claims 27-29, or the polypeptide of any one of claims 30-32.
A method of preventing or treating a disease or disorder in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 26, the vaccine of any one of claims 27-29, or the polypeptide of any one of claims 30-32.
The method of claim 33 or 34, wherein the disease or disorder is cancer.
The method of claim 35, wherein the cancer is head and neck squamous cell carcinoma (HNSCC) or lung cancer.
The method of any one of claims 33-36, further comprising administering to the subject one or more additional therapeutic agents.
The method of claim 37, wherein the one or more additional therapeutic agents are anti-cancer therapeutic agents.
A method of making a vaccine comprising mixing a nucleic acid of any one of claims 1-25 with a lipid nanoparticle formulation, thereby producing a vaccine.
A nucleic acid comprising a sequence set forth in any one of SEQ ID NOs: 1, 2 and 8-33.
The nucleic acid of claim 40, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 10 or 20.
The nucleic acid of claim 40, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 11 or 21.
The nucleic acid of claim 40, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 8, 9, 18 or 19.
The nucleic acid of claim 40, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 14, 15, 24 or 25.
The nucleic acid of claim 40, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 26 or 27.
The nucleic acid of claim 40, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 28 or 29.
The nucleic acid of claim 40, wherein the nucleic acid comprises a sequence set forth in SEQ ID NO: 30, 31, 32 or 33.
The nucleic acid of any one of claims 40-47, wherein the nucleic acid is an mRNA.