Cancer vaccines and their uses

Abstract

This article provides nucleic acids, pharmaceutical compositions, and vaccines that express cancer antigens (e.g., MAGE-A4). It also provides methods for treating cancer.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to International Application No. PCT / CN2024 / 070610, filed on January 4, 2024, which is incorporated herein by reference in its entirety for all purposes. Technical Field

[0003] This disclosure relates to compositions and methods for treating cancer, and in particular to vaccines that treat tumor growth and provide protection against tumor growth. Background Technology

[0004] Melanoma is a skin cancer that develops in melanocytes after a DNA mutation, often secondary to excessive sun exposure. People with fair skin and light hair who live in environments with high sun exposure face the greatest risk. Clinically, melanomas present as irregular shapes, colors, and asymmetry. Sometimes, melanomas exhibit ulceration and bleeding, which is associated with a poor prognosis. Fine-needle aspiration biopsy typically reveals nests of atypical melanocytes accumulating and coalescing at the dermal-epidermal junction. The depth of the melanoma is the most important prognostic factor.

[0005] In the United States, malignant melanoma is now the fifth most common cancer among men and the seventh most common cancer among women, with approximately 50,000 new cases diagnosed each year. About 15% of these cases result in death. At diagnosis, approximately 80% present with localized disease, 15% with regional disease, and 5% with distant metastases.

[0006] Melanoma most commonly metastasizes to the skin and lungs, but sometimes to the small intestine (the most common site of metastasis). When there is diffuse metastasis, chemotherapy options are available. Immunotherapy such as PD-1 or PD-L1 therapy has also been used to treat melanoma. However, morbidity, recurrence, and mortality remain unacceptably high, and much work remains to be done to address fundamental questions regarding melanoma prevention, detection, diagnosis, metastatic dormancy and progression, and response and resistance to targeted and immuno-based therapies.

[0007] Lung cancer is a leading cause of cancer death worldwide and the third most common cancer in the United States. Lung cancer often presents with no signs or symptoms in its early stages. Signs and symptoms typically appear in later stages of the disease. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for more than 80% of all lung cancer cases. Common types include adenocarcinoma and squamous cell carcinoma. Adenosquamous carcinoma and sarcomatoid carcinoma are two less common types of NSCLC.

[0008] Lung cancer treatment includes surgery, radiofrequency ablation, radiotherapy, chemotherapy, targeted drug therapy, and immunotherapy. Targeting single-molecule abnormalities or cancer pathways has achieved good clinical responses, which have moderately impacted survival in some cancers. However, this approach to cancer treatment remains rudimentary, and many challenges need to be addressed to improve treatment outcomes.

[0009] Therefore, there is an unmet need to develop new models and therapies that can improve treatment outcomes and prolong survival for cancer patients. Summary of the Invention

[0010] In one aspect, this article provides a nucleic acid encoding a polypeptide comprising: (a) an Igκ light chain signal peptide (SP) sequence; (b) a MAGE-A4 antigen sequence; and (c) a human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) sequence.

[0011] In some embodiments, the Igκ light chain signal peptide (SP) sequence is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 3.

[0012] In some embodiments, the nucleic acid described herein comprises a nucleic acid encoding an 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 shown in SEQ ID NO: 8, 9, 17 or 18.

[0013] In some embodiments, the MITD sequence contains at least 80% of the same amino acid sequence as shown in SEQ ID NO: 4.

[0014] In some embodiments, the nucleic acid described herein comprises a nucleic acid encoding a MITD sequence, wherein the nucleic acid encoding the MITD sequence is at least 80% identical to the sequence shown in SEQ ID NO: 13, 14, 22 or 23.

[0015] In some embodiments, the MAGE-A4 antigen sequence contains at least 80% of the same amino acid sequence as shown in SEQ ID NO: 5.

[0016] In some embodiments, the nucleic acid described herein comprises a nucleic acid encoding a MAGE-A4 antigen sequence, wherein the nucleic acid encoding the MAGE-A4 antigen sequence is at least 80% identical to the sequence shown in SEQ ID NO: 10, 11, 19 or 20.

[0017] In some implementations, the Igκ light chain signal peptide (SP) sequence, the antigen sequence, and / or the MITD sequence are linked via a linker.

[0018] In some implementations, the connector contains the sequence shown in SEQ ID NO: 6.

[0019] In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding a adapter, wherein the nucleic acid sequence encoding the adapter is at least 80% identical to the sequence shown in SEQ ID NO: 12 or 21.

[0020] In some implementations, the nucleic acid described herein contains a stop codon.

[0021] In some implementations, the nucleic acid described herein contains at least 80% identical nucleotide sequences to the sequences shown in SEQ ID NO: 1, 2, 15 or 16.

[0022] In some implementations, the nucleic acid is mRNA.

[0023] In some implementations, the mRNA contains at least one chemical modification.

[0024] In some embodiments, the mRNA comprises a 5' UTR and / or a 3' UTR. In some embodiments, the 5' UTR comprises at least 80% identical to the sequence shown in SEQ ID NO: 27. In some embodiments, the 3' UTR comprises at least 80% identical to the sequence shown in SEQ ID NO: 29 or 31.

[0025] In some embodiments, the chemical modification is selected from pseudouridine, N1-methyl-pseudouridine, m7G(5')ppp(5')(2'-OMeA)pG, uridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deazo-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, 2'-O-methyluridine.

[0026] In some implementations, the nucleic acid includes a capped enhancement sequence.

[0027] In some implementations, the capped enhancement sequence includes the sequence shown in SEQ ID NO: 25.

[0028] In some implementations, the nucleic acid contains multiple (A) sequences.

[0029] In some implementations, the multiple (A) sequence comprises the sequence shown in SEQ ID NO: 32.

[0030] In one respect, this document provides pharmaceutical compositions comprising any of the nucleic acids described herein.

[0031] In one respect, this article provides vaccines formulated in lipid nanoparticles (LNPs) comprising any of the nucleic acids described herein.

[0032] In some implementations, the lipid nanoparticles comprise cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids.

[0033] In some implementations, the cationic lipids are ionizable cationic lipids, the non-cationic lipids are neutral lipids, and the sterols are cholesterol.

[0034] In one aspect, this article provides a polypeptide comprising: (a) an Igκ light chain signal peptide (SP) sequence; (b) a MAGE-A4 antigen sequence; and (c) a human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) sequence.

[0035] In some embodiments, the polypeptide described herein contains at least 80% of the same sequence as the sequence shown in SEQ ID NO: 7.

[0036] In one aspect, this document provides a method for inducing an immune response in a subject, comprising administering to the subject an effective amount of any of the pharmaceutical compositions described herein, any of the vaccines described herein, or any of the peptides described herein.

[0037] In one aspect, this document provides methods for preventing or treating a disease or ailment in a subject, comprising administering to the subject an effective amount of any of the pharmaceutical compositions described herein, any of the vaccines described herein, or any of the peptides described herein.

[0038] In some implementations, the disease or symptom is cancer.

[0039] In some implementation schemes, the cancer is melanoma, lung cancer, esophageal cancer, head and neck cancer, stomach cancer, bladder urothelial carcinoma, hepatocellular carcinoma, triple-negative breast cancer (TNBC), or ovarian cancer.

[0040] In some implementations, the method further includes administering one or more additional therapeutic agents to the subject.

[0041] In some implementations, one or more additional therapeutic agents are anticancer therapeutic agents.

[0042] In one aspect, this article provides a method for preparing a vaccine, which includes mixing any of the nucleic acids described herein with a lipid nanoparticle formulation to produce a vaccine.

[0043] In one respect, this article provides nucleic acids comprising the sequences shown in any one of SEQ ID NO: 1, 2 and 8-31.

[0044] In some implementations, the nucleic acid comprises the sequence shown in SEQ ID NO: 10 or 19.

[0045] In some implementations, the nucleic acid comprises the sequence shown in SEQ ID NO: 11 or 20.

[0046] In some implementations, the nucleic acid comprises the sequence shown in SEQ ID NO: 8, 9, 17 or 18.

[0047] In some embodiments, the nucleic acid comprises the sequence shown in SEQ ID NO: 13, 14, 22 or 23. In some embodiments, the nucleic acid comprises the sequence shown in SEQ ID NO: 24 or 25.

[0048] In some implementations, the nucleic acid comprises the sequence shown in SEQ ID NO: 26 or 27.

[0049] In some embodiments, the nucleic acid comprises the sequence shown in SEQ ID NO: 28, 29, 30, or 31. In some embodiments, the nucleic acid is mRNA.

[0050] 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 pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All disclosures, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0051] Other features and advantages of the invention will become apparent from the following detailed description, accompanying drawings, and claims. Attached Figure Description

[0052] Figure 1 Western blot results showing the expression level of MAGE-A4 protein. MAGE-A4-1 and MAGE-A4-2 are different mRNA sequences encoding the MAGE-A4 antigen.

[0053] Figure 2 The study demonstrated the MAGE-A4 antigen-specific immune response in the spleen of mice under a single-dose regimen.

[0054] Figure 3 This study demonstrates the MAGE-A4 antigen-specific immune response in mouse lymph nodes under a single-dose regimen.

[0055] Figure 4 Tolerance to MAGE-A4 mRNA-LNP immunization was demonstrated in mice.

[0056] Figure 5 The MAGE-A4 mRNA-LNP vaccine was shown to inhibit tumor growth.

[0057] Figure 6 The sequence selected in this disclosure is shown. Invention Details

[0059] The use of mRNA technology allows for the induction of a wide range of secreted, membrane-bound, and intracellular proteins in humans. mRNA encoding antigens is an attractive technological platform for tumor-associated antigen (TAA) vaccination because mRNA vaccines can deliver multiple antigens (e.g., TAAs) in a single molecule, enabling the rapid fabrication of vaccines specific to each individual subject, and the antigens (e.g., TAAs) are endogenously translated and incorporated into natural cellular antigen processing and presentation pathways. Furthermore, this mRNA-based vaccine technology overcomes challenges typically associated with DNA-based vaccines, such as the risk of genome integration or the high doses and devices (e.g., electroporation) required for administration.

[0060] This disclosure relates to nucleic acids and pharmaceutical compositions or vaccines (e.g., mRNA vaccines) containing such nucleic acids, wherein the nucleic acid encodes a polypeptide comprising (a) an Igκ light chain signal peptide (SP) sequence; (b) a cancer antigen, such as MAGE-A4; and (c) a human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD).

[0061] This disclosure also relates to polypeptides comprising (a) an Igκ light chain signal peptide (SP) sequence; (b) a cancer antigen, such as MAGE-A4; and (c) a human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) sequence.

[0062] This disclosure also relates to methods for inducing an immune response or treating cancer by administering a vaccine (e.g., an mRNA cancer vaccine) formulated as lipid nanoparticles to a subject.

[0063] The present invention further relates to a method of treating cancer by combining anticancer immunotherapy with the administration of the aforementioned vaccine (e.g., an mRNA cancer vaccine).

[0064] Nucleic acid

[0065] In one aspect, this disclosure relates to nucleic acids encoding polypeptides comprising: (a) an Igκ light chain signal peptide (SP) sequence; (b) a MAGE-A4 antigen sequence; and (c) a human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD).

[0066] Signal peptides (SPs), also known as signal sequences, are short peptides that can influence protein targeting pathways and promote protein secretion or specific post-translational modifications (such as glycosylation). Therefore, SPs derived from highly secreted proteins can be used to improve the protein secretion levels of recombinant proteins in cell lines and for ectopic expression of endogenous genes.

[0067] In some embodiments, the nucleic acid described herein encodes a polypeptide comprising an immunoglobulin κ (Igκ) light chain signal peptide (SP) sequence. As used herein, “Igκ signal peptide (SP)” refers to a sequence derived from the immunoglobulin κ (Igκ) light chain. In some embodiments, the Igκ signal peptide (SP) is the human Igκ signal peptide (SP). As used herein, “Igκ signal peptide (SP) sequence” refers to the full-length sequence of the Igκ signal peptide (SP) or a sequence variant thereof, for example, a sequence that is about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the full-length sequence of the Igκ signal peptide (SP). An example full-length sequence of the Igκ signal peptide (SP) is shown in SEQ ID NO: 3. In some embodiments, Igκ SP has about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the same amino acid sequence as shown in SEQ IDNO: 3.

[0068] In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding an Igκ signal peptide (SP) sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA). In some embodiments, the nucleic acid sequence encoding the Igκ signal peptide (SP) sequence comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 8 or 9. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid sequence encoding the Igκ signal peptide (SP) sequence comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 17 or 18.

[0069] In one aspect, this document provides a nucleic acid comprising about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 8 or 9. In some embodiments, the nucleic acid encodes an Igκ signal peptide (SP) sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA).

[0070] In one aspect, this document provides a nucleic acid comprising about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 17 or 18. In some embodiments, the nucleic acid encodes an Igκ signal peptide (SP) sequence. In some embodiments, the nucleic acid is DNA.

[0071] Genetic modification of vaccines by linking antigens to lysosomal or endosomal targeting signals has been used to deliver antigens into MHC class II processing compartments to improve CD4 expression. + T cell responses. Combining the N-terminal leader peptide with an MHC class I transport signaling pathway (MITD) attached to an antigen (e.g., a cancer antigen) can improve the presentation of MHC class I and II epitopes in human cells. Such chimeric fusion proteins exhibit a maturation-dependent subcellular distribution pattern in both immature and mature immune cells (e.g., dendritic cells (DCs)), mimicking the dynamic transport properties of MHC molecules. Linking an antigen (e.g., a cancer antigen) to the MITD transport signal allows CD8... + and CD4 + Simultaneous multi-epitope amplification of T cells leads to different CD8... + T cell-specific and broadly variable Ag-specific CD4 + Library.

[0072] In some embodiments, the nucleic acid described herein encodes a polypeptide containing an MHC class I transport signal (MITD) sequence. As used herein, “MHC class I transport signal (MITD)” refers to a sequence derived from an MHC class I transport signal (MITD). In some embodiments, the MITD is a human MITD. As used herein, “MHC class I transport signal (MITD) sequence” refers to the full-length sequence of an MHC class I transport signal or a sequence variant thereof, for example, a sequence that is approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the full-length sequence of an MHC class I transport signal (MITD). An example full-length sequence of an MHC class I transport signal (MITD) is shown in SEQ ID NO: 4. In some embodiments, the MHC class I transport signal (MITD) has about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 4.

[0073] In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding the MITD sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA). In some embodiments, the nucleic acid sequence encoding the MITD sequence comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 13 or 14. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid sequence encoding the MITD sequence comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 22 or 23.

[0074] In one aspect, this document provides nucleic acids comprising approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical sequences to those shown in SEQ ID NO: 13 or 14. In some embodiments, the nucleic acid encodes a MITD sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA).

[0075] In one aspect, this document provides nucleic acids comprising approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical sequences to those shown in SEQ ID NO: 22 or 23. In some embodiments, the nucleic acid encodes a MITD sequence. In some embodiments, the nucleic acid is DNA.

[0076] In some embodiments, the Igκ signal peptide (SP) is located at the N-terminus of an antigen sequence (e.g., a cancer antigen sequence such as MAGE-A4). In some embodiments, the Igκ signal peptide (SP) sequence is located at the N-terminus of a MITD sequence. In some embodiments, the antigen sequence (e.g., a cancer antigen sequence such as MAGE-A4) is located at the N-terminus of a MITD sequence. In some embodiments, the nucleic acid encoding polypeptide described herein comprises, from N-terminus to C-terminus, an Igκ signal peptide (SP) sequence, an antigen sequence (e.g., a cancer antigen sequence such as MAGE-A4 sequence), and a MITD sequence.

[0077] In some embodiments, the Igκ signal peptide (SP) sequence and the cancer antigen sequence (e.g., the MAGE-A4 antigen sequence); the cancer antigen sequence (e.g., the MAGE-A4 antigen 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 may be used herein. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 6.

[0078] In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding an adapter sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA). In some embodiments, the nucleic acid sequence encoding the adapter sequence comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 12. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid sequence encoding the adapter sequence comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 21.

[0079] In one aspect, this document provides a nucleic acid comprising approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 12. In some embodiments, the nucleic acid encodes an adapter sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA).

[0080] In one aspect, this document provides a nucleic acid comprising approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 21. In some embodiments, the nucleic acid encodes an adapter sequence. In some embodiments, the nucleic acid is DNA.

[0081] In some embodiments, the nucleic acid described herein comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the same nucleotide sequence as the amino acid sequence shown in SEQ ID NO: 1 or 2. In some embodiments, the nucleic acid is RNA (e.g., mRNA).

[0082] In some embodiments, the nucleic acid described herein comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence shown in SEQ ID NO: 15 or 16. In some embodiments, the nucleic acid is DNA.

[0083] In some embodiments, the nucleic acid described herein contains a stop codon (e.g., TAA, TGA, or TAG). In some embodiments, the nucleic acid described herein contains a TAA (UAA in the case of RNA) stop codon. In some embodiments, the nucleic acid described herein contains a TGA (UGA in the case of RNA) stop codon. In some embodiments, the nucleic acid described herein contains a TAG (UAG in the case of RNA) stop codon.

[0084] In some embodiments, the stop codon is located at the 3' end of the nucleic acid described herein. In some embodiments, the stop codon is located at the 3' end of the nucleic acid encoding an antigen (e.g., a cancer antigen, such as MAGE-A4) and / or MITD.

[0085] Stop codons were initially identified through mutations in bacteriophage T4. The first identified stop codon was TAG (UAG in the case of RNA), the amber codon. The second stop codon, TAA (UAA in the case of RNA), was called the ochre codon. The third stop codon, TGA (UGA in the case of RNA), was called the opal or brown codon.

[0086] Translation of mRNA into a polypeptide terminates when the releasing factor eRF1 interacts with the UAA, UAG, or UGA stop codon at the ribosomal A site and another releasing factor eRF3 hydrolyzes GTP and stimulates the polypeptide-releasing activity of eRF1. However, at low frequencies, near-homologous tRNAs (nc-tRNAs; tRNAs with a one-base-pair mismatch in their anticodons) outperform eRF1 in decoding the stop codon, leading to continued translational elongation. When the stop codon is located at a normal site at the end of the open reading frame (ORF), elongation continues into the 3'-untranslated region (3'-UTR) of the mRNA, resulting in a C-terminal elongated polypeptide product. This type of event is called stop codon readthrough or nonsense repression.

[0087] In some embodiments, the nucleic acid disclosed herein is messenger RNA (mRNA). “Ms. RNA” (mRNA) refers to any nucleic acid that encodes (at least one) a polypeptide (a naturally occurring, non-natural, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide.

[0088] 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, and a multiple A tail. The nucleic acids disclosed herein can function as mRNA but can be distinguished from wild-type mRNAs by their functional and / or structural design features designed to overcome existing problems in the efficient expression of peptides using nucleic acid-based therapies.

[0089] In some embodiments, the nucleic acids described herein contain a cap sequence. In some embodiments, the cap sequence is a 5' cap sequence. 5' capping in mature mRNA is necessary to protect mRNA from degradation, promote ribosome recruitment, gene expression, and self- and non-self recognition. Several variants of the 5' cap structure have been found in nature. The 5' cap of eukaryotic mRNA contains 7-methylguanosine (m7G) via a 5'-5'-triphosphate bridge (m7GpppN) through a series of enzymatic capping reactions involving RNA triphosphatase, guanylate transferase, and S-adenosylmethionine. To further enhance translation efficiency, additional methylation can be introduced at the first nucleotide (cap1: m7GpppNmpN) or both the first and second nucleotides (cap2: m7GpppNmpNm). 5' capping modification improves translation initiation by recruiting translation initiation factors, protects synthesized mRNA from exonuclease degradation, and avoids excessive activation of the innate immune response. mRNA capping can be performed during IVT reactions by replacing part of the guanosine triphosphate (GTP) substrate with a cap analog. Alternatively, vaccinia capping enzyme (VCC) and a methyl donor can be used as substrates to cap mRNA in the second enzymatic reaction.

[0090] In some embodiments, the nucleic acid described herein comprises a capping enhancement sequence. As used herein, a “capping enhancement sequence” is a sequence that enhances the function of the 5' cap of RNA (e.g., mRNA). In some embodiments, the capping enhancement sequence is an RNA (e.g., mRNA) sequence. In some embodiments, the capping enhancement sequence comprises about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 25. In some embodiments, the DNA sequence encodes the capping enhancement sequence. In some embodiments, the sequence comprises about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 24.

[0091] This document also provides nucleic acid sequences that are approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 24. In some embodiments, the nucleic acid encodes a capped enhancement sequence. In some embodiments, the nucleic acid is a DNA sequence.

[0092] This document also provides nucleic acid sequences that are approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 25. In some embodiments, the nucleic acid encodes a capped enhancement sequence. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) sequence.

[0093] In some embodiments, the capping enhancement sequence is located at the 3' of the 5' cap of the nucleic acid described herein. In some embodiments, the capping enhancement sequence is located at the 5' of the 5' cap of the nucleic acid described herein. In some embodiments, the capping enhancement sequence is located at the 3' of the 5' UTR of the nucleic acid described herein. In some embodiments, the capping enhancement sequence is located at the 5' of the 5' UTR of the nucleic acid described herein.

[0094] In some implementations, the nucleic acid described herein includes a 3' untranslated region (3' UTR) and / or a 5' UTR. The untranslated region (UTR) is responsible for transcriptional regulation and mRNA stability. These regions strongly influence translation efficiency because the sequences used are involved in translational machine recognition, recruitment, and mRNA transport.

[0095] 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 approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 28 or 30. In some embodiments, the 3' UTR sequence is an RNA (e.g., mRNA) sequence. In some embodiments, the 3' UTR comprises approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 29 or 31.

[0096] This document also provides nucleic acid sequences that are approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 28 or 30. In some embodiments, the nucleic acid encodes a 3' UTR. In some embodiments, the nucleic acid is a DNA sequence.

[0097] This document also provides nucleic acid sequences that are approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 29 or 31. In some embodiments, the nucleic acid is a 3' UTR. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) sequence.

[0098] 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 approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 26. In some embodiments, the 5' UTR sequence is an RNA (e.g., mRNA) sequence. In some embodiments, the 5' UTR comprises approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 27.

[0099] This document also provides a nucleic acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 26. In some embodiments, the nucleic acid encodes a 5' UTR. In some embodiments, the nucleic acid is a DNA sequence.

[0100] This document also provides a nucleic acid sequence that is approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 27. In some embodiments, the nucleic acid is a 5' UTR. In some embodiments, the nucleic acid is an RNA (e.g., mRNA) sequence.

[0101] In some embodiments, the nucleic acids described herein contain a multiple (A) sequence. The addition of a multiple (A) sequence (e.g., a 3' multiple (A) tail) improves mRNA stability and translational activity because it protects the mRNA from nuclease degradation by multiple (A)-binding proteins (PABPs). This tail can be added to the transcript by inserting the multiple (A) sequence into a DNA template or through an enzymatic reaction with the RNA sequence. Tail size optimization is an important factor for mRNA stability and expression. Longer multiple A tails can improve mRNA stability and translation. However, this effect is not linear, and the optimal tail size depends on the cell type.

[0102] In some embodiments, the multiple (A) sequence may be in the DNA sequence described herein and may be transcribed into the RNA (e.g., mRNA) sequence described herein. In some embodiments, the multiple (A) sequence is added after transcription of the RNA (e.g., mRNA) sequence. In some embodiments, the multiple (A) sequence comprises about or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 32.

[0103] This document also provides nucleic acid sequences that are approximately or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 32. In some embodiments, the nucleic acid is a multiple (A) sequence.

[0104] In some implementations, the multiple (A) sequence is located at the 3' end of the nucleic acid described herein.

[0105] On the one hand, the nucleic acids described in this article from 5' to 3' include:

[0106] (a) Capped enhancement sequence;

[0107] (b) 5' UTR;

[0108] (c) Igκ light chain signal peptide (SP) sequence;

[0109] (d) Antigen sequence (e.g., melanoma preferentially expressed antigen (PRAME));

[0110] (e) Human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) sequence;

[0111] (f) 3' UTR; and

[0112] (g)Optional multiple (A) sequences.

[0113] In some implementations, the nucleic acid is DNA. In some implementations, the nucleic acid is RNA. In some implementations, the nucleic acid described herein is codon-optimized.

[0114] Cancer antigens

[0115] The nucleic acids described herein encode polypeptides containing antigens. In some embodiments, the antigen is a cancer antigen. In some embodiments, the antigen is the full length or a portion of a cancer antigen. In some embodiments, the cancer antigen is melanoma-associated antigen 4 (MAGE-A4).

[0116] Melanoma-associated antigen 4 (MAGE-A4) is one of the most frequently expressed cancer-testis antigens (expressed in approximately 67% of synovial sarcomas and 34% of synovial sarcomas or myxoid / round cell liposarcomas (MRCLS)). MAGE-A tumor-associated antigens are among the first human tumor-associated antigens identified at the molecular level. They belong to the larger cancer / testis antigen (CTA) family, whose expression is consistently detected in cancers of diverse histological origins and germ cells. The MAGE-A subfamily comprises 12 highly homologous genes located on chromosome Xq28. Specific gene products have been identified by immunohistochemistry in cancers of diverse histological origins, including high percentages of non-small cell lung cancer (NSCLC), bladder cancer, esophageal cancer, and head and neck cancer, as well as sarcomas. These antigens are also frequently expressed in Reed-Sternberg cells of triple-negative breast cancer, myeloma, and Hodgkin's disease, with the highest frequency detected in synovial sarcomas. In healthy tissues, expression of specific members of this family has been observed in spermatogonia, placenta, and fetal ovaries. Detailed reviews of the MAGE-A4 antigen can be found, for example, in Zajac et al., Front Med (Lausanne). 2017; 4: 18; and ASCO 2021 – MAGE-A4: Expectations from one of the most promising targets in Oncology, which are incorporated herein by reference in their entirety. MAGE-A4 is expressed in a variety of solid tumors such as melanoma, lung cancer, esophageal cancer, head and neck cancer, gastric cancer, urothelial carcinoma of the bladder, hepatocellular carcinoma, triple-negative breast cancer (TNBC), and ovarian cancer. An overview of MAGE-A4 expression can be found on websites such as The Human Protein Atlas.

[0117] As used herein, “antigen sequence” refers to the full-length sequence of a cancer antigen or a sequence variant thereof, for example, a sequence that is approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the full-length sequence of a cancer antigen. An example full-length sequence of MAGE-A4 is shown in SEQ ID NO: 5. In some embodiments, MAGE-A4 has Uniprot ID No. P43358. In some embodiments, MAGE-A4 comprises an amino acid sequence that is approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 5.

[0118] In some embodiments, the nucleic acid described herein comprises a nucleic acid sequence encoding a MAGE-A4 antigen sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA). In some embodiments, the nucleic acid sequence encoding the MAGE-A4 antigen sequence comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 10 or 11. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid sequence encoding the MAGE-A4 antigen sequence comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 19 or 20.

[0119] In one aspect, this document provides a nucleic acid comprising approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 10 or 11. In some embodiments, the nucleic acid encodes a MAGE-A4 antigen sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA).

[0120] In one aspect, this document provides a nucleic acid comprising approximately or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 19 or 20. In some embodiments, the nucleic acid encodes a MAGE-A4 antigen sequence. In some embodiments, the nucleic acid is RNA (e.g., mRNA).

[0121] In some implementations, the antigen is a personalized cancer antigen (e.g., a personalized MAGE-A4 antigen sequence) or a portion thereof.

[0122] In some embodiments, a cancer antigen sequence (e.g., a MAGE-A4 antigen sequence) is a sequence of an antigenic epitope. As used herein, an epitope, also known as an antigenic determinant, is a portion of an antigen that is recognized by the immune system, particularly by antibodies, B cells, or T cells, when appropriate. Epitopes can include B-cell epitopes (e.g., predicted B-cell reactive epitopes) and T-cell epitopes (e.g., predicted T-cell reactive epitopes). A B-cell epitope (e.g., a predicted B-cell reactive epitope) is a peptide sequence required for recognition by B cells that produce specific antibodies. A B-cell epitope (e.g., a predicted B-cell reactive epitope) refers to a specific region of an antigen that is recognized by antibodies. A T-cell epitope (e.g., a predicted T-cell reactive epitope) is a peptide sequence that associates with a protein on an APC and is 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 an APC, where they bind to MHC molecules, including, for example, MHC class II and / or MHC class I molecules. The portion of an antibody that binds to an epitope is called the complementary site. Based on structure and interaction with the complementary site, an epitope can be a conformational epitope or a linear epitope. A linear or continuous epitope is determined by the primary amino acid sequence of a specific region of a protein. This sequence, which interacts with the antibody, is located continuously in adjacent positions on the protein, and the epitope can typically be mimicked by a single peptide. A conformational epitope is an epitope determined by the conformational structure of the native protein. These epitopes can be continuous or discontinuous (i.e., they can be components of an epitope that can be located on different parts of the protein and are close to each other in the folded native protein structure).

[0123] In one respect, this article provides a polypeptide comprising (a) the Igκ light chain signal peptide (SP) sequence described herein; (b) the MAGE-A4 antigen sequence described herein; and (c) the human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) sequence described herein.

[0124] In one aspect, this document provides a polypeptide comprising about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence identical to that of the protein translation product of the nucleic acid described herein. The translation product may be a full-length or partial translation of the nucleic acid described herein. In another aspect, this document provides a polypeptide comprising about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence shown in SEQ ID NO: 7.

[0125] Chemical modification

[0126] In some embodiments, the nucleic acids described herein comprise one or more chemically modified nucleobases. Modified polynucleotides comprise the nucleic acids described herein (or, interchangeably, "polynucleotides" herein). Modified nucleic acids can be chemically modified and / or structurally modified. When the nucleic acids of this disclosure are chemically and / or structurally modified, the polynucleotide may be referred to as a "modified nucleic acid."

[0127] This disclosure provides modified nucleosides and nucleotides encoding nucleic acids (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprising (a) an Igκ light chain signal peptide (SP) sequence; (b) a cancer antigen sequence (e.g., a MAGE-A4 antigen sequence); and (c) a human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) sequence. “Nucleoside” refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof combined with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a “nucleobase”). “Nucleoside” refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method (e.g., chemical, enzymatic, or recombinant) to include one or more modified or non-natural nucleosides. Nucleic acids may contain one or more regions of linked nucleosides. Such regions may have variable backbone linkages. The linkage may be a standard phosphodiester bond, in which case the polynucleotide will contain the nucleotide region.

[0128] The modified nucleic acids disclosed herein may contain a variety of different modifications. In some embodiments, the modified polynucleotide contains one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotide introduced into the cell may exhibit one or more desired properties, such as, for example, improved protein expression, reduced innate immune response, or reduced degradation in the cell, compared to unmodified polynucleotides.

[0129] In some embodiments, the nucleic acids disclosed herein (e.g., nucleic acids encoding one or more peptide epitopes) are structurally modified. As used herein, a “structural” modification is a modification in which two or more linked nucleosides are inserted, deleted, replicated, reversed, or randomized in a polynucleotide without significant chemical modification to the nucleotide itself. Because chemical bonds must be broken and rearranged to achieve structural modification, structural modification is chemical in nature and is therefore a chemical modification. However, structural modification will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” can be chemically modified to “AT-5meC-G”.

[0130] In some embodiments, the nucleic acids disclosed herein are chemically modified. As used herein with respect to nucleic acids, the term "chemically modified" or, where appropriate, "chemically modified" refers to a modification of one or more of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribose or deoxyribonucleoside at their position, pattern, percentage, or group. Generally, these terms are not intended to refer to ribonucleotide modifications in the naturally occurring 5' end mRNA cap portion.

[0131] In some embodiments, the nucleic acid of this disclosure may have uniform chemical modifications of all or any of the same nucleoside types, or may have a group of modifications resulting from only downward titration of the same initial modification in all or any of the same nucleoside types, or may have chemical modifications of all or any of the same nucleoside types at a measured percentage but with random incorporation, such as where all uridines are replaced by uridine analogs (e.g., pseudouridine or 5-methoxyuridine). In another embodiment, the polynucleotide may have two, three, or four uniform chemical modifications of the same nucleoside types throughout the polynucleotide (e.g., all uridines and all cytosines are modified in the same manner).

[0132] Modified nucleotide base pairings include not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides (including non-standard or modified bases), wherein the arrangement of hydrogen bond donors and acceptors allows hydrogen bonds to form between non-standard and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairings is the base pairing between modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker can be incorporated into the polynucleotides of this disclosure.

[0133] Those skilled in the art will understand that, unless otherwise stated, nucleic acid sequences shown in this disclosure will be represented as “T” in representative DNA sequences, but in the case where the sequence represents RNA, “T” will be replaced by “U”.

[0134] In some embodiments, the nucleotides and nucleosides of this disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications may include modifications at the sugar, backbone, or nucleobase portions of the nucleotides and / or nucleosides as recognized in the art.

[0135] In some embodiments, the naturally occurring modified nucleotides or nucleosides disclosed herein are nucleotides or nucleosides generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleosides can be found, in particular, in the widely recognized MODOMICS database.

[0136] In some embodiments, the non-naturally occurring modified nucleotides or nucleosides of this disclosure are nucleotides or nucleosides generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, in particular, in the published U.S. Applications 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 herein by reference for this purpose.

[0137] Therefore, the nucleic acids disclosed herein (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) may comprise standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

[0138] In some embodiments, the nucleic acids of this disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) comprise various (more than one) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, specific regions of the nucleic acid contain one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0139] In some implementations, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms exhibit reduced degradation in cells or organisms, relative to unmodified nucleic acids containing standard nucleotides and nucleosides.

[0140] In some implementations, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms can exhibit a reduced innate immune response compared to unmodified nucleic acids containing standard nucleotides and nucleosides.

[0141] In some implementations, nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise non-natural modified nucleotides introduced during or after nucleic acid synthesis to achieve a desired function or property. Modifications can be present at internucleotide linkages, purine or pyrimidine bases, or sugars. Modifications can be introduced chemically or using polymerases at the ends of the chain or anywhere else in the chain. Any region of the nucleic acid can be chemically modified.

[0142] This disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., DNA nucleic acids or RNA nucleic acids, such as mRNA nucleic acids).

[0143] In some embodiments, the modified nucleotides in the nucleic acid (e.g., RNA nucleic acid, such as mRNA nucleic acid) comprise 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (ψ). In some embodiments, the modified nucleotides in the nucleic acid (e.g., RNA nucleic acid, such as mRNA nucleic acid) comprise 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some embodiments, the polyribonucleotide comprises at least two (e.g., two, three, four, or more) combinations of any of the aforementioned modified nucleotides, including but not limited to chemical modifications.

[0144] In some implementations, the modified nucleobases in the nucleic acid (e.g., RNA nucleic acid, such as mRNA nucleic acid) include m 1 A(1-methyladenosine); m 2 A (2-methyladenosine); Am (2'-O-methyladenosine); ms 2 m 6 A(2-methylthio-N) 6 -methyladenosine); i 6 A(N 6 -Isopentenyl adenosine); ms 2 i6A(2-methylthio-N) 6 (Isopentenyl adenosine); io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine); ms 2 io 6 A(2-methylthio-N) 6 -(cis-hydroxyisopentenyl)adenosine); g 6 A(N 6 -glycylcarbamoyladenosine); t 6 A(N 6 (-Threonylcarbamoyladenosine); ms 2 t 6A(2-methylthio-N) 6 -Threonylcarbamoyladenosine); m 6 t 6 A(N 6 -Methyl-N 6 -Threonylcarbamoyladenosine); hn 6 A(N 6 -hydroxyn-valinecarbamoyladenosine); ms 2 hn 6 A(2-methylthio-N) 6 -hydroxyvaline carbamoyl adenosine); Ar(p)(2'-O-ribosyl adenosine (phosphate)); I(inosine); m 1 I(1-methylinosine); m 1 Im(1,2'-O-dimethylinosine); m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C(2-thiocytidine); ac 4 C(N 4 -acetylcytidine); f 5 C(5-formylcytidine); m 5 Cm(5,2'-O-dimethylcytidine); ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine); k 2 C (lysicytoside); m 1 G(1-methylguanosine); m 2 G(N 2 -methylguanosine); m 7 G(7-methylguanosine); Gm(2'-O-methylguanosine); m 2 2G(N 2 N 2 -dimethylguanosine); m 2 Gm(N 2 ,2'-O-dimethylguanosine); m 2 2Gm(N 2 N 2 ,2'-O-trimethylguanosine); Gr(p)(2'-O-ribosylguanosine (phosphate)); yW (huaitin); o2yW (peroxyhuaitin); OHyW (hydroxyhuaitin); OHyW (Incompletely modified hydroxyhuaitoside); imG (huaitoside); mimG (methylhuaitoside); Q (huaitoside); oQ (epoxyhuaitoside); galQ (galactosylhuaitoside); manQ (mannosylhuaitoside); preQ0 (7-cyano-7-deazoguanosine); preQ1 (7-aminomethyl-7-deazoguanosine); G +(Ancient purine); D (dihydrouridine); m 5 Um(5,2'-O-dimethyluridine); s 4 U(4-thiouridine); m 5 s 2 U(5-methyl-2-thiouridine); s 2 Um (2-thio-2'-O-methyluridine); acp 3 U(3-(3-amino-3-carboxypropyl)uridine); ho 5 U(5-hydroxyuridine); mo 5 U(5-methoxyuridine); cmo 5 U (uridine 5-hydroxyacetic acid); mcmo 5 U (uridine 5-hydroxyacetic acid methyl ester); chm 5 U(5-(carboxyhydroxymethyl)uridine); mchm 5 U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm 5 U(5-methoxycarbonylmethyluridine); mcm 5 Um(5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine); nm 5 s 2 U(5-aminomethyl-2-thiouridine); mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine); mnm 5 se 2 U(5-methylaminomethyl-2-selenoside); ncm 5 U(5-carbamoylmethyluridine); ncm 5 Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm 5 U(5-carboxymethylaminomethyluridine); cmnm 5 Um(5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm 5 s 2 U(5-carboxymethylaminomethyl-2-thiouridine); m 6 2A(N 6 N 6 -dimethyladenosine); Im(2'-O-methylinosine); m 4 C(N 4 -methylcytidine); m 4 Cm(N 4 ,2'-O-dimethylcytidine); hm5 C(5-hydroxymethylcytidine); m 3 U(3-methyluridine); cm 5 U(5-carboxymethyluridine); m 6 Am(N 6 ,2'-O-dimethyladenosine); m 6 2Am(N 6 N 6 (,O-2'-trimethyladenosine); m 2,7 G(N 2 ,7-Dimethylguanosine); m 2,2,7 G(N 2 N 2 ,7-Trimethylguanosine); m 3 Um(3,2'-O-dimethyluridine); m 5 D(5-methyldihydrouridine); f 5 Cm(5-formyl-2'-O-methylcytidine); m 1 Gm(1,2'-O-dimethylguanosine); m 1 Am(1,2'-O-dimethyladenosine); τm 5 U(5-Taurine methyluridine); τm 5 s 2 U (5-Tauratemethyl-2-thiouridine); imG-14 (4-Demethylwyoside); imG2 (Isowyoside); or ac 6 A(N 6 -acetyl adenosine.

[0145] In some embodiments, the RNA nucleic acid of this disclosure includes 1-methyl-pseudouridine (m1ψ) substitution at one or all uridine sites of the nucleic acid.

[0146] In some embodiments, the RNA nucleic acid of this disclosure comprises a 1-methyl-pseudouridine (m1ψ) substitution at one or more uridine sites and a 5-methylcytidine substitution at one or more cytidine sites. In some embodiments, the nucleic acid (e.g., mRNA) described herein comprises an N1-methyl-pseudouridine substitution at one or more sites and an m7G(5')ppp(5')(2'-OMeA)pG substitution at one or more sites.

[0147] In some embodiments, the RNA nucleic acid of this disclosure includes pseudouridine (ψ) substitution at one or all of the uridine sites of the nucleic acid.

[0148] In some embodiments, the RNA nucleic acid of this disclosure comprises pseudouridine (ψ) substitution at one or more or all uridine sites of the nucleic acid and 5-methylcytidine substitution at one or more or all cytidine sites of the nucleic acid.

[0149] In some embodiments, the RNA nucleic acid of this disclosure contains uridine at one or more or all of the uridine sites of the nucleic acid.

[0150] In some implementations, for a specific modification, the nucleic acid (e.g., RNA nucleic acid, such as mRNA nucleic acid) is uniformly modified (e.g., completely modified, modified throughout the entire sequence). For example, the nucleic acid can be uniformly modified with 1-methyl-pseudouridine, which means that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, the nucleic acid can be uniformly modified by replacing any type of nucleoside residue present in the sequence with modified residues (such as those shown above).

[0151] The nucleic acids of this disclosure can be modified partially or completely along the entire length of the molecule. For example, one or more or all of a given type of nucleotide (e.g., purine or pyrimidine, or any one or more of A, G, U, and C) in the nucleic acids of this disclosure or in a predetermined sequence region thereof (e.g., in mRNAs including or excluding multi-A tails) can be uniformly modified. In some embodiments, all nucleotides X in the nucleic acids of this disclosure (or in one sequence region thereof) are modified nucleotides, wherein X can be any of nucleotides A, G, U, and C, or combinations of A+G, A+U, A+C, G+U, G+C, U+C, A+G+C, G+U+C, or A+G+C.

[0152] Nucleic acids may contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10%). (up to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). It will be understood that any remaining percentage is calculated from the presence of the unmodified A, G, U, or C.

[0153] Nucleic acids may contain a minimum of 1% and a maximum of 100% or any intermediate percentage of modified nucleotides, 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, nucleic acids may contain modified pyrimidines, such as 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 modified uracil (e.g., 5-substituted uracil). The modified uracil may be replaced with a compound having a single unique structure or with multiple compounds having different structures (e.g., two, three, four, 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 modified cytosine (e.g., 5-substituted cytosine). The modified cytosine can be replaced with a compound having a single unique structure, or with multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0154] In some embodiments, the nucleic acid may contain any useful linker between nucleosides. Such linkers (including backbone modifications) that can be used in the compositions of this disclosure include, but are not limited to, the following: 3'-alkylphosphonates, 3'-aminophosphamides, olefin-containing backbones, aminoalkylphosphamides, aminoalkyl phosphate triesters, boron phosphates, —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2—, —CH2—NH—CH2—, chiral phosphates, chiral thiophosphates, methylacetyl and thiomethylacetyl backbones, and methylene (methylimino) compounds. Methylene methylacetyl and thiomethylacetyl backbones, methylene imino and methylene hydrazine backbones, morpholino linkages, —N(CH3)—CH2—CH2—, oligonucleotides with heteroatomic nucleoside linkages, hypophosphite, aminophosphate, dithiophosphate, thiophosphate nucleoside linkages, thiophosphate, triphosphate, PNA, siloxane backbones, aminosulfonate backbones, sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thioalkylphosphonates, thioalkyl phosphate triesters, and thioaminophosphates.

[0155] Modified nucleosides and nucleotides (e.g., building block molecules) that can be incorporated into nucleic acids (e.g., RNA or mRNA, as described herein) can be modified on the sugars of ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substituents at the 2' position include, but are not limited to, H, halogens, and optionally substituted C. 1-6 Alkyl; optionally substituted C 1-6 Alkyl group; optionally substituted C6-10 aryl group; optionally substituted C 3-8 cycloalkyl; optionally substituted C 3-8 Cycloalkoxy; optionally substituted C 6-10 aryloxy group; optionally substituted C 6-10 aryl-C1-6 alkoxy, optionally substituted C 1-12 (Heterocyclic)O group; sugar (e.g., ribose, pentose, or any sugar described herein); polyethylene glycol (PEG), —O(CH2CH2O) n CH2CH2OR, where R is H or an optionally substituted alkyl group, and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20); “locked” nucleic acid (LNA), wherein the 2'-hydroxyl group is connected to the 4'-carbon of the same ribose via a C1-6 alkylene or C1-6 heteroalkylene bridge, wherein exemplary bridges include methylene, propene, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acids.

[0156] Typically, RNA comprises a glycosylribose, which is a 5-membered ring with oxygen. Exemplary, non-limiting modified nucleotides include the substitution of oxygen in the ribose (e.g., with S, Se, or alkylene groups such as methylene or ethylene); the addition of a double bond (e.g., replacing the ribose with a cyclopentenyl or cyclohexenyl group); ring contraction of the ribose (e.g., forming a 4-membered ring of cyclobutane or oxetane); ring expansion of the ribose (e.g., forming a 6- or 7-membered ring with an additional carbon or heteroatom, such as anhydride hexitol, atroitol, mannitol, cyclohexyl, cyclohexenyl, and morpholino groups that also have an aminophosphate backbone); polycyclic forms (e.g., tricyclic; and "unlocked" forms, such as glycol nucleic acids (GNAs). (e.g., R-GNA or S-GNA, where the ribose is replaced by an ethylene glycol unit attached to a phosphodiester bond), threononucleotides (TNA, where the ribose is replaced by α-L-threofuranyl-(3'→2')), and peptide nucleotides (PNA, where the 2-amino-ethyl-glycine linker replaces the ribose and phosphodiester backbone). The sugar group may also contain one or more carbons having a stereochemical configuration opposite to the corresponding carbon in the ribose. Thus, polynucleotide molecules may include nucleotides containing, for example, arabinose as the sugar. Such sugar modifications are described, for example, in International Patent Publications WO2013052523 and WO2014093924, the contents of each of which are incorporated herein by reference in their entirety for this purpose.

[0157] The nucleic acids disclosed herein may include combinations of modifications to sugars, nucleobases, and / or nucleoside linkages. These combinations may include any one or more modifications described herein.

[0158] Pharmaceutical compositions and vaccine formulations

[0159] This article provides compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of cancer in humans (e.g., subjects or patients) and other mammals.

[0160] On the one hand, this article provides pharmaceutical compositions and vaccines comprising the nucleic acids described herein.

[0161] 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, one or more excipients may be used to formulate cancer vaccines to: (1) increase stability; (2) increase cell transfection; (3) allow sustained or delayed release (e.g., formulation from a depot); (4) alter biodistribution (e.g., targeting specific tissues or cell types); (5) increase the translation of encoded proteins in vivo; and / or (6) alter the release profile of encoded proteins (antigens) in vivo. In addition to conventional excipients (such as any and all solvents, dispersion media, diluents or other liquid media, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives), excipients may include, but are not limited to, lipids, liposomes, lipid nanoparticles, polymers, lipid complexes, core-shell nanoparticles, peptides, proteins, cells transfected with a cancer vaccine (e.g., for transplantation into subjects), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0162] In some embodiments, the vaccine composition comprises at least one additional active substance, such as a therapeutic active substance, a prophylactic active substance, or a combination of both. The vaccine composition may be sterile, pyrogen-free, or sterile and pyrogen-free. General considerations for the formulation and / or manufacture of pharmaceutical preparations (such as vaccine compositions) can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference in its entirety for this purpose).

[0163] In some embodiments, the cancer vaccine is administered to a human, a human patient, or a subject. For the purposes of this disclosure, the phrase "active ingredient" generally refers to a cancer vaccine or a nucleic acid contained therein, such as RNA (e.g., mRNA) encoding an antigenic polypeptide.

[0164] The formulations of the vaccine compositions described herein can be prepared by any method known in or subsequently developed in the field of pharmacology. Typically, such preparation methods include the following steps: associating an active ingredient (e.g., a nucleic acid, such as mRNA) with an excipient and / or one or more other auxiliary ingredients, and then, if desired and / or expected, separating, shaping, and / or packaging the product into desired single-dose or multi-dose units.

[0165] Formulations of any of the compositions disclosed herein may include one or more components other than those described above. For example, lipid compositions may include one or more permeability enhancer molecules, carbohydrates, polymers, surface modifiers (e.g., surfactants), or other components. For example, permeability enhancer molecules may be those described in U.S. Patent Application Publication No. 2005 / 0222064. Carbohydrates may include monosaccharides (e.g., glucose) and polysaccharides (e.g., glycogen and its derivatives and analogues).

[0166] Polymers may be included in or used for encapsulating or partially encapsulating the pharmaceutical compositions disclosed herein (e.g., pharmaceutical compositions in the form of lipid nanoparticles). Polymers may be biodegradable and / or biocompatible. Polymers may be selected from, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrene, polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyarylates.

[0167] In some embodiments, the compositions disclosed herein can be formulated into lipid nanoparticles (LNPs). Therefore, this disclosure also provides vaccines comprising (i) a lipid composition containing a delivery agent, and (ii) the nucleic acids described herein. In such vaccines, the lipid compositions disclosed herein may encapsulate the nucleic acids described herein.

[0168] Nanoparticle compositions are typically on the order of micrometers or smaller and may include lipid bilayers. These compositions include lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipid complexes. For example, a nanoparticle composition may be a liposome with a lipid bilayer having a diameter of 500 nm or less.

[0169] Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers may be functionalized and / or cross-linked with each other. The lipid bilayer may contain one or more ligands, proteins, or channels.

[0170] In one embodiment, the vaccine comprises ionizable lipids, structural lipids, phospholipids, and nucleic acids (e.g., mRNA) as described herein. In some embodiments, the LNP comprises ionizable lipids, PEG-modified lipids, phospholipids, and structural lipids.

[0171] The ratio of the lipid composition to the vaccine can be from about 10:1 to about 60:1 (wt / wt). In some embodiments, the ratio of the lipid composition to the nucleic acid can 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, or 32:1. The ratios are 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.

[0172] In some embodiments, the vaccine (e.g., a nucleic acid cancer vaccine) may be contained 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, about 5:1 to about 15:1, about 5:1 to about 20:1, about 5:1 to about 25:1, about 5:1 to about 30:1, about 5:1 to about 35:1, about 5:1 to about 40:1, about 5:1 to about 45:1, about 5:1 to about 50:1, about 5:1 to about 55:1, about 5:1 to about 60:1, about 5:1 to about 70:1, about 10:1 to about 15:1, about 10:1 to about 20:1, about 10:1 to about 25:1, about 10:1 to about 30:1, about 10:1 to about 35:1, about 10:1 to about 40:1, about 10:1 to about 45:1, about 10:1 to about 50:1, about 10:1 to about 55:1, about 10:1 to about 60:1, about 10:1 to about 70:1, about 15:1 to about 20:1, about 15:1 to about 25:1, about 15:1 to about 30:1, about 15:1 to about 35:1, about 15:1 to about 40:1, about 15:1 to about 45:1, about 15:1 to about 50:1, about 15:1 to about 55:1, about 15:1 to about 60:1, or about 15:1 to about 70:1.

[0173] In some implementations, the vaccine (e.g., a nucleic acid cancer vaccine) may be contained in lipid nanoparticles at a concentration of approximately 0.1 mg / ml to 2 mg / ml, such concentration 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.

[0174] As generally defined herein, the term "lipid" refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids can be naturally occurring or synthetic. Examples of lipid classes include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerides, glycerophospholipids, sphingolipids, glycolipids, polyketides, and isopentenyl lipids. In some cases, the amphiphilic nature of some lipids leads them to form liposomes, vesicles, or membranes in aqueous media.

[0175] In some embodiments, lipid nanoparticles (LNPs) may comprise ionizable lipids. As used herein, the term "ionizable lipid" has its common meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, ionizable lipids may be positively or negatively charged. Ionizable lipids may be positively charged, in which case they may be referred to as "cationic lipids." In some embodiments, ionizable lipid molecules may comprise amine groups and may be referred to as ionizable amino lipids. As used herein, a "charged moiety" is a chemical moiety carrying a form of electronic charge such as monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cation (i.e., positively charged). Examples of positively charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridine groups, guanidine groups, and imidazole groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively charged groups or their precursors include carboxylates, sulfonates, sulfates, phosphonates, phosphates, hydroxyl groups, etc. In some cases, the charge of the charged moiety can vary with environmental conditions; for example, changes in pH can alter the charge of the moiety and / or cause it to become charged or uncharged. Typically, the charge density of the molecule can be selected as desired. Ionizable lipids may also be compounds disclosed in International Publications: WO2017075531, WO2015199952, WO2013086354, or WO2013116126, or selected from the formula CLI-CLXXXXII of U.S. Patent No. 7,404,969; each of these documents is incorporated herein by reference in its entirety for this purpose.

[0176] It should be understood that the terms "charged" or "charged portion" do not refer to "partially negative charge" or "partially positive charge" on a molecule. The terms "partially negative charge" and "partially positive charge" are given their common meaning in the art. A "partially negative charge" can arise when a functional group contains a bond that becomes polarized, causing the electron density to be drawn toward the bond's atom, resulting in a partially negative charge on the atom. Those skilled in the art will generally recognize that bonds can become polarized in this way.

[0177] In some embodiments, the ionizable lipid is an ionizable amino lipid, which is 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 connected via a connector structure. In addition to these, the ionizable lipid may also be a lipid containing cyclic amine groups.

[0178] The vaccines disclosed herein are typically formulated as lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise 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.

[0179] In some embodiments, the lipid nanoparticles comprise 20-60% of ionizable amino lipids in a molar ratio. For example, the lipid nanoparticles may comprise 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% of ionizable amino lipids in a molar ratio. In some embodiments, the lipid nanoparticles comprise 20%, 30%, 40%, 50%, or 60% of ionizable amino lipids in a molar ratio.

[0180] In some embodiments, the lipid nanoparticles comprise a molar ratio of 5-25% non-cationic lipids. For example, the lipid nanoparticles 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 lipids. In some embodiments, the lipid nanoparticles comprise a molar ratio of 5%, 10%, 15%, 20%, or 25% non-cationic lipids.

[0181] In some embodiments, the lipid nanoparticles contain 25-55% sterols in a molar ratio. For example, the lipid nanoparticles may contain 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% sterols. In some embodiments, the lipid nanoparticles contain 25%, 30%, 35%, 40%, 45%, 50%, or 55% sterols in a molar ratio.

[0182] In some embodiments, the lipid nanoparticles comprise 0.5-15% of PEG-modified lipids in a molar ratio. For example, the lipid nanoparticles may comprise 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 a molar ratio. In some embodiments, the lipid nanoparticles comprise 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of PEG-modified lipids in a molar ratio.

[0183] In some embodiments, the lipids may be cleavable lipids, such as those described in PCT Publication No. WO2012170889, which is incorporated herein by reference in its entirety for this purpose. In one embodiment, the lipids may be synthesized by methods known in the art and / or as described in PCT Publication No. WO2013086354; the contents of which are incorporated herein by reference in their entirety for this purpose.

[0184] Nanoparticle compositions can be characterized using 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 nanoparticle compositions. Dynamic light scattering or potentiometry (e.g., potentiometric titration) can be used to measure the zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure various properties of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0185] The size of nanoparticles can help combat biological responses, such as, but not limited to, inflammation, or can enhance the biological effects of polynucleotides. As used herein, in the context of nanoparticle compositions, “size” or “average size” refers to the average diameter of the nanoparticle composition.

[0186] The relative amounts of the active ingredient (e.g., in a nucleic acid cancer vaccine), pharmaceutically acceptable excipients, and / or any additional ingredients in a vaccine composition can vary depending on the identity, body size, and / or condition of the subject being treated, and further on the route of administration of the composition. For example, the composition may contain 0.1% to 99% (w / w) of the active ingredient. For instance, the composition may contain 0.1% to 100%, such as 0.5% to 50%, 1% to 30%, 5% to 80%, or at least 80% (w / w) of the active ingredient.

[0187] In some implementations, this document provides a package containing a vaccine containing about 0.1 mg to about 1 mg of the nucleic acid (e.g., mRNA) described herein.

[0188] Treatment

[0189] In one respect, this article provides a method for inducing an immune response in a subject, which includes administering to the subject an effective amount of the pharmaceutical composition or vaccine described herein.

[0190] In one respect, this article provides methods for preventing or treating a disease or condition in a subject, which include administering to the subject an effective amount of the pharmaceutical composition or vaccine described herein.

[0191] The vaccines described herein (e.g., nucleic acid cancer vaccines) can be used as therapeutic or prophylactic agents in pharmaceuticals to prevent and / or treat cancer. In some embodiments, the cancer vaccines of this disclosure are used to provide prophylactic protection against cancer. Prophylactic protection against cancer can be achieved after administration of the cancer vaccines of this disclosure. The vaccine can be administered once, twice, three times, four times, or more, but administration of a single vaccine (optionally followed by a single booster dose) may be sufficient. It may also be desirable to administer the vaccine to an individual with cancer to achieve a therapeutic response. The dosage may need to be adjusted accordingly.

[0192] In some implementations, the disease or symptom is cancer. A non-limiting list of cancers that a cancer vaccine can treat is shown below. Peptide epitopes or antigens can be derived from any antigen of these cancers or tumors. Such epitopes can be called cancer antigens or tumor antigens. Cancer cells can differentially express cell surface molecules at different stages of tumor progression. For example, cancer cells can express cell surface antigens in a benign state but downregulate that particular cell surface antigen during metastasis. Therefore, it is envisioned that tumor antigens or cancer antigens can cover antigens generated at any stage of cancer progression. The methods of this disclosure can be adapted to accommodate these variations. For example, several different cancer vaccines can be generated for a particular patient. For example, a first vaccine can be used at the start of treatment. At subsequent time points, new cancer vaccines can be generated and administered to the patient to address the issue of different expressed antigens.

[0193] Cancer or tumor includes, but is not limited to, growths, malignant tumors, metastases, or any disease or condition characterized by uncontrolled cell growth that would make it to be considered cancerous. Cancer can be primary or metastatic. Specific cancers that can be treated according to this disclosure include, but are not limited to, those listed below (for a review of such conditions, see Fishman et al., 1985, Medicine, 2d Ed., JB Lippincott Co., Philadelphia). Cancers that can be treated using the methods and compositions described above include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; breast cancer (e.g., triple-negative breast cancer (TNBC)); cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematologic malignancies, including acute lymphoblastic leukemia and myeloid leukemia; multiple myeloma; AIDS-related leukemia and adult T-cell leukemia lymphoma; intraepithelial vegetations, including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphoma, including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; and oral cancer. This includes squamous cell carcinoma; ovarian cancer, including those originating 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, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including reproductive tumors such as seminoma, non-seminomatous tumors, and teratomas; tumors with high tumor mutation burden; choriocarcinoma; stromal tumors and germ cell tumors; thyroid cancer, including adenocarcinoma and medullary carcinoma; and kidney cancer, including adenocarcinoma and Wilms' tumor. In some embodiments, the cancer is any of the following: melanoma, bladder cancer, bladder urothelial carcinoma, hepatocellular carcinoma, HPV-negative head and neck squamous cell carcinoma (HNSCC), NSCLC, SCLC, MSI-high tumors, or TMB (tumor mutation burden)-high cancers.

[0194] In some implementations, the cancer is one in which MAGE-A4 expression is increased. In some implementations, the cancer is melanoma, lung cancer, esophageal cancer, head and neck cancer, gastric cancer, bladder urothelial carcinoma, hepatocellular carcinoma, triple-negative breast cancer (TNBC), or ovarian cancer.

[0195] Once a vaccine (e.g., a nucleic acid cancer vaccine) is synthesized, it is administered to patients. In some implementations, the vaccine is administered according to a schedule for up to two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, one and a half years, two years, three years, or four years. The schedule can be the same or varied. In some implementations, the schedule is weekly for the first three weeks, then monthly thereafter. The schedule can be determined or modified by someone skilled in the art (e.g., a physician) based on criteria for the individual patient or subject (e.g., weight, age, cancer type, etc.).

[0196] Vaccines can be administered via any route. In some implementations, vaccines are administered via intradermal, intramuscular, intravascular, intratumoral, and / or subcutaneous routes.

[0197] In some implementations, the nucleic acid cancer vaccine may also be administered in conjunction with one or more additional therapeutic agents. In some implementations, the one or more additional therapeutic agents are anticancer therapeutic agents. The nucleic acid cancer vaccine and other therapeutic agents may be administered simultaneously or sequentially. When other therapeutic agents are administered simultaneously, they may be administered in the same or separate formulations, but at the same time. When the administration of other therapeutic agents and the nucleic acid cancer vaccine is time-separated, the other therapeutic agents are administered sequentially to each other and sequentially with the nucleic acid cancer vaccine. The time interval between the administration of these compounds may be minutes, or it may be longer, such as hours, days, weeks, or months. Other therapeutic agents include, but are not limited to, anticancer therapeutic agents, adjuvants, cytokines, antibodies, antigens, etc.

[0198] At any point in treatment, patients can be examined to determine if the mutations in the vaccine are still appropriate. Based on this analysis, the vaccine can be adjusted or reconfigured to include one or more different mutations or to remove one or more mutations.

[0199] In some implementations, a cancer vaccine containing a nucleic acid (e.g., RNA polynucleotide) as described herein may be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotide may be translated in vivo to produce an antigenic polypeptide.

[0200] Vaccines (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) can be induced to translate polypeptides (e.g., antigens or immunogens) in cells, tissues, or organisms. In exemplary embodiments, such translation occurs in vivo, although embodiments in which such translation occurs in vitro, in cultures, or outside the body are contemplated. In exemplary embodiments, cells, tissues, or organisms are contacted with an effective amount of a composition containing a cancer vaccine, which contains a polynucleotide having at least one translatable region encoding an antigenic polypeptide.

[0201] An “effective amount” of a cancer RNA vaccine can be provided, at least in part, based on the target tissue, target cell type, method of administration, physical characteristics of the polynucleotide (e.g., the size and extent of the modified nucleoside), other components of the cancer vaccine, and other determinants. Typically, an effective amount of a cancer vaccine composition provides an induced or enhanced immune response as a function of antigen production in cells, preferably more efficiently than compositions containing corresponding unmodified polynucleotides encoding the same antigen or peptide antigen. Increased antigen production can be demonstrated by increased cell transfection (the percentage of cells transfected with the cancer vaccine), increased protein translation from the polynucleotide, reduced nucleic acid degradation (as demonstrated, for example, by the increased duration of protein translation from the modified polynucleotide), or altered antigen-specific immune responses in host cells.

[0202] Vaccines (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) can be administered prophylactically or therapeutically to healthy individuals or during active cancer, either in the early stages of cancer or after the onset of symptoms, as part of an active immunization program. In some embodiments, the amount of the RNA vaccine of this disclosure delivered to cells, tissues, or subjects can be an amount effective for immunoprophylaxis.

[0203] In some implementations, the vaccine may be administered prophylactically approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some implementations, the vaccine may be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. In some implementations, the vaccine may be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more. The vaccine may be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months or more. In some implementations, the subject has not been diagnosed with cancer or does not have detectable levels of cancer-related biomarkers.

[0204] In some implementations, the vaccine may be administered therapeutically approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some implementations, the vaccine may be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. In some implementations, the vaccine may be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more. The vaccine may be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months or more. In some implementations, the subject has been diagnosed with cancer or has detectable levels of cancer-related markers.

[0205] In some implementations, the vaccine is administered to the subject once. In some implementations, the vaccine is administered to the subject three times.

[0206] Vaccines (e.g., nucleic acid cancer vaccines such as mRNA cancer vaccines) can be administered together with other prophylactic or therapeutic compounds besides checkpoint inhibitors. As a non-limiting example, the prophylactic or therapeutic compound can be an immune enhancer or booster. As used herein, when referring to a composition such as a vaccine, the term "booster" means the additional administration of a prophylactic (vaccine) composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time between the initial application of the preventative composition and the application of the stimulant can 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 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 99 years or more. In an exemplary embodiment, the time between the initial application of the preventive composition and the application of the reinforcing agent may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

[0207] In some embodiments, the nucleic acid (e.g., mRNA) vaccine composition can be delivered at doses 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. Administer doses of mg / kg subject weight / day, once or more daily, once or more weekly, once or more monthly, etc., to achieve the desired therapeutic, diagnostic, preventative, or imaging effect (see, for example, the unit dose range described in International Publication No. WO2013078199, which is incorporated herein by reference in its entirety). In some implementations, nucleic acid (e.g., mRNA) vaccines are administered at doses sufficient to deliver approximately 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, etc. Nucleic acid (e.g., mRNA) is administered at dose levels sufficient to deliver between 10 μg and 400 μg to the subject. In some embodiments, the nucleic acid (e.g., mRNA) vaccine is administered at dose levels sufficient to deliver between 10 μg and 400 μg of mRNA vaccine to the subject.In some implementations, nucleic acid (e.g., mRNA) vaccines are administered at a dose level sufficient to deliver 10 mg of nucleic acid (e.g., mRNA) to the subject.

[0208] Vaccines (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) can be used in a variety of contexts depending on the severity of the cancer or the extent or level of unmet medical needs. As a non-limiting example, cancer vaccines can be used to treat cancer at any stage.

[0209] In some embodiments, vaccines (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) and / or checkpoint inhibitors can be used to treat PD-L1 positive tumors. In other embodiments, cancer vaccines and / or checkpoint inhibitors can 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 combination of the present invention in the treatment of PD-1 “negative” tumors is contemplated. Mechanistically, tumors exhibit an adaptive component to PD-L1 expression; that is, a tumor may initially be PD-L1 negative but upregulate PD-L1 expression in response to IFN-γ secreted by invasive tumor lymphocytes. This has been clinically translated, resulting in higher response rates in PD-L1 negative tumors to a combination of PD-1 and CTLA-4 blockade than to a single-agent PD-1 inhibitor in both cutaneous melanoma and lung cancer. An aspect of the present invention relates to the use of a combination of a personalized cancer vaccine and a PD-1 inhibitor to induce PD-L1 expression in PD-L1-low tumors.

[0210] In some embodiments, vaccines (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) and / or checkpoint inhibitors can be used to treat tumors with high tumor mutational burden. Therefore, in some embodiments, TMB can be tested in a cohort of subjects, and subjects with TMB values ​​exceeding a threshold level can be treated with the combination therapy of the present invention.

[0211] This document provides pharmaceutical compositions comprising cancer vaccine and RNA vaccine compositions and / or complexes, optionally in combination with one or more pharmaceutically acceptable excipients. Vaccines (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) may be formulated or administered alone, or in combination with one or more other components described herein.

[0212] In other embodiments, the vaccines described herein (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) can be combined with any other therapies available for treating a patient. For example, a patient can be treated with a cancer vaccine and an anticancer agent. Therefore, in one embodiment, the methods of this disclosure can be used in combination with one or more cancer therapeutic agents, such as with an anticancer agent, a conventional cancer vaccine, chemotherapy, radiotherapy, etc. (e.g., simultaneously or as part of an overall treatment procedure). Cancer treatment parameters that can vary include, but are not limited to, dosage, timing of administration, or duration of administration or therapy; and cancer treatment can vary in dosage, timing, or duration. Another treatment for cancer is surgery, which can be used alone or in combination with any prior treatment. Any agent or therapy known to be useful for the prevention or treatment of cancer, or that has been used or is currently used for the prevention or treatment of cancer (e.g., conventional cancer vaccines, chemotherapy, radiotherapy, surgery, hormone therapy, and / or biotherapy / immunotherapy) can be used in combination with the compositions of this disclosure according to the disclosure described herein. A person skilled in the medical field can determine the appropriate treatment for a subject.

[0213] Examples of such agents (i.e., anticancer agents) include, but are not limited to, DNA interacting agents, including, but not limited to, alkylating agents (e.g., nitrogen mustard, such as chlorambucil, cyclophosphamide, ifosfamide, dichloromethyldiethylamine, melphalan, uracil mustard; aziridine, such as thiotepa; methanesulfonates, such as busulfan; nitrosoureas, such as carmustine, lomustine, streptozotocin; platinum complexes, such as cisplatin, carboplatin; bioreducing alkylating agents, such as mitomycin and procarbazine, dacarbazine, and hexamethylmelamine); DNA strand breaking agents, such as bleomycin; intercalating topoisomerase II inhibitors, such as intercalating agents, such as acridine, daunorubicin, and daunorubicin). Rubibrine, idarubicin, mitoxantrone, and non-intercalating agents such as etoposide and teniposide; non-intercalating topoisomerase II inhibitors such as etoposide and teniposide; DNA minor groove binding agents such as procainamide; antimetabolites, including but not limited to folic acid antagonists such as methotrexate and trimethoprim; pyrimidine antagonists such as fluorouracil, fludeoxyuridine, CB3717, azacitidine, and fluorouridine; purine antagonists such as mercaptopurine, 6-thioguanine, and pentostatin; glycosyl-modified analogs such as cytarabine and fludarabine; and ribonucleotide reductase inhibitors such as hydroxyurea; microtubule interactors, including but not limited to colchicine, vincristine, and cyclophosphamide. Cladosine (both are alkaloids), as well as paclitaxel and cyclophosphamide; hormones, including but not limited to estrogens, conjugated estrogens, ethinyl estradiol and diethylstilbestrol, chlorestradiol and diethylstilbestrol; progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; and androgens, such as testosterone, testosterone propionate; fluoxytestosterone, methyltestosterone; corticosteroids, such as prednisone, dexamethasone, methylprednisolone and prednisolone; luteinizing hormone-releasing hormone agents or gonadotropin-releasing hormone antagonists, such as leuprorelin acetate and goserelin acetate; anti-hormone antigens, including but not limited to anti-estrogens such as tamoxifen, and anti-androgens such as flutamide; And anti-adrenergic agents, such as mitotane and aminoglutethimide; cytokines, including but not limited to IL-1α, IL-1β, 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 uterine globin (US Patent No. 5,696,092);Anti-angiogenic agents, including but not limited to VEGF inhibitors (e.g., other neutralizing antibodies), soluble receptor constructs, tyrosine kinase inhibitors, antisense strategies, RNA aptamers and ribozymes targeting VEGF or VEGF receptors, immunotoxins and coagulation ligands, tumor vaccines, and antibodies.

[0214] Specific examples of anticancer agents that may be used according to the methods of this disclosure include, but are not limited to: acivitine; ararubicin; acodazole hydrochloride; acroline; adorine; interleukin; hexamethylmelamine; ambroxol; amyronone acetate; aminoglutethimide; acridine; anastrozole; atrazolyl; asparaginase; triamcinolone; azacitidine; atepa; azomycin; palmastat; benzothioprine; bicalutamide; bisaminoglycan hydrochloride; dimethylsulfonic acid. Nefenadine; Bleomycin sulfate; Buquina sodium; Brompirimidine; Busulfan; Actinomycin C; Calotestosterone; Carbetamide; Carbetin; Carboplatin; Carmustine; Carrubicin hydrochloride; Carzelazine; Sildenafil; Chlorisac; Sirloin; Cisplatin; Cladribine; Clinapordine mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Daunorubicin hydrochloride; Decitabine; Dextromethorphan; Dexamethasone; Dezaguanine sulfonate; Diazinon; Docetaxel; Doxorubicin; Doxorubicin hydrochloride; Droloxifen; Droloxifen citrate; Drotahistamine propionate; Dazomycin; Edatraxa; Eflunomide hydrochloride; Exalucin; Enloplatin; Enpromethazine; Epiloperidol; Epirubicin hydrochloride; Ibuproazole; Exorubicin hydrochloride; Estrostimine; Estrostimine sodium phosphate; Ethanidazole; Etoposide; Etoposide phosphate Etoprine; Fazodazole hydrochloride; Fazalabin; Feniveryl Aamine; Fluorouracil; Fludarabine phosphate; Fluorouracil; Flucitabine; Phosphorione; Fostracin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Imofocin; Interleukin II (including recombinant interleukin II or rIL2), Interferon α-2a; Interferon α-2b; Interferon α-n1; Interferon α-n3; Interferon β-Ia; Interferon γ-I b; Isopropylplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprorelin acetate; Riazol hydrochloride; Lometraxo sodium; Lomustine; Loxoanthraquinone hydrochloride; Masrophenone; Maytansine; Nitrogen mustard hydrochloride; Medroxyprogesterone acetate; Meropenem; Minoril; Mercaptopurine; Methotrexate; Methotrexate sodium; Chlorpheniramine; Meutipate; Mitocarcin; Mitocarcin; Mitocarcin; Mitocarcin; Mitocarcin; Mitocarcin; Mitocarcin; Mitocarcin; Mitocarcin; Mitocarcin Totan; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogamycin; Omaplatin; Oxysoxuron; Paclitaxel; Pegaspargase; Perymycin; Pendimethalin; Pyromycin Sulfate; Pephosphatamide; Piperabromide; Piperabromide; Pirroantrone Hydrochloride; Procaycin; Promethene; Porphyrom sodium; Pofibromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazosulfan; Lipoadenosine; Lotamethasone; Safungo; Safungosulfan Hydrochloride; Semustine; Citricazone; Sparfosate Sodium; Sparsomycin; Spiramustine; Spiramustine; Spiramustine; Streptomycin; Streptozotocin; Sulfonamide; Talimycin; Ticogallan Sodium; Tegafur; Tiloantrone Hydrochloride; Temopofen;Teniposide; Tiroxicon; Testrolide; Thiomipurine; Thioguanine; Thiotepa; Thiazofuranoline; Tirazamine; Toremifene Citrate; Tritoprolone Acetate; Tricerebrolysine Phosphate; Trimethotraxate; Trimethotraxate Glucuronate; Triptorelin; Tobaccochloride Hydrochloride; Uracil Nitrogen Mustard; Uretoprolone; Vaportide; Vertepofen; Vincristine Sulfate; Vincristine Sulfate; Vinpicrocin Sulfate; Vinpocetine Sulfate; Vinpocetine Sulfate; Vinpocetine Sulfate; Vinpocetine Sulfate; Vinpocetine Sulfate; Vinpocetine Sulfate; Vorticillium; Zonipram; Netsistatin; and Zorubicin Hydrochloride.

[0215] Other anticancer drugs that can be used with the compositions and methods of the present invention include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; angiogenesis inhibitors; anti-dorsal morphogenetic protein-1; ara-CDP-DL-PTBA; BCR / ABL antagonists; CaRest M3; CARN 700; casein kinase inhibitors (ICOS); clotrimazole; cyclophosphamide A; cyclophosphamide B; cobustatin A4; crambescidin 816; cantharidin 8; curacin A; dehydrodidemnin B; mesotheliin B; dihydro-5-azacytidine; dihydropaclitaxel; pyruvicin SA; kahalalide F; spirotinic acid triacetate; leuprolide + estrogen + progesterone; lissoclinamide 7; monophosphoryl lipid A + mycobacterial cell wall SK; N-acetyl-binarin; N-substituted benzamide; O6-benzylguanine; placetin A; placetin B; platinum complex; platinum compound; platinum-triamine complex; rhenium Re 186 etidronate; RH retinoic acid; rubiginone B1; SarCNU; muscle phytosterol A; shamoxetine; senescence-related inhibitor 1; spicamycin D; tamustine; 5-fluorouracil; thrombopoietin; thymotrione; thyroid-stimulating hormone; variolin B; thalidomide; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; zanoterone; zebuline; and benzylvin C.

[0216] This disclosure also covers the combined administration of compositions containing vaccines (e.g., nucleic acid cancer vaccines, such as mRNA cancer vaccines) with radiotherapy, including the use of X-rays, gamma rays, and other radiation sources to destroy cancer cells. In some embodiments, the radiotherapy is administered as external beam radiation or teletherapy, wherein the radiation is directed from a distant source. In other embodiments, the radiotherapy is administered as internal therapy or brachytherapy, wherein the radiation source is placed close to the cancer cells or tumor mass to be positioned inside the body.

[0217] In some implementations, an appropriate anticancer regimen is selected (e.g., by a physician) based on the type of cancer. For example, a combination containing a cancer vaccine may be administered to a patient with ovarian cancer in a prophylactic or therapeutically effective amount, in combination with one or more other agents suitable for the treatment of ovarian cancer, including but not limited to intraperitoneal radiation therapy such as P32 therapy, whole abdominal and pelvic radiation therapy, combinations of cisplatin (Taxol) or docetaxel and cisplatin or carboplatin, combinations of cyclophosphamide and cisplatin, combinations of cyclophosphamide and carboplatin, combinations of 5-FU and folate, etoposide, liposomal doxorubicin, gemcitabine, or topotecan. Cancer therapies and their dosages, routes of administration, and recommended uses are known in the art and have been described in literature such as Physician's Desk Reference (56th edition, 2002).

[0218] In some embodiments, the cancer therapeutic agent is a cytokine. In still other embodiments, the cancer therapeutic agent is a vaccine comprising a population-based tumor-specific antigen.

[0219] In some implementations, the vaccine described herein elicits an antigen-specific immune response. In some implementations, the immune response is a T-cell-mediated immune response.

[0220] Antigen-specific immune responses can be detected and measured in any suitable site, location, or organ of the subject. In some embodiments, antigen-specific immune responses are detected and measured in the subject's spleen. In some embodiments, antigen-specific immune responses are detected and measured in the subject's lymph nodes.

[0221] In some implementations, the antigen-specific immune response is measured by the level of IFN-γ in the subject. In some implementations, the level of IFN-γ is increased to approximately 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 times.

[0222] In some implementations, the antigen-specific immune response lasts for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, or 21 days or longer. In some implementations, the antigen-specific immune response lasts for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or longer.

[0223] In some implementations, administration of the vaccine described herein does not cause significant weight loss in subjects.

[0224] In some embodiments, administration of the vaccine described herein inhibits tumor growth in subjects. In some embodiments, administration of the vaccine described herein reduces tumor volume in subjects. In some embodiments, tumor volume is reduced by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, tumor volume is reduced by approximately 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 times or more.

[0225] Methods of producing vaccines

[0226] On one hand, this article provides a method for preparing a vaccine, which includes mixing the nucleic acid described herein with a lipid nanoparticle formulation to produce a vaccine.

[0227] The vaccine disclosed herein may contain at least one nucleic acid (e.g., RNA polynucleotides, such as mRNA (messenger RNA) or mmRNA (modified mRNA)). For example, the mRNA is transcribed in vitro from template DNA (referred to as the "in vitro transcription template"). In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region containing an open reading frame and encoding a 3' UTR and multiple A tails. In some embodiments, the in vitro transcription template encodes a capped enhancement sequence. The specific nucleic acid sequence composition and length of the in vitro transcription template will depend on the mRNA encoded by the template.

[0228] In some implementations, the nucleic acid comprises about 15 to about 3,000 nucleotides. For example, polynucleotides 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, approximately 50 to 300, approximately 50 to 400, approximately 50 to 500, approximately 50 to 600, approximately 50 to 700, approximately 50 to 800, approximately 50 to 900, approximately 50 to 1000, approximately 50 to 1200, approximately 50 to 1400, approximately 50 to 1500, approximately 50 to 1800, approximately 50 to 2000, approximately 50 to 2500, approximately 50 to 3000, approximately 100 to 200, approximately 100 to 300, approximately 100 to 400, approximately 100 to 500 Approximately 100 to 600, approximately 100 to 700, approximately 100 to 800, approximately 100 to 900, approximately 100 to 1000, approximately 100 to 1200, approximately 100 to 1400, approximately 100 to 1500, approximately 100 to 1800, approximately 100 to 2000, approximately 100 to 2500, approximately 100 to 3000, approximately 200 to 300, approximately 200 to 400, approximately 200 to 500, approximately 200 to 600, approximately 200 to 700, approximately 200 to 800 Approximately 200 to 900, approximately 200 to 1000, approximately 200 to 1500, approximately 200 to 3000, approximately 500 to 1000, approximately 500 to 1500, approximately 500 to 2000, approximately 500 to 2500, approximately 500 to 3000, approximately 1000 to 1500, approximately 1000 to 2000, approximately 1000 to 2500, approximately 1000 to 3000, approximately 1500 to 3000, approximately 2500 to 3000 or approximately 2000 to 3000 nucleotides.

[0229] In other respects, this disclosure relates to methods for preparing or generating nucleic acid vaccines (e.g., mRNA cancer vaccines) via in vitro transcription (IVT) methods.

[0230] In one aspect, this disclosure relates to a method for preparing a vaccine, which includes mixing the nucleic acid described herein with a lipid nanoparticle formulation to produce a vaccine.

[0231] In vitro transcription (IVT) allows template-guided synthesis of RNA molecules of virtually any sequence. In some embodiments, the RNA molecules described herein (e.g., mRNA) can be transcribed from corresponding DNA molecules described herein (e.g., DNA molecules encoding the same amino acid sequence). In some embodiments, the DNA molecules described herein are in a vector. In some embodiments, the vector is a plasmid. RNA molecules that can be synthesized using IVT methods range in size from short oligonucleotides to long nucleic acid polymers of several thousand bases. IVT methods allow the synthesis of large quantities of RNA transcripts (e.g., micrograms to milligrams). 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 DC: ASM Press, 2007. 262-299, each of which is incorporated herein by reference for this purpose. Typically, IVT utilizes a DNA template with a promoter sequence upstream of the sequence of interest. The most common promoter sequences are of phage origin (e.g., T7, T3, or SP6 promoter sequences), but many other promoter sequences are acceptable, including those designed de novo. Transcription of the DNA template is usually best achieved using an RNA polymerase corresponding to a specific phage promoter sequence. Exemplary RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase. IVT typically begins at dsDNA, but can occur on a single strand.

[0232] It should be understood that the nucleic acid vaccines of this disclosure (e.g., mRNA cancer vaccines), such as mRNA encoding cancer antigens, can be prepared using any suitable synthetic method. For example, in some embodiments, the mRNA vaccines of this disclosure are prepared using IVT from a single bottom-strand DNA serving as a template and a complementary oligonucleotide acting as a promoter. The single bottom-strand DNA can serve as a DNA template for in vitro transcription of RNA and can be obtained from, for example, plasmids, PCR products, or chemical synthesis. In some embodiments, the single bottom-strand DNA is linearized from a circular template. The single bottom-strand DNA template typically includes a promoter sequence, such as a bacteriophage promoter sequence, to facilitate IVT. Methods for preparing RNA using a single bottom-strand DNA and a top-strand promoter complementary oligonucleotide are known in the art. Exemplary methods include, but are not limited to, annealing the DNA bottom-strand template with a top-strand promoter complementary oligonucleotide (e.g., T7 promoter complementary oligonucleotide, T3 promoter complementary oligonucleotide, or SP6 promoter complementary oligonucleotide) and then performing IVT using an RNA polymerase corresponding to the promoter sequence, such as T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase.

[0233] The IVT method can also be performed using a double-stranded DNA template. For example, in some embodiments, the double-stranded DNA template is prepared by extending complementary oligonucleotides using strand extension techniques available in the art to generate a complementary DNA strand. In some embodiments, a single bottom-strand DNA template containing a promoter sequence and a 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 both the bottom-strand promoter sequence and 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, for example, 3 to 10 cycles. In some embodiments, the double-stranded DNA template is synthesized, in whole or in part, by chemical synthesis methods. The double-stranded DNA template can be transcribed in vitro as described herein.

[0234] On the other hand, the nucleic acid cancer vaccine of this disclosure, containing mRNA encoding, for example, a cancer antigen (e.g., MAGE-A4), can be prepared using two DNA strands complementary in overlapping portions of their sequences, leaving single-stranded overhangs (i.e., sticky ends) when the complementary portions are annealed. These single-stranded overhangs can be extended to form double-stranded DNA by using another strand as a template. In some cases, this primer extension method can allow for the incorporation of a larger ORF into the template DNA sequence, for example, compared to the size incorporated into the template DNA sequence obtained by top-strand DNA synthesis methods. In the primer extension method, a portion of the 3F end of the first strand (in the 5'-3' orientation) is complementary to a portion of the 3' end of the second strand (in the 3'-5' orientation). In some such embodiments, a single first-strand DNA may contain a promoter sequence (e.g., T7, T3, or SP6), optionally a 5'-UTR, and some or all of the ORF (e.g., a portion of the 5' end of the ORF). In some embodiments, a single second-stranded DNA may contain some or all of the complementary sequence of the ORF (e.g., a portion complementary to the 3' end of the ORF), and optionally a 3'-UTR, a termination sequence, and / or multiple (A) tails. Methods for preparing 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, for example, 3 to 10 cycles. Such double-stranded DNA can be transcribed in vitro as described herein.

[0235] On the other hand, the nucleic acid vaccines of this disclosure containing mRNA encoding, for example, a cancer antigen (e.g., MAGE-A4) can be prepared using synthetic double-stranded linear DNA molecules (such as gBlocks® (Integrated DNA Technologies, Coralville, Iowa)) as double-stranded DNA templates. An advantage of such synthetic double-stranded linear DNA molecules is that they provide a longer template from which mRNA can be generated. For example, gBlocks® can be 45-1000 nucleotides (e.g., 125-750 nucleotides). In some embodiments, the synthetic double-stranded linear DNA template comprises a full-length 5'-UTR, a full-length 3'-UTR, or both. The full-length 5'-UTR can be up to 100 nucleotides long, for example, about 40-60 nucleotides. The full-length 3'-UTR can be up to 300 nucleotides long, for example, about 100-150 nucleotides.

[0236] To facilitate the generation of longer constructs, two or more double-stranded linear DNA molecules and / or gene fragments with designed overlapping sequences on their 3' strands can be assembled using methods known in the art. For example, the Gibson Assembly™ Method (Synthetic Genomics, Inc., La Jolla, Calif.) can be performed using a mesophilic exonuclease that cleaves the bases from the 5' end of the double-stranded DNA fragment, followed by annealing of the newly formed complementary single-stranded 3' end, polymerase-dependent extension to fill any single-strand gaps, and finally covalent ligation of the DNA segments by a DNA ligase.

[0237] On the other hand, the nucleic acid cancer vaccine of this disclosure, comprising mRNA encoding, for example, a cancer antigen (e.g., MAGE-A4), can be prepared using the chemical synthesis of RNA. For example, the method involves annealing a first polynucleotide comprising an open reading frame encoding a polypeptide and a second polynucleotide comprising a 5'-UTR to a complementary polynucleotide conjugated to a solid support. The 3' end of the second polynucleotide is then ligated to the 5' end 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' end of a third polynucleotide comprising a 3'-UTR is then ligated to the 3' end 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 mRNA encoding the polypeptide of interest. In some embodiments, the mRNA is between 30 and 1000 nucleotides.

[0238] In some embodiments, the template DNA encoding the nucleic acid (e.g., mRNA) cancer vaccine of this disclosure includes an open reading frame (ORF) encoding one or more peptide epitopes. In some embodiments, the template DNA contains an ORF of up to 1000 nucleotides, such as about 10-350, 30-300, or about 50-250 nucleotides. In some embodiments, the template DNA contains an ORF of about 150 nucleotides. In some embodiments, the template DNA contains an ORF of about 200 nucleotides.

[0239] In some embodiments, IVT transcripts are purified from components of the IVT reaction mixture after the reaction has occurred. For example, the crude IVT mixture can be treated with an RNase-free DNase to digest the original template. Nucleic acids (such as mRNA) can be purified using methods known in the art, including but not limited to precipitation with organic solvents or column-based purification methods. Commercial kits are available for purifying RNA, such as MEGACLEAR. TMKits (Ambion, Austin, Tex.). Nucleic acids (e.g., mRNA) can be quantified using methods known in the art, including but not limited to commercially available instruments such as NanoDrop. Purified nucleic acids (e.g., mRNA) can be analyzed, for example, by agarose gel electrophoresis to confirm that the nucleic acids are of appropriate size and / or to confirm that the nucleic acids have not been degraded.

[0240] Methods for preparing vaccines (e.g., mRNA vaccines) by in vitro transcription of nucleic acids are described in, for example, US Patent Publications US20220125899A1, US20190351040A1 and US20180318409A1, the entire contents of which are incorporated herein by reference.

[0241] In some embodiments, in vitro transcribed nucleic acids (e.g., mRNA) are mixed with a mixture of lipids to produce the vaccine described herein. In some embodiments, the lipid mixture comprises ionizable cationic lipids, phospholipids, cholesterol, and PEG-conjugated lipids. In some embodiments, the lipid mixture is dissolved in ethanol. In some embodiments, prior to preparation, the nucleic acid (e.g., mRNA) is dissolved in a sterile, RNase-free buffer (e.g., sodium acetate buffer).

[0242] Lipid nanoparticles can be produced using any method known in the art and described herein. In some embodiments, LNPs are prepared by rapidly mixing an ethanol phase containing a lipid mixture with an aqueous phase containing RNA at 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) using a microfluidic mixing device. In some embodiments, LNPs are prepared by rapidly mixing an ethanol phase containing a lipid mixture with an aqueous phase containing RNA at a ratio of one volume of ethanol-soluble lipid mixture to three volumes of RNA using a microfluidic mixing device. In some embodiments, the RNA-containing lipid nanoparticles are subjected to dialysis and / or ultrafiltration. Any suitable method for preparing LNPs known in the art can be used in the methods described herein. Example

[0243] The invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0244] Example 1: Design and immunogenicity of MAGE-A4 mRNA-LNP vaccine

[0245] method

[0246] Antigen design:

[0247] The Igκ light chain signal peptide (SP) sequence was fused to the N-terminus of the full-length MAGE-A4 antigen, and the human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) was fused to the C-terminus of the target antigen.

[0248] Using a proprietary codon optimization algorithm, different nucleotide sequences encoding target antigens (with SP and MITD) can be designed, which can improve mRNA stability and translation efficiency, and further improve protein expression.

[0249] plasmid design

[0250] The nucleotide sequence of the full-length antigen was cloned into the pUC57-kan plasmid backbone (Genscript), which contained optimized 5'-untranslated region (UTR), 3'-UTR, and multiple A tails. The plasmid was then extracted after amplification in *E. coli*.

[0251] mRNA preparation

[0252] The plasmid was linearized using BspQI restriction enzyme (New England Biolabs, R0712L). After purification using the QIAquick Gel Extraction Kit (QIAGEN, catalog number: 278704), the linearized plasmid DNA was transcribed in vitro into mRNA containing modified mRNA molecules (N1-methyl-pseudouridine, Hongene, catalog number: R5-064) and m7G(5')ppp(5')(2'-OMeA)pG (Hongene, catalog number: ON-134) according to the manufacturer's instructions. The transcribed mRNA was then purified using the Monarch® RNA Cleanup Kit (NEW ENGLAND BioLabs, catalog number: T2050L) according to the manufacturer's instructions. The purified mRNA was then stored at -80°C.

[0253] mRNA-LNP preparation

[0254] A lipid mixture containing ionizable cationic lipids, phospholipids, cholesterol, and PEG-conjugated lipids was dissolved in ethanol. Prior to preparation, mRNA was dissolved in sterile, RNase-free 10 mM sodium acetate buffer (pH 4.0). Lipid nanoparticles were prepared by rapidly mixing the ethanol-containing lipid mixture with the RNA-containing aqueous phase at a total flow rate of 20 mL / min using a microfluidic mixing apparatus (one volume of lipid mixture in ethanol and three volumes of RNA in 10 mM sodium acetate buffer), followed by direct mixing and dilution with 10 volumes of 1x phosphate-buffered saline (PBS). The RNA-containing lipid nanoparticles were dialyzed and ultrafiltered using an Amicon Ultra-15 Millipore centrifuge filter or tangential flow filtration. The final lipid nanoparticle product was stored in 10% sucrose (w / v), 5 mM Tris buffer (pH 8.0), and filtered aseptically through a 0.22 μm filter. The mRNA-LNP was stored at -80°C until use.

[0255] In vitro protein expression

[0256] The target mRNA was mixed with lipofectamine and transfected into the HEK293T cell line. Cell lysates were collected and target antigen expression was measured using Western blotting.

[0257] For in vitro expression assays, HEK293T cells were harvested by trypsin-EDTA digestion and seeded into 24-well plates (300,000 cells / well). 1 μg mRNA was mixed with 1 μL Lipofectamine (Invitrogen, LMRNA015) and incubated at room temperature for 15 min. The mixture was then transfected into HEK293T cells. After 24 hours of incubation, cells were washed and cell lysates were prepared using RIPA (Pierce, catalog #89901) with a mixture of protease and phosphatase inhibitors (Thermo Scientific, catalog #78441). After incubation on ice, the tubes were rotated at >12,000 rpm and the supernatant was collected. Total protein concentration was quantified using a BCA protein assay kit (Pierce, catalog #23225). 10 μg protein was loaded onto SDS-PAGE, and MAGE-A4 protein expression levels were measured by Western blotting.

[0258] Immunogenicity in mice

[0259] The target encapsulated mRNA-LNP was injected intramuscularly (IM) into two sites of the gastrocnemius muscle in female C57BL / 6 mice (Shanghai BK). The mRNA dose was 10 μg / mouse. The dosing schedule was indicated for each experiment.

[0260] Mice were euthanized at different time points after immunization, and spleens, as well as inguinal and popliteal lymph nodes, were collected for IFN-γ enzyme-linked immunospot (ELISPOT) assays.

[0261] ELISPOT measurement

[0262] Antigen-specific immunogenicity was measured using an ELISPOT assay with stimulation from an in vitro target peptide library. Briefly, mouse spleens or lymph nodes were ground using a sterile syringe plunger and filtered through a 70 μm cell filter to separate single-cell suspensions. Single spleen cells or lymphocytes were seeded into IFN-γ ELISPOT plates (Mabtech, catalog number #3321-4AST-10) and incubated with the target peptide library for 40–44 hours. IFN-γ spots were then determined according to the manufacturer's instructions.

[0263] Research on the generation of transgenic cell lines and their anti-tumor efficacy

[0264] The full-length wild-type antigen was cloned into the genomic DNA of the mouse colon cancer MC38 cell line using the CRISPR-Cas9 method. In short, the full-length wild-type human MAGE-A4 gene was inserted into a cloning vector containing a puromycin resistance gene. Then, the ROSA26-sgRNA plasmid expressing sgRNA targeting the ROSA26 genomic locus, along with the Cas9 protein, was co-electroplated into MC38 cells with the MAGE-A4 cloning vector. Transgenic MC38 cells expressing human MAGE-A4 were treated with puromycin to enrich the cell line, and single-cell clones were selected using limiting dilution. The MAGE-A4-MC38 transgenic cell clones were confirmed by qPCR sequencing and protein expression, and passed the mycoplasma test, before in vivo studies were conducted.

[0265] For in vivo efficacy studies, on day 0, 5x10 5One MAGE-A4-MC38 transgenic cancer cell line was inoculated into the right flank of female C57BL / 6 mice (Beijing Vital River). MAGE-A4 mRNA-LNP was administered intramuscularly at a dose of 10 μg / mouse, with a volume of 0.1 mL / mouse. Two dosing regimens were used to evaluate antitumor efficacy. The prophylactic dosing regimen began before cancer cell inoculation and was administered on days -10, -7, -3, 4, 11, 18, 25, and 32. The therapeutic dosing regimen began after cancer cell inoculation and was administered on days 1, 4, 7, 11, 18, 25, and 32. Mouse body weight and tumor volume were measured three times weekly.

[0266] result

[0267] Two mRNAs (SEQ ID NO: 1 and 2) encoding the full-length MAGE-A4 antigen, with different nucleotide sequences, can be translated into protein products with the expected molecular weight. The nucleotide sequences of SEQ ID NO: 1 and 2 are shown below:

[0268] MAGE-A4-1

[0269]

[0270] >MAGE-A4-2

[0271]

[0272] The two mRNAs showed similar protein expression levels. Figure 1 ).

[0273] In a single-dose kinetic assay, MAGE-A4 mRNA-LNP (MAGE-A4-2 encoded by SEQ ID NO: 2) was injected into C57BL / 6 mice via IM at day 0, and T cell responses in the spleen and lymph nodes were measured at days 7 and 14. Figure 2 and Figure 3 As shown, a single dose of MAGE-A4 mRNA-LNP vaccine administered via IM in C57BL / 6 mice induced antigen-specific immune responses in both the spleen and lymph nodes.

[0274] In the antitumor efficacy study, MAGE-A4-MC38 transgenic tumor cells were inoculated into C57BL / 6 mice (D0), and 10 μg / mouse of MAGE-A4 mRNA-LNP was administered via two different dosing regimens (prophylactic: day -10, day -7, day -3, day 4, day 11, day 18, day 25, and day 32; therapeutic: day 1, day 4, day 7, day 11, day 18, day 25, and day 32). Each dosing regimen induced transient but recoverable weight loss. Mice tolerated mRNA-LNP immunization ( Figure 4 In the PBS-treated group, all mice developed tumors. MAGE-A4 mRNA-LNP effectively inhibited tumor growth in both prophylactic and therapeutic administration regimens, with tumor growth inhibition rates (TGI%) of 66.2% and 60.2%, respectively. Figure 5 ).

[0275] Other implementation plans

[0276] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is determined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the claims of this application.

Claims

1. A nucleic acid encoding a polypeptide, said polypeptide comprising: (a) Igκ light chain signal peptide (SP) sequence; (b) MAGE-A4 antigen sequence; and (c) Human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) sequence.

2. The nucleic acid according to claim 1, wherein the Igκ light chain signal peptide (SP) sequence is at least 80% identical to the amino acid sequence shown in SEQ ID NO:

3.

3. The nucleic acid according to claim 1 or 2, comprising 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 shown in SEQ ID NO: 8, 9, 17 or 18.

4. The nucleic acid according to any one of claims 1-3, wherein the MITD sequence comprises at least 80% identical amino acid sequence to that shown in SEQ ID NO:

4.

5. The nucleic acid according to any one of claims 1-4, comprising a nucleic acid encoding the MITD sequence, wherein the nucleic acid encoding the MITD sequence is at least 80% identical to the sequence shown in SEQ ID NO: 13, 14, 22 or 23.

6. The nucleic acid according to any one of claims 1-5, wherein the MAGE-A4 antigen sequence comprises at least 80% identical amino acid sequence to that shown in SEQ ID NO:

5.

7. The nucleic acid according to any one of claims 1-6, comprising a nucleic acid encoding the MAGE-A4 antigen sequence, wherein the nucleic acid encoding the MAGE-A4 antigen sequence is at least 80% identical to the sequence shown in SEQ ID NO: 10, 11, 19 or 20.

8. The nucleic acid according to any one of claims 1-7, wherein the Igκ light chain signal peptide (SP) sequence, the antigen sequence and / or the MITD sequence are linked via a adapter.

9. The nucleic acid according to claim 8, wherein the adapter comprises the sequence shown in SEQ ID NO:

6.

10. The nucleic acid according to claim 8 or 9, comprising a nucleic acid sequence encoding the adapter, wherein the nucleic acid sequence encoding the adapter is at least 80% identical to the sequence shown in SEQ ID NO: 12 or 21.

11. The nucleic acid according to any one of claims 1-10, comprising a stop codon.

12. The nucleic acid according to any one of claims 1-11, comprising at least 80% identical nucleotide sequences to the sequence shown in SEQ ID NO: 1, 2, 15 or 16.

13. The nucleic acid according to any one of claims 1-12, wherein the nucleic acid is mRNA.

14. The nucleic acid according to claim 13, wherein the mRNA comprises at least one chemical modification.

15. The nucleic acid according to claim 13 or 14, wherein the mRNA comprises a 5' UTR and / or a 3' UTR.

16. The nucleic acid according to claim 14 or 15, 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-deazo-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-methyluridine.

17. The nucleic acid of claim 15, wherein the 5' UTR comprises at least 80% identical to the sequence shown in SEQ ID NO:

27.

18. The nucleic acid according to claim 15 or 17, wherein the 3' UTR comprises at least 80% identical to the sequence shown in SEQ ID NO: 29 or 31.

19. The nucleic acid according to any one of claims 15-18, wherein the nucleic acid comprises a capping enhancement sequence.

20. The nucleic acid of claim 19, wherein the capped enhancement sequence comprises the sequence shown in SEQ ID NO:

25.

21. The nucleic acid according to any one of claims 13-20, wherein the nucleic acid comprises multiple (A) sequences.

22. The nucleic acid according to claim 21, wherein the multiple (A) sequence comprises the sequence shown in SEQ ID NO:

32.

23. A pharmaceutical composition comprising a nucleic acid according to any one of claims 1-22.

24. A vaccine comprising a nucleic acid according to any one of claims 1-22 formulated in lipid nanoparticles (LNPs).

25. The vaccine of claim 24, wherein the lipid nanoparticles comprise cationic lipids, PEG-modified lipids, sterols, and non-cationic lipids.

26. The vaccine of claim 25, wherein the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol.

27. Polypeptides, which include: (a) Igκ light chain signal peptide (SP) sequence; (b) MAGE-A4 antigen sequence; and (c) Human major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD) sequence.

28. The polypeptide of claim 27, comprising at least 80% identical sequence to the sequence shown in SEQ ID NO:

7.

29. A method for inducing an immune response in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 23, the vaccine according to any one of claims 24-26, or the polypeptide according to claim 27 or 28.

30. A method for preventing or treating a disease or ailment of a subject, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 23, the vaccine according to any one of claims 24-26, or the polypeptide according to claim 27 or 28.

31. The method according to claim 29 or 30, wherein the disease or condition is cancer.

32. The method of claim 31, wherein the cancer is melanoma, lung cancer, esophageal cancer, head and neck cancer, gastric cancer, bladder urothelial carcinoma, hepatocellular carcinoma, triple-negative breast cancer (TNBC), or ovarian cancer.

33. The method according to any one of claims 29-32, further comprising administering one or more additional therapeutic agents to the subject.

34. The method of claim 33, wherein the one or more additional therapeutic agents are anticancer therapeutic agents.

35. A method for preparing a vaccine, comprising mixing a nucleic acid according to any one of claims 1-22 with a lipid nanoparticle formulation to produce a vaccine.

36. Nucleic acid comprising the sequence shown in any one of SEQ ID NO: 1, 2 and 8-31.

37. The nucleic acid according to claim 36, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 10 or 19.

38. The nucleic acid according to claim 36, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 11 or 20.

39. The nucleic acid according to claim 36, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 8, 9, 17 or 18.

40. The nucleic acid of claim 36, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 13, 14, 22 or 23.

41. The nucleic acid according to claim 36, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 24 or 25.

42. The nucleic acid according to claim 36, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 26 or 27.

43. The nucleic acid according to claim 36, wherein the nucleic acid comprises the sequence shown in SEQ ID NO: 28, 29, 30 or 31.

44. The nucleic acid according to any one of claims 36-43, wherein the nucleic acid is mRNA.

Images