Polypeptide for preventing or treating tumors, RNA molecule, and medical use

Abstract

A composition for preventing or treating tumors, comprising at least one antigenic peptide derived from a tumor-associated antigen (TAA), and at least one antigenic peptide derived from KRAS. Further provided are an RNA molecule encoding the antigenic peptides, a polynucleotide, a vector, a lipid nanoparticle, a pharmaceutical composition, and a medical use.

Description

Peptides, RNA molecules used for the prevention or treatment of tumors and their medicinal applications Technical Field

[0001] This disclosure pertains to the field of biomedicine, specifically relating to tumor-associated antigens, RNA molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and pharmaceutical uses. Background Technology

[0002] KRAS mutations are a class of tumor-specific antigens (TSA) with potential therapeutic applications. KRAS mutations can help regulate the immunosuppressive effects of the tumor microenvironment, such as upregulating PD-L1 expression to reduce immune signal recognition, downregulating MHC class I molecule expression to decrease antigen presentation, and enhancing the secretion of immunosuppressive cytokines and chemokines. KRAS mutations in tumors are primarily single-gene mutations, with 98% of mutations occurring at codons 12, 13, and 61. The cancer types with the highest KRAS mutation rates are pancreatic cancer, colorectal cancer, and non-small cell lung cancer.

[0003] Tumor vaccines targeting KRAS include peptide vaccines, viral vaccines, RNA vaccines, and dendritic cell (DC) vaccines, most of which are in the clinical development stage. Most vaccines target the three cancer types with the highest KRAS mutation rates, and their applications include tumor treatment and recurrence prevention. Furthermore, most employ a strategy of combining these vaccines with immune checkpoint inhibitors, adjuvants, and chemotherapy drugs.

[0004] However, existing KRAS tumor vaccines still have some limitations. First, the immunogenicity of the vaccines needs to be improved to ensure more effective activation of the immune system. Second, current vaccines have insufficient patient coverage, making it difficult to meet the needs of a wider patient population. Furthermore, epitope competition between different antigens may also affect vaccine efficacy. Therefore, there is an urgent clinical need to develop a universal KRAS tumor vaccine with higher immunogenicity, broader patient coverage, and the ability to avoid epitope competition. Summary of the Invention

[0005] This disclosure relates to tumor-associated antigens, RNA molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and pharmaceutical uses.

[0006] In one aspect, this disclosure provides a composition comprising at least one RNA molecule, wherein the RNA molecule encodes at least one antigenic peptide derived from a tumor-associated antigen (TAA) and at least one antigenic peptide derived from KRAS. In some embodiments, the composition comprises 1-10 RNA molecules. In some embodiments, the composition comprises 1, 2, 3, 4, or 5 RNA molecules. In some embodiments, the RNA molecule comprises an open reading frame (ORF) encoding a target protein. In some embodiments, the ORF encoding at least one TAA-derived antigenic peptide and the ORF encoding at least one KRAS-derived antigenic peptide are located on the same RNA molecule. In some embodiments, the ORF encoding at least one TAA-derived antigenic peptide and the ORF encoding at least one KRAS-derived antigenic peptide are located on different RNA molecules.

[0007] In some embodiments, at least one antigenic peptide derived from TAA comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, OAS1, hTERT, and / or survivin. In some embodiments, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, or SEQ ID NO:8. In some embodiments, the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10. In some embodiments, the antigenic peptide derived from TPX2 comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:9. In some embodiments, the antigenic peptide derived from C12orf32 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:11. In some embodiments, the antigenic peptide derived from OAS1 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:12. In some embodiments, the antigenic peptide derived from hTERT comprises the amino acid sequence shown in any one of SEQ ID NO:17-20 or any combination thereof. In some implementations, the antigenic peptide derived from survivin contains an amino acid sequence as shown in SEQ ID NO:21 and / or 22.

[0008] In some embodiments, at least one KRAS-derived antigenic peptide has a G12 and / or G13 mutation. In some embodiments, the KRAS-derived antigenic peptide has a G12A, G12C, G12D, G12R, G12S, G12V, and / or G13D mutation. In some embodiments, the KRAS-derived antigenic peptide comprises at least one of the sequences shown in SEQ ID NO:13-16.

[0009] In some implementations, the KRAS-derived antigenic peptide synergistically enhances the level of immune response and / or antitumor activity in vivo with the TAA-derived antigenic peptide.

[0010] In another aspect, this disclosure provides a composition comprising at least one polypeptide, wherein the polypeptide comprises at least one antigenic peptide derived from a tumor-associated antigen (TAA) and at least one antigenic peptide derived from a KRAS. In some embodiments, the composition comprises 1-10 polypeptides. In some embodiments, the composition comprises 1, 2, 3, 4, or 5 polypeptides. In some embodiments, at least one TAA-derived antigenic peptide and at least one KRAS-derived antigenic peptide are located on the same polypeptide. In some embodiments, at least one TAA-derived antigenic peptide and at least one KRAS-derived antigenic peptide are located on different polypeptides.

[0011] In some embodiments, at least one antigenic peptide derived from TAA comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, OAS1, hTERT, and / or survivin. In some embodiments, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, or SEQ ID NO:8. In some embodiments, the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10. In some embodiments, the antigenic peptide derived from TPX2 comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:9. In some embodiments, the antigenic peptide derived from C12orf32 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:11. In some embodiments, the antigenic peptide derived from OAS1 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:12. In some embodiments, the antigenic peptide derived from hTERT comprises the amino acid sequence shown in any one of SEQ ID NO:17-20 or any combination thereof. In some implementations, the antigenic peptide derived from survivin contains an amino acid sequence as shown in SEQ ID NO:21 and / or 22.

[0012] In some embodiments, at least one KRAS-derived antigenic peptide has a G12 and / or G13 mutation. In some embodiments, the KRAS-derived antigenic peptide has a G12A, G12C, G12D, G12R, G12S, G12V, and / or G13D mutation. In some embodiments, the KRAS-derived antigenic peptide comprises at least one of the sequences shown in SEQ ID NO:13-16.

[0013] In some implementations, the KRAS-derived antigenic peptide synergistically enhances the level of immune response and / or antitumor activity in vivo with the TAA-derived antigenic peptide.

[0014] In another aspect, this disclosure provides the use of ECT2, TOP2A, TPX2, C12orf32, and / or OAS1 proteins, or immunogenic fragments thereof, in the preparation of pharmaceutical compositions for treating or preventing cancer. In some embodiments, the cancer is a KRAS-mutated malignant tumor. In some embodiments, the cancer is selected from pancreatic cancer, colorectal cancer, and / or lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer.

[0015] In some embodiments, the immunogenic fragment comprises a high-affinity HLA sequence, exhibits an enhanced immune response level after immunization, and / or has antitumor activity.

[0016] In some embodiments, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7 or SEQ ID NO:8; the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10; the antigenic peptide derived from TPX2 comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:9; the antigenic peptide derived from C12orf32 comprises the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:11; and the antigenic peptide derived from OAS1 comprises the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:12.

[0017] In some implementations, the ECT2, TOP2A, TPX2, C12orf32, and OAS1 proteins contain the amino acid sequences shown in SEQ ID NO:126, 129, 132, 135, and 138, respectively.

[0018] In some embodiments, the pharmaceutical composition further comprises at least one KRAS-derived antigenic peptide. In some embodiments, the at least one KRAS-derived antigenic peptide has a G12 and / or G13 mutation. In some embodiments, the KRAS-derived antigenic peptide has a G12A, G12C, G12D, G12R, G12S, G12V, and / or G13D mutation. In some embodiments, the KRAS-derived antigenic peptide comprises at least one of the sequences shown in SEQ ID NO:13-16.

[0019] In some embodiments, the antigenic peptide further comprises at least one source of hTERT and / or survivin. In some embodiments, the hTERT-derived antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; the survivin-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:21 and / or 22.

[0020] In some embodiments, the pharmaceutical composition is a tumor vaccine. In some embodiments, the pharmaceutical composition is an mRNA vaccine. In some embodiments, the pharmaceutical composition is a recombinant protein vaccine.

[0021] In another aspect of this disclosure, a nucleic acid molecule is provided comprising (a) an open reading frame (ORF) encoding a target protein; wherein the target protein comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, and OAS1. In some embodiments, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4; the antigenic peptide derived from TPX2 comprises the amino acid sequence shown in SEQ ID NO:3; the antigenic peptide derived from C12orf32 comprises the amino acid sequence shown in SEQ ID NO:5; and the antigenic peptide derived from OAS1 comprises the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:10; the antigenic peptide derived from TPX2 comprises the amino acid sequence shown in SEQ ID NO:9; the antigenic peptide derived from C12orf32 comprises the amino acid sequence shown in SEQ ID NO:11; and the antigenic peptide derived from OAS1 comprises the amino acid sequence shown in SEQ ID NO:12.

[0022] In some embodiments, the target protein further includes one or more antigenic peptides derived from hTERT and / or survivin. In some embodiments, the hTERT-derived antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; the survivin-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:21 or 22.

[0023] In some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the RNA molecule is mRNA.

[0024] In another aspect of this disclosure, a polypeptide is provided comprising one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, and OAS1. In some embodiments, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4; the antigenic peptide derived from TPX2 comprises the amino acid sequence shown in SEQ ID NO:3; the antigenic peptide derived from C12orf32 comprises the amino acid sequence shown in SEQ ID NO:5; the antigenic peptide derived from OAS1 comprises the amino acid sequence shown in SEQ ID NO:6; in some embodiments, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:10; the antigenic peptide derived from TPX2 comprises the amino acid sequence shown in SEQ ID NO:9; the antigenic peptide derived from C12orf32 comprises the amino acid sequence shown in SEQ ID NO:11; and the antigenic peptide derived from OAS1 comprises the amino acid sequence shown in SEQ ID NO:12.

[0025] In another aspect of this disclosure, a polypeptide is provided comprising an amino acid sequence as shown in or having at least 90% sequence identity with any one of SEQ ID NO:44, 47, 56, 59, 62, 65 and 68.

[0026] In another aspect of this disclosure, an RNA molecule is provided comprising or having at least 90% sequence identity with any one of SEQ ID NO:46, 49, 58, 61, 64, 67 and 70.

[0027] In another aspect of this disclosure, a DNA molecule is provided comprising or having at least 90% sequence identity with any one of SEQ ID NO:45, 48, 57, 60, 63, 66 and 69.

[0028] In another aspect of this disclosure, a polynucleotide is provided that encodes a polypeptide or RNA molecule of any of the foregoing.

[0029] In another aspect of this disclosure, a carrier is provided that comprises a DNA molecule or polynucleotide of any of the foregoing.

[0030] In another aspect of this disclosure, a host cell is provided that contains any of the aforementioned polynucleotides, or a vector.

[0031] In another aspect of this disclosure, a lipid nanoparticle is provided that comprises any of the foregoing RNA molecules, compositions, DNA molecules, polynucleotides, or carriers.

[0032] In another aspect of this disclosure, a pharmaceutical composition is provided comprising any of the foregoing RNA molecules, compositions, peptides, DNA molecules, polynucleotides, carriers, or lipid nanoparticles or any combination thereof. In some embodiments, the pharmaceutical composition comprises pharmaceutically acceptable excipients, diluents, or excipients.

[0033] In another aspect of this disclosure, a tumor vaccine is provided comprising the pharmaceutical composition of any of the foregoing claims. In some embodiments, the tumor vaccine is a universal pancreatic cancer, colorectal cancer, and / or non-small cell lung cancer vaccine.

[0034] In another aspect of this disclosure, the use of any of the foregoing RNA molecules, compositions, peptides, DNA molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, or tumor vaccines is provided in (1) the preparation of a medicament that induces an immune response in a subject, or (2) the preparation of a medicament for the prevention and / or treatment of cancer. In some embodiments, the cancer is a KRAS-mutated malignant tumor. In some embodiments, the cancer is pancreatic cancer, colorectal cancer, or lung cancer (e.g., non-small cell lung cancer).

[0035] In another aspect of this disclosure, a method for preparing an antigenic peptide is provided, comprising: expressing the polynucleotide or vector of any of the preceding items in a host cell to produce the antigenic peptide.

[0036] In another aspect of this disclosure, a method for preparing an RNA molecule is provided, comprising: transcribing an RNA molecule using a polynucleotide or vector as a template, as described above. In some embodiments, the method further comprises adding a 5' cap to the 5' end of the RNA molecule.

[0037] In another aspect of this disclosure, a method for inducing an immune response in a desired subject is provided, comprising administering to the subject an effective amount of any of the foregoing RNA molecules, compositions, peptides, DNA molecules, polynucleotides, carriers, lipid nanoparticles, or pharmaceutical compositions.

[0038] In another aspect of this disclosure, there is provided the use of a molecule targeting ECT2, TOP2A, TPX2, C12orf32, and / or OAS1 in the preparation of a medicament for the prevention and / or treatment of cancer. In some embodiments, the molecule modulates the activity of ECT2, TOP2A, TPX2, C12orf32, and / or OAS1. In some embodiments, the cancer is a KRAS-mutated malignant tumor. In some embodiments, the cancer is selected from pancreatic cancer, colorectal cancer, and / or lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer.

[0039] Antigenic peptides of tumor-associated antigens (TAAs)

[0040] In some embodiments, this disclosure provides a polypeptide comprising an antigenic peptide selected from ECT2, TOP2A, TPX2, C12orf32, or OAS1, or any combination thereof.

[0041] In some embodiments, ECT2, TOP2A, TPX2, C12orf32, and OAS1 are derived from vertebrates (e.g., mammals, such as humans or cattle). In some embodiments, ECT2, TOP2A, TPX2, C12orf32, and OAS1 are derived from humans. In some embodiments, ECT2, TOP2A, TPX2, C12orf32, and OAS1 are each independently derived from wild-type ECT2, TOP2A, TPX2, C12orf32, and OAS1 peptides.

[0042] In some embodiments, the antigenic peptide derived from ECT2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:1 and / or SEQ ID NO:2. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TPX2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:3. In some embodiments, the antigenic peptide derived from C12orf32 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:5. In some embodiments, the antigenic peptide derived from OAS1 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:6.

[0043] In some embodiments, ECT2, TOP2A, TPX2, C12orf32, and the OAS1-derived antigenic peptide each contain at least M consecutive amino acids. In some embodiments, M is less than 50. In some embodiments, M is less than 40. In some embodiments, M is less than 35. In some embodiments, M is less than 30. In some embodiments, M is less than 25. In some embodiments, M is less than 20. In some embodiments, M is less than 15. In some embodiments, M is selected from 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35.

[0044] In some embodiments, the ECT2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, containing at least 9-31 consecutive amino acids. In some embodiments, the ECT2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the ECT2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:7 or 8, or having at least 80% sequence identity with it.

[0045] In some embodiments, the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4, which contains at least 11-31 consecutive amino acids. In some embodiments, the antigenic peptide derived from TOP2A further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence as shown in SEQ ID NO:10 or having at least 80% sequence identity with it.

[0046] In some embodiments, the TPX2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:3, which contains at least 11-31 consecutive amino acids. In some embodiments, the TPX2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the TPX2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:9 or having at least 80% sequence identity with it.

[0047] In some embodiments, the C12orf32-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:5, which contains at least 9-31 consecutive amino acids. In some embodiments, the C12orf32-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the C12orf32-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% sequence identity with it.

[0048] In some embodiments, the OAS1-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:6, which contains at least 10-31 consecutive amino acids. In some embodiments, the OAS1-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the OAS1-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% sequence identity with it.

[0049] In some embodiments, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NO:1, 2, 3, 4, 5, 6 or having at least 80% sequence identity with it, or any combination thereof.

[0050] In some embodiments, the polypeptide comprises an amino acid sequence as shown in or having at least 80% sequence identity with any of SEQ ID NO:7, 8, 9, 10, 11, 12, or any combination thereof.

[0051] In some embodiments, the polypeptide further comprises one or more antigenic peptides derived from hTERT and / or survivin. In some embodiments, the hTERT and / or survivin are each independently derived from wild-type hTERT and / or survivin polypeptides.

[0052] In some embodiments, the hTERT-derived antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or having at least 80% sequence identity with it, or any combination thereof. In some embodiments, the survivin-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:21 or 22 or having at least 80% sequence identity with it, or any combination thereof.

[0053] In some embodiments, the antigenic peptides in the polypeptide are arbitrarily linked directly by peptide bonds or by linkers.

[0054] In some implementations, the linker is a peptide linker.

[0055] In some implementations, the connector is as follows: (GS)a(GGS)b(GGGS)c(GGGGS)d(GGGGG)e, where a, b, c, d, and e are independent integers greater than or equal to 0; or the connector is selected from: (EAAAK)3(SEQ ID NO: 94), (EAAAR)3(SEQ ID NO: 95), (EGGGK)3(SEQ ID NO: 96), (EGGGR)3(SEQ ID NO: 97), (DAAAR)3(SEQ ID NO: 98), (DAAAK)3(SEQ ID NO: 99), (DGGGR)3(SEQ ID NO: 100) or (DGGGK)3(SEQ ID NO: 101); or the connector is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS(SEQ ID NO: 102) or GGSGGGGSGG(SEQ ID NO: 102). NO: 103); or the connector is (GxZy)i, where Z is selected from P, A, L or S, x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to GGPPG (SEQ ID NO: 104), GAGPG (SEQ ID NO: 105) or GPLS (SEQ ID NO: 106); or the connector is (AxYz)i, where x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to AAY.

[0056] In some implementations, the connector is EAAAK (SEQ ID NO: 107), AAY, or GGPPG (SEQ ID NO: 104).

[0057] In some embodiments, the polypeptide further comprises a signal peptide. In some embodiments, the signal peptide is fused to the polypeptide of this disclosure directly or via a linker (e.g., a linker having the amino acid sequence GGSGGGGSGG (SEQ ID NO: 103)). In some embodiments, the signal peptide has a length of about 15 to 30 amino acids. In some embodiments, the signal peptide is located at the N-terminus of the polypeptide. In some embodiments, the signal peptide allows the transport of an RNA-encoded polypeptide or protein to a defined cellular compartment, such as the cell surface, endoplasmic reticulum (ER), or endosome-lysosome compartment. In some embodiments, the signal peptide sequence includes, but is not limited to, signal peptide sequences derived from sequences encoding human MHC class I complexes (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3). In some embodiments, the signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 92 or having at least 80% sequence identity with it.

[0058] In some embodiments, the polypeptide further comprises a MITD domain. The MITD domain may correspond to the transmembrane and cytoplasmic domains of MHC class I molecules, also known as an MHC class I transport domain. In some embodiments, the MITD domain enhances the processing and presentation of the antigenic peptide. In some embodiments, the MITD domain comprises an amino acid sequence as shown in SEQ ID NO:93 or having at least 80% sequence identity with it.

[0059] In some embodiments, the polypeptides disclosed herein also include tag sequences, which, exemplarily, include, but are not limited to, protease cleavage site sequences, such as thrombin cleavage sites (LVPRGS (SEQ ID NO: 141)); protein tags, such as 6×His-tags (HHHHHH (SEQ ID NO: 142)) and streptomycin tag II (WSHPGFEK (SEQ ID NO: 143)); said sequences are not essential for the function of the polypeptide, such as for inducing an immune response. Those skilled in the art will recognize such sequences and, where appropriate, understand that they are not included in the disclosed polypeptides.

[0060] In some embodiments, the polypeptide also includes a His tag.

[0061] In some embodiments, the polypeptide comprises an amino acid sequence as shown in or having at least 80% sequence identity with any of SEQ ID NO:44, 47, 56, 59, 62, 65, 68.

[0062] Composition

[0063] This disclosure provides a composition comprising at least one polypeptide, wherein the polypeptide comprises at least one antigenic peptide derived from a tumor-associated antigen (TAA) and at least one antigenic peptide derived from KRAS.

[0064] In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides.

[0065] In some embodiments, the at least one TAA-derived antigenic peptide and the at least one KRAS-derived antigenic peptide are located on the same polypeptide.

[0066] In some embodiments, the at least one TAA-derived antigenic peptide and the at least one KRAS-derived antigenic peptide are located on different polypeptides.

[0067] In some embodiments, the antigenic peptide derived from TAA and / or the antigenic peptide derived from KRAS has a length of 9-50 amino acids. In some specific embodiments, the antigenic peptide derived from TAA and / or the antigenic peptide derived from KRAS has a length of 9-35 amino acids.

[0068] In some embodiments, the at least one TAA-derived antigenic peptide comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin.

[0069] In some embodiments, ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin are derived from vertebrates (e.g., mammals, such as humans or cattle). In some embodiments, ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin are derived from humans, such as hTERT. In some embodiments, ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin are each independently derived from wild-type ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and survivin peptides.

[0070] In some embodiments, the antigenic peptide derived from ECT2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:1 and / or SEQ ID NO:2. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TPX2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:3. In some embodiments, the antigenic peptide derived from C12orf32 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:5. In some embodiments, the antigenic peptide derived from OAS1 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:6.

[0071] In some embodiments, the antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, and OAS1 each contain at least M consecutive amino acids. In some embodiments, M is less than 50. In some embodiments, M is less than 40. In some embodiments, M is less than 35. In some embodiments, M is less than 30. In some embodiments, M is less than 25. In some embodiments, M is less than 20. In some embodiments, M is less than 15. In some embodiments, M is selected from 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35.

[0072] In some embodiments, the antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, and OAS1 each contain 9 to 31 amino acids. In other embodiments, the length is 9 to 30, 9 to 29, 9 to 28, 9 to 27, 9 to 26, 9 to 25, 9 to 24, 9 to 23, 9 to 22, 9 to 21, 9 to 20, 9 to 19, 9 to 18, 10 to 31, 10 to 30, 10 to 29, 10 to 28, 10 to 27, 10 to 26, 10 to 25, 10 to 24, 10 to 23, 10 to 22, 1... 0 to 21, 10 to 20, 11 to 31, 11 to 30, 11 to 29, 11 to 28, 11 to 27, 11 to 26, 11 to 25, 11 to 24, 11 to 23, 11 to 22, 11 to 21, 11 to 20, 12 to 22, 12 to 21, 12 to 20, 13 to 22, 13 to 21, 13 to 20, 14 to 19, 15 to 18, or 16 to 17 amino acids.

[0073] In some embodiments, the ECT2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, containing at least 9-31 consecutive amino acids. In some embodiments, the ECT2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the ECT2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:7 or 8, or having at least 80% sequence identity with it.

[0074] In some embodiments, the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4, which contains at least 11-31 consecutive amino acids. In some embodiments, the antigenic peptide derived from TOP2A further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence as shown in SEQ ID NO:10 or having at least 80% sequence identity with it.

[0075] In some embodiments, the TPX2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:3, which contains at least 11-31 consecutive amino acids. In some embodiments, the TPX2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the TPX2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:9 or having at least 80% sequence identity with it.

[0076] In some embodiments, the C12orf32-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:5, which contains at least 9-31 consecutive amino acids. In some embodiments, the C12orf32-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the C12orf32-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% sequence identity with it.

[0077] In some embodiments, the OAS1-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:6, which contains at least 10-31 consecutive amino acids. In some embodiments, the OAS1-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the OAS1-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% sequence identity with it.

[0078] In some embodiments, the amino acid sequence derived from the hTERT antigen peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or having at least 80% sequence identity with it, or any combination thereof.

[0079] In some embodiments, the amino acid sequence derived from the survivin antigen peptide comprises an amino acid sequence as shown in or having at least 80% sequence identity with any of SEQ ID NO:21 and / or 22, or any combination thereof.

[0080] In some embodiments, the at least one TAA-derived antigenic peptide comprises:

[0081] (1) One or more antigenic peptides derived from ECT2; preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0082] (2) One or more antigenic peptides derived from TOP2A; preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10;

[0083] (3) One or more antigenic peptides derived from TPX2; preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:3; more preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:9;

[0084] (4) One or more antigenic peptides derived from C12orf32; preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11; and

[0085] (5) One or more antigenic peptides derived from OAS1; preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:12.

[0086] In some embodiments, the at least one TAA-derived antigenic peptide comprises:

[0087] (1) One or more antigenic peptides derived from ECT2; preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0088] (2) One or more antigenic peptides derived from TOP2A; preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10;

[0089] (3) One or more antigenic peptides derived from TPX2; preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:3; more preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:9;

[0090] (4) One or more antigenic peptides derived from C12orf32; preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11;

[0091] (5) One or more antigenic peptides derived from OAS1; preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:12.

[0092] (6) One or more antigenic peptides derived from hTERT; preferably, the antigenic peptides derived from hTERT comprise an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; and

[0093] (7) One or more antigenic peptides derived from survivin, preferably, the antigenic peptides derived from survivin contain an amino acid sequence as shown in SEQ ID NO:21 or 22.

[0094] In some embodiments, the at least one KRAS-derived antigenic peptide has a G12 and / or G13 mutation.

[0095] In some embodiments, the KRAS-derived antigenic peptide contains G12A, G12C, G12D, G12R, G12S, G12V and / or G13D mutations.

[0096] In some embodiments, the KRAS-derived antigenic peptide comprises 10 to 30, 15 to 25, or 20 to 25 amino acids in length. In some aspects, the KRAS-derived antigenic peptide comprises 20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, the KRAS-derived antigenic peptide comprises 25 amino acids in length.

[0097] In some embodiments, the KRAS-derived antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:13-16 or having at least 80% sequence identity with it, or any combination thereof.

[0098] In some embodiments, the KRAS-derived antigenic peptide synergistically enhances the level of immune response and / or antitumor activity in vivo with the TAA-derived antigenic peptide.

[0099] In some embodiments, the composition comprises a first polypeptide and a second polypeptide.

[0100] In some embodiments, in the composition:

[0101] The first polypeptide contains:

[0102] (1) One or more antigenic peptides derived from ECT2;

[0103] (2) One or more antigenic peptides derived from TOP2A;

[0104] (3) One or more antigenic peptides derived from TPX2;

[0105] (4) One or more antigenic peptides derived from C12orf32;

[0106] (5) One or more antigenic peptides derived from OAS1;

[0107] (6) One or more antigenic peptides derived from hTERT; and

[0108] (7) One or more antigenic peptides derived from survivin,

[0109] The second polypeptide contains:

[0110] (1) One or more antigenic peptides derived from KRAS and having a G12D mutation;

[0111] (2) One or more antigenic peptides derived from KRAS and having a G12V mutation;

[0112] (3) One or more antigenic peptides derived from KRAS and possessing the G13D mutation; and

[0113] (4) One or more antigenic peptides derived from KRAS and having a G12C mutation.

[0114] In some embodiments, in the composition:

[0115] The first polypeptide contains:

[0116] (1) One or more antigenic peptides derived from ECT2;

[0117] (2) One or more antigenic peptides derived from TOP2A;

[0118] (3) One or more antigenic peptides derived from TPX2;

[0119] (4) One or more antigenic peptides derived from C12orf32; and

[0120] (5) One or more antigenic peptides derived from OAS1;

[0121] The second polypeptide contains:

[0122] (1) One or more antigenic peptides derived from KRAS and having a G12D mutation;

[0123] (2) One or more antigenic peptides derived from KRAS and having a G12V mutation;

[0124] (3) One or more antigenic peptides derived from KRAS and having a G13D mutation;

[0125] (4) One or more antigenic peptides derived from KRAS and having a G12C mutation;

[0126] (7) One or more antigenic peptides derived from hTERT; and

[0127] (8) One or more antigenic peptides derived from survivin.

[0128] In some embodiments, the composition comprises a first polypeptide, a second polypeptide, and a third polypeptide.

[0129] In some embodiments, in the composition:

[0130] The first polypeptide contains:

[0131] (1) One or more antigenic peptides derived from ECT2;

[0132] (2) One or more antigenic peptides derived from TOP2A;

[0133] (3) One or more antigenic peptides derived from TPX2;

[0134] (4) One or more antigenic peptides derived from C12orf32; and

[0135] (5) One or more antigenic peptides derived from OAS1;

[0136] The second polypeptide contains:

[0137] (1) One or more antigenic peptides derived from KRAS and having a G12D mutation;

[0138] (2) One or more antigenic peptides derived from KRAS and having a G12V mutation;

[0139] (3) One or more antigenic peptides derived from KRAS and possessing the G13D mutation; and

[0140] (4) One or more antigenic peptides derived from KRAS and having a G12C mutation;

[0141] The third polypeptide contains:

[0142] (1) One or more antigenic peptides derived from hTERT; and

[0143] (2) One or more antigenic peptides derived from survivin.

[0144] In some embodiments, the antigenic peptides in the polypeptide (e.g., the first polypeptide, the second polypeptide, and / or the third polypeptide) are arbitrarily linked directly by peptide bonds or by linkers.

[0145] In some implementations, the linker is a peptide linker.

[0146] In some implementations, the connector is as follows: (GS)a(GGS)b(GGGS)c(GGGGS)d(GGGGG)e, where a, b, c, d, and e are independent integers greater than or equal to 0; or the connector is selected from: (EAAAK)3(SEQ ID NO: 94), (EAAAR)3(SEQ ID NO: 95), (EGGGK)3(SEQ ID NO: 96), (EGGGR)3(SEQ ID NO: 97), (DAAAR)3(SEQ ID NO: 98), (DAAAK)3(SEQ ID NO: 99), (DGGGR)3(SEQ ID NO: 100) or (DGGGK)3(SEQ ID NO: 101); or the connector is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS(SEQ ID NO: 102) or GGSGGGGSGG(SEQ ID NO: 102). NO: 103); or the connector is (GxZy)i, where Z is selected from P, A, L or S, x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to GGPPG (SEQ ID NO: 104), GAGPG (SEQ ID NO: 105) or GPLS (SEQ ID NO: 106); or the connector is (AxYz)i, where x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to AAY.

[0147] In some implementations, the connector is EAAAK (SEQ ID NO: 107), AAY, or GGPPG (SEQ ID NO: 104).

[0148] In some embodiments, the polypeptide (e.g., the first polypeptide, the second polypeptide, and / or the third polypeptide) further comprises a signal peptide. In some embodiments, the signal peptide is fused to the polypeptide of this disclosure (e.g., the first polypeptide, the second polypeptide, and / or the third polypeptide) directly or via a linker (e.g., a linker having the amino acid sequence GGSGGGGSGG (SEQ ID NO: 103)). In some embodiments, the signal peptide has a length of about 15 to 30 amino acids. In some embodiments, the signal peptide is located at the N-terminus of the polypeptide (e.g., the first polypeptide, the second polypeptide, and / or the third polypeptide). In some embodiments, the signal peptide allows the transport of an RNA-encoded polypeptide or protein to a defined cellular compartment, such as the cell surface, endoplasmic reticulum (ER), or endosome-lysosome compartment. In some embodiments, the signal peptide sequence includes, but is not limited to, signal peptide sequences derived from sequences encoding human MHC class I complexes (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3). In some embodiments, the signal peptide comprises an amino acid sequence as shown in SEQ ID NO:92 or having at least 80% sequence identity with it.

[0149] In some embodiments, the polypeptide (e.g., a first polypeptide, a second polypeptide, and / or a third polypeptide) further comprises a MITD domain. The MITD domain may correspond to the transmembrane and cytoplasmic domains of MHC class I molecules, also referred to as an MHC class I transport domain. In some embodiments, the MITD domain enhances the processing and presentation of the antigenic peptide. In some embodiments, the MITD domain comprises an amino acid sequence as shown in SEQ ID NO:93 or having at least 80% sequence identity with it.

[0150] In some embodiments, the polypeptides disclosed herein also include tag sequences, which, exemplarily, include, but are not limited to, protease cleavage site sequences, such as thrombin cleavage sites (LVPRGS (SEQ ID NO: 141)); protein tags, such as 6×His-tags (HHHHHH (SEQ ID NO: 142)) and streptomycin tag II (WSHPGFEK (SEQ ID NO: 143)); said sequences are not essential for the function of the polypeptide, such as for inducing an immune response. Those skilled in the art will recognize such sequences and, where appropriate, understand that they are not included in the disclosed polypeptides.

[0151] In some embodiments, the polypeptide (e.g., a first polypeptide, a second polypeptide, and / or a third polypeptide) also contains a His tag.

[0152] In some embodiments, the composition comprises a first polypeptide and a second polypeptide, and optionally a third polypeptide, the composition being selected from any group thereof:

[0153] (a) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it.

[0154] (b) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:50 or having at least 90% sequence identity with it.

[0155] (c) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:59 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it.

[0156] (d) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it, and the third polypeptide comprises an amino acid sequence as shown in SEQ ID NO:71 or having at least 90% sequence identity with it.

[0157] (e) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it.

[0158] (f) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:53 or having at least 90% sequence identity with it.

[0159] (g) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:59 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it.

[0160] (h) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it, and the third polypeptide comprises an amino acid sequence as shown in SEQ ID NO:71 or having at least 90% sequence identity with it.

[0161] (i) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:47 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it.

[0162] (j) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:62 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:68 or having at least 90% sequence identity with it.

[0163] (k) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:47 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it.

[0164] (l) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:62 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it.

[0165] In some embodiments, the polypeptide comprises an amino acid sequence as shown in or having at least 90% sequence identity with any of SEQ ID NO:44, 47, 56, 59, 62, 65 and 68.

[0166] Nucleic acid molecules

[0167] This disclosure provides a nucleic acid molecule (e.g., RNA) that includes an open reading frame (ORF) encoding at least one target protein. In some embodiments, the target protein comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, and OAS1.

[0168] In some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid molecule is an RNA molecule.

[0169] In some embodiments, the antigenic peptide derived from ECT2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:1 and / or SEQ ID NO:2. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TPX2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:3. In some embodiments, the antigenic peptide derived from C12orf32 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:5. In some embodiments, the antigenic peptide derived from OAS1 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:6.

[0170] In some embodiments, the ECT2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, containing at least 9-31 consecutive amino acids. In some embodiments, the ECT2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the ECT2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:7 or 8, or having at least 80% sequence identity with it.

[0171] In some embodiments, the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4, which contains at least 11-31 consecutive amino acids. In some embodiments, the antigenic peptide derived from TOP2A further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence as shown in SEQ ID NO:10 or having at least 80% sequence identity with it.

[0172] In some embodiments, the TPX2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:3, which contains at least 11-31 consecutive amino acids. In some embodiments, the TPX2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the TPX2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:9 or having at least 80% sequence identity with it.

[0173] In some embodiments, the C12orf32-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:5, which contains at least 9-31 consecutive amino acids. In some embodiments, the C12orf32-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the C12orf32-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% sequence identity with it.

[0174] In some embodiments, the OAS1-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:6, which contains at least 10-31 consecutive amino acids. In some embodiments, the OAS1-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the OAS1-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% sequence identity with it.

[0175] In some embodiments, the target protein comprises an amino acid sequence as shown in any one of SEQ ID NO:1, 2, 3, 4, 5, 6 or having at least 80% sequence identity with it, or any combination thereof.

[0176] In some embodiments, the target protein comprises an amino acid sequence as shown in any one of SEQ ID NO:7, 8, 9, 10, 11, 12 or having at least 80% sequence identity with it, or any combination thereof.

[0177] In some embodiments, the target protein further comprises one or more antigenic peptides derived from hTERT and / or survivin. In some embodiments, the hTERT and / or survivin are each independently derived from wild-type hTERT and / or survivin polypeptides.

[0178] In some embodiments, the hTERT-derived antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or having at least 80% sequence identity with it, or any combination thereof. In some embodiments, the survivin-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:21 or 22 or having at least 80% sequence identity with it, or any combination thereof.

[0179] In some implementations, the antigenic peptides in the target protein are arbitrarily linked directly by peptide bonds or by linkers.

[0180] In some implementations, the linker is a peptide linker.

[0181] In some implementations, the connector is as follows: (GS)a(GGS)b(GGGS)c(GGGGS)d(GGGGG)e, where a, b, c, d, and e are independent integers greater than or equal to 0; or the connector is selected from: (EAAAK)3(SEQ ID NO: 94), (EAAAR)3(SEQ ID NO: 95), (EGGGK)3(SEQ ID NO: 96), (EGGGR)3(SEQ ID NO: 97), (DAAAR)3(SEQ ID NO: 98), (DAAAK)3(SEQ ID NO: 99), (DGGGR)3(SEQ ID NO: 100) or (DGGGK)3(SEQ ID NO: 101); or the connector is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS(SEQ ID NO: 102) or GGSGGGGSGG(SEQ ID NO: 102). NO: 103); or the connector is (GxZy)i, where Z is selected from P, A, L or S, x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to GGPPG (SEQ ID NO: 104), GAGPG (SEQ ID NO: 105) or GPLS (SEQ ID NO: 106); or the connector is (AxYz)i, where x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to AAY.

[0182] In some implementations, the connector is EAAAK (SEQ ID NO: 107), AAY, or GGPPG (SEQ ID NO: 104).

[0183] In some embodiments, the target protein further comprises a signal peptide. In some embodiments, the signal peptide is fused to the polypeptide of this disclosure directly or via a linker (e.g., a linker having the amino acid sequence GGSGGGGGSGG (SEQ ID NO: 103)). In some embodiments, the signal peptide has a length of about 15 to 30 amino acids. In some embodiments, the signal peptide is located at the N-terminus of the polypeptide. In some embodiments, the signal peptide allows the transport of an RNA-encoded polypeptide or protein to a defined cellular compartment, such as the cell surface, endoplasmic reticulum (ER), or endosome-lysosome compartment. In some embodiments, the signal peptide sequence includes, but is not limited to, a signal peptide sequence derived from sequences encoding human MHC class I complexes (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3). In some embodiments, the signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 92 or having at least 80% sequence identity with it (MRVTAPRTLILLLSGALALTETWAGS).

[0184] In some embodiments, the target protein further comprises a MITD domain. The MITD domain may correspond to the transmembrane and cytoplasmic domains of MHC class I molecules, also known as an MHC class I transport domain. In some embodiments, the MITD domain enhances the processing and presentation of the antigenic peptide. In some embodiments, the MITD domain comprises an amino acid sequence as shown in SEQ ID NO:93 or having at least 80% sequence identity with it.

[0185] In some embodiments, the target protein comprises an amino acid sequence as shown in or having at least 80% sequence identity with any of SEQ ID NO:44, 47, 56, 59, 62, 65, 68.

[0186] In some embodiments, the nucleic acid molecule (e.g., an RNA molecule) further comprises an untranslated region element (UTR). In some embodiments, the UTR is derived from the UTR of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. In some embodiments, the aforementioned genes are human genes.

[0187] In some embodiments, the untranslated region element (UTR) includes a 5' UTR and a 3' UTR. In some embodiments, the 3' UTR and 5' UTR are of the same or different origins, for example, from the same or different genes. For example, the 3' UTR originates from the 3' UTR of gene ACTG1, and the 5' UTR originates from the 5' UTR of gene ACTG1. Another example is that the 3' UTR originates from the 3' UTR of gene CTSB, and the 5' UTR originates from the 5' UTR of gene CHCHD10. In some embodiments, the 5' UTR and 3' UTR originate from the same or different species.

[0188] In some embodiments, the 5'UTR is located upstream of the ORF. In some embodiments, the 5'UTR is located at the 5' end of the ORF. In some embodiments, the 5'UTR is selected from the 5'UTR or a derived sequence of any of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. In some embodiments, the 5'UTR is derived from or is a derived sequence of the 5'UTR of the gene ACTG1. In some embodiments, the 5'UTR contains the sequence shown in SEQ ID NO:33 or has at least 80% identity with it.

[0189] In some embodiments, the 3'UTR is located downstream of the ORF. In some embodiments, the 3'UTR is located at the 3' end of the ORF. In some embodiments, the 3'UTR is selected from the 3'UTR or a derived sequence of any of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9. In some embodiments, the 3'UTR is derived from or is a derived sequence of the 3'UTR of the gene CTSB. In some embodiments, the 3'UTR contains the sequence shown in SEQ ID NO:35 or has at least 80% identity with it.

[0190] In some implementations, the nucleic acid molecule (e.g., an RNA molecule) contains a 5' UTR and a 3' UTR, wherein:

[0191] The 5'UTR is selected from the 5'UTR or a derivative sequence of any of the following genes: ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. The 3'UTR is selected from the 3'UTR or a derivative sequence of any of the following genes: ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9.

[0192] In some embodiments, the nucleic acid molecule (e.g., an RNA molecule) contains a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following:

[0193] The 5'UTR is derived from or is a 5'UTR of the ACTG1 gene or a derivative thereof, and the 3'UTR is derived from or is a 3'UTR of the CTSB gene or a derivative thereof.

[0194] In some embodiments, the nucleic acid molecule (e.g., RNA molecule) disclosed herein contains a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following:

[0195] The 5'UTR contains or is a nucleotide sequence as shown in SEQ ID NO:33 or a nucleotide sequence having at least 80% sequence identity with it, and / or the 3'UTR contains or is a nucleotide sequence as shown in SEQ ID NO:35 or a nucleotide sequence having at least 80% sequence identity with it;

[0196] In some embodiments, the nucleic acid molecule (e.g., an RNA molecule) also includes a poly-A tail.

[0197] In some embodiments, the poly-A tail in the nucleic acid molecule is located downstream of the 3' UTR. In some embodiments, the poly-A tail in the nucleic acid molecule is located at the 3' end of the 3' UTR. In some embodiments, the poly-A tail is located at the 3' end of the nucleic acid molecule. In some embodiments, the poly-A tail is at least about 50, 100, 150, 200, 300, 400, or 500 nucleotides long.

[0198] In some implementations, the poly-A tail includes, but is not limited to, tails selected from 120A, Poly A-3070, HGH polyA, SV40polyA, BGH polyA, rbGlob polyA, or SV40late polyA.

[0199] In some embodiments, the poly-A tail is selected from 120A or Poly A-3070, which comprises or has at least 80% sequence identity with the sequence shown in SEQ ID NO:36.

[0200] In some embodiments, the nucleic acid molecules (e.g., RNA molecules) disclosed herein further include: (e) a 5' cap structure (5'Cap).

[0201] In some embodiments, the 5' cap structure in the nucleic acid molecule is located upstream of the 5' UTR. In some embodiments, the 5' cap structure in the nucleic acid molecule is located at the 5' end of the 5' UTR. In some embodiments, the 5' cap structure is a cap structure known to those skilled in the art, such as Cap0 (methylation of the first base, e.g., m7GpppN), Cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap2 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphate thioester modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deazo-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0202] In some implementations, 5'-cap structures (such as Cap0 or Cap1) are formed using chemical RNA synthesis or in vitro RNA transcription (co-transcriptional capping).

[0203] In some embodiments, a capping enzyme (e.g., a vaccinia virus capping enzyme and / or a cap-dependent 2'-O methyltransferase) is used to form a 5'-cap structure (such as Cap0 or Cap1) via enzymatic capping. In some embodiments, an immobilized capping enzyme is used to add a 5' cap structure (Cap0 or Cap1). The capping methods and means described in WO2016 / 193226 are incorporated herein by reference in their entirety.

[0204] In some implementations, the 5' cap structure includes, but is not limited to, ARCA, 3'OMe-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0205] In some implementations, the nucleic acid molecule is an RNA molecule.

[0206] In some embodiments, the RNA comprises a nucleotide sequence as shown in any one of SEQ ID NO:46, 49, 58, 61, 64, 67, 70, or a nucleotide sequence having at least 80% sequence identity with such sequences.

[0207] In some implementations, the RNA molecule may be mRNA.

[0208] In some embodiments, this disclosure provides a target protein encoded by any of the aforementioned nucleic acid molecules. The target protein is produced in vitro or in vivo.

[0209] RNA composition

[0210] This disclosure provides a composition comprising at least one RNA molecule, wherein the RNA molecule encodes at least one antigenic peptide derived from a tumor-associated antigen (TAA) and at least one antigenic peptide derived from KRAS.

[0211] In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA molecules.

[0212] In some implementations, the RNA molecule contains an open reading frame (ORF) encoding the target protein.

[0213] In some embodiments, the ORF encoding at least one antigenic peptide derived from TAA and the ORF encoding at least one antigenic peptide derived from KRAS are located on the same RNA molecule.

[0214] In some embodiments, the ORF encoding at least one antigenic peptide derived from TAA and the ORF encoding at least one antigenic peptide derived from KRAS are located on different RNA molecules.

[0215] In some embodiments, the antigenic peptide derived from TAA and / or the antigenic peptide derived from KRAS has a length of 9-50 amino acids. In some specific embodiments, the antigenic peptide derived from TAA and / or the antigenic peptide derived from KRAS has a length of 9-35 amino acids.

[0216] In some embodiments, the at least one TAA-derived antigenic peptide comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin.

[0217] In some embodiments, ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin are derived from vertebrates (e.g., mammals, such as humans or cattle). In some embodiments, ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin are derived from humans, such as hTERT. In some embodiments, ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin are each independently derived from wild-type ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and survivin peptides.

[0218] In some embodiments, the antigenic peptide derived from ECT2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:1 and / or SEQ ID NO:2. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TPX2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:3. In some embodiments, the antigenic peptide derived from C12orf32 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:5. In some embodiments, the antigenic peptide derived from OAS1 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:6.

[0219] In some embodiments, the antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, and OAS1 each contain at least M consecutive amino acids. In some embodiments, M is less than 50. In some embodiments, M is less than 40. In some embodiments, M is less than 35. In some embodiments, M is less than 30. In some embodiments, M is less than 25. In some embodiments, M is less than 20. In some embodiments, M is less than 15. In some embodiments, M is selected from 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35.

[0220] In some embodiments, the antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, and OAS1 each contain 9 to 31 amino acids. In other embodiments, the length is 9 to 30, 9 to 29, 9 to 28, 9 to 27, 9 to 26, 9 to 25, 9 to 24, 9 to 23, 9 to 22, 9 to 21, 9 to 20, 9 to 19, 9 to 18, 10 to 31, 10 to 30, 10 to 29, 10 to 28, 10 to 27, 10 to 26, 10 to 25, 10 to 24, 10 to 23, 10 to 22, 1... 0 to 21, 10 to 20, 11 to 31, 11 to 30, 11 to 29, 11 to 28, 11 to 27, 11 to 26, 11 to 25, 11 to 24, 11 to 23, 11 to 22, 11 to 21, 11 to 20, 12 to 22, 12 to 21, 12 to 20, 13 to 22, 13 to 21, 13 to 20, 14 to 19, 15 to 18, or 16 to 17 amino acids.

[0221] In some embodiments, the ECT2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, containing at least 9-31 consecutive amino acids. In some embodiments, the ECT2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the ECT2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:7 or 8, or having at least 80% sequence identity with it.

[0222] In some embodiments, the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4, which contains at least 11-31 consecutive amino acids. In some embodiments, the antigenic peptide derived from TOP2A further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence as shown in SEQ ID NO:10 or having at least 80% sequence identity with it.

[0223] In some embodiments, the TPX2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:3, which contains at least 11-31 consecutive amino acids. In some embodiments, the TPX2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the TPX2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:9 or having at least 80% sequence identity with it.

[0224] In some embodiments, the C12orf32-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:5, which contains at least 9-31 consecutive amino acids. In some embodiments, the C12orf32-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the C12orf32-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% sequence identity with it.

[0225] In some embodiments, the OAS1-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:6, which contains at least 10-31 consecutive amino acids. In some embodiments, the OAS1-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the OAS1-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% sequence identity with it.

[0226] In some embodiments, the amino acid sequence derived from the hTERT antigen peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or having at least 80% sequence identity with it, or any combination thereof.

[0227] In some embodiments, the amino acid sequence derived from the Survivin antigen peptide comprises an amino acid sequence as shown in or having at least 80% sequence identity with any of SEQ ID NO:21 and / or 22, or any combination thereof.

[0228] In some embodiments, the at least one TAA-derived antigenic peptide comprises:

[0229] (1) One or more antigenic peptides derived from ECT2; preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0230] (2) One or more antigenic peptides derived from TOP2A; preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10;

[0231] (3) One or more antigenic peptides derived from TPX2; preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:3; more preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:9;

[0232] (4) One or more antigenic peptides derived from C12orf32; preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11; and

[0233] (5) One or more antigenic peptides derived from OAS1; preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:12.

[0234] In some embodiments, the at least one TAA-derived antigenic peptide comprises:

[0235] (1) One or more antigenic peptides derived from ECT2; preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8.

[0236] (2) One or more antigenic peptides derived from TOP2A; preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10;

[0237] (3) One or more antigenic peptides derived from TPX2; preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:3; more preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:9;

[0238] (4) One or more antigenic peptides derived from C12orf32; preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11;

[0239] (5) One or more antigenic peptides derived from OAS1; preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:12.

[0240] (6) One or more antigenic peptides derived from hTERT; preferably, the antigenic peptides derived from hTERT comprise an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; and

[0241] (7) One or more antigenic peptides derived from survivin, preferably, the antigenic peptides derived from survivin contain an amino acid sequence as shown in SEQ ID NO:21 or 22.

[0242] In some embodiments, the at least one KRAS-derived antigenic peptide has a G12 and / or G13 mutation.

[0243] In some embodiments, the KRAS-derived antigenic peptide contains G12A, G12C, G12D, G12R, G12S, G12V and / or G13D mutations.

[0244] In some embodiments, the KRAS-derived antigenic peptide comprises 10 to 30, 15 to 25, or 20 to 25 amino acids in length. In some aspects, the KRAS-derived antigenic peptide comprises 20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, the KRAS-derived antigenic peptide comprises 25 amino acids in length.

[0245] In some embodiments, the KRAS-derived antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:13-16 or having at least 80% sequence identity with it, or any combination thereof.

[0246] In some embodiments, the KRAS-derived antigenic peptide synergistically enhances the level of immune response and / or antitumor activity in vivo with the TAA-derived antigenic peptide.

[0247] In some embodiments, the composition comprises a first RNA molecule and a second RNA molecule, the first RNA molecule and the second RNA molecule each comprising an open reading frame (ORF) encoding a target protein.

[0248] In some embodiments, in the composition:

[0249] The first target protein encoded by the first RNA molecule includes:

[0250] (1) One or more antigenic peptides derived from ECT2;

[0251] (2) One or more antigenic peptides derived from TOP2A;

[0252] (3) One or more antigenic peptides derived from TPX2;

[0253] (4) One or more antigenic peptides derived from C12orf32;

[0254] (5) One or more antigenic peptides derived from OAS1;

[0255] (6) One or more antigenic peptides derived from hTERT; and

[0256] (7) One or more antigenic peptides derived from survivin,

[0257] The second target protein encoded by the second RNA molecule includes:

[0258] (1) One or more antigenic peptides derived from KRAS and having a G12D mutation;

[0259] (2) One or more antigenic peptides derived from KRAS and having a G12V mutation;

[0260] (3) One or more antigenic peptides derived from KRAS and possessing the G13D mutation; and

[0261] (4) One or more antigenic peptides derived from KRAS and having a G12C mutation.

[0262] In some embodiments, in the composition:

[0263] The first target protein encoded by the first RNA molecule includes:

[0264] (1) One or more antigenic peptides derived from ECT2;

[0265] (2) One or more antigenic peptides derived from TOP2A;

[0266] (3) One or more antigenic peptides derived from TPX2;

[0267] (4) One or more antigenic peptides derived from C12orf32; and

[0268] (5) One or more antigenic peptides derived from OAS1;

[0269] The second target protein encoded by the second RNA molecule includes:

[0270] (1) One or more antigenic peptides derived from KRAS and having a G12D mutation;

[0271] (2) One or more antigenic peptides derived from KRAS and having a G12V mutation;

[0272] (3) One or more antigenic peptides derived from KRAS and having a G13D mutation;

[0273] (4) One or more antigenic peptides derived from KRAS and having a G12C mutation;

[0274] (7) One or more antigenic peptides derived from hTERT; and

[0275] (8) One or more antigenic peptides derived from survivin.

[0276] In some embodiments, the composition comprises a first RNA molecule, a second RNA molecule, and a third RNA molecule, wherein the first RNA molecule, the second RNA molecule, and the third RNA molecule each contain an open reading frame (ORF) encoding a target protein.

[0277] In some embodiments, in the composition:

[0278] The first target protein encoded by the first RNA molecule includes:

[0279] (1) One or more antigenic peptides derived from ECT2;

[0280] (2) One or more antigenic peptides derived from TOP2A;

[0281] (3) One or more antigenic peptides derived from TPX2;

[0282] (4) One or more antigenic peptides derived from C12orf32; and

[0283] (5) One or more antigenic peptides derived from OAS1;

[0284] The second target protein encoded by the second RNA molecule includes:

[0285] (1) One or more antigenic peptides derived from KRAS and having a G12D mutation;

[0286] (2) One or more antigenic peptides derived from KRAS and having a G12V mutation;

[0287] (3) One or more antigenic peptides derived from KRAS and possessing the G13D mutation; and

[0288] (4) One or more antigenic peptides derived from KRAS and having a G12C mutation;

[0289] The third target protein encoded by the third RNA molecule includes:

[0290] (1) One or more antigenic peptides derived from hTERT; and

[0291] (2) One or more antigenic peptides derived from survivin.

[0292] In some embodiments, the antigenic peptides in the target proteins (e.g., the first target protein, the second target protein, and / or the third target protein) encoded by the RNA molecule are directly linked by peptide bonds or linked by linkers.

[0293] In some implementations, the linker is a peptide linker.

[0294] In some implementations, the connector is as follows: (GS)a(GGS)b(GGGS)c(GGGGS)d(GGGGG)e, where a, b, c, d, and e are independent integers greater than or equal to 0; or the connector is selected from: (EAAAK)3(SEQ ID NO: 94), (EAAAR)3(SEQ ID NO: 95), (EGGGK)3(SEQ ID NO: 96), (EGGGR)3(SEQ ID NO: 97), (DAAAR)3(SEQ ID NO: 98), (DAAAK)3(SEQ ID NO: 99), (DGGGR)3(SEQ ID NO: 100) or (DGGGK)3(SEQ ID NO: 101); or the connector is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS(SEQ ID NO: 102) or GGSGGGGSGG(SEQ ID NO: 102). NO: 103); or the connector is (GxZy)i, where Z is selected from P, A, L or S, x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to GGPPG (SEQ ID NO: 104), GAGPG (SEQ ID NO: 105) or GPLS (SEQ ID NO: 106); or the connector is (AxYz)i, where x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to AAY.

[0295] In some implementations, the connector is EAAAK (SEQ ID NO: 107), AAY, or GGPPG (SEQ ID NO: 104).

[0296] In some embodiments, the target protein (e.g., a first target protein, a second target protein, and / or a third target protein) further comprises a signal peptide. In some embodiments, the signal peptide is fused to the target protein (e.g., a first target protein, a second target protein, and / or a third target protein) of this disclosure, either directly or via a linker (e.g., a linker having the amino acid sequence GGSGGGGSGG (SEQ ID NO: 103)). In some embodiments, the signal peptide has a length of about 15 to 30 amino acids. In some embodiments, the signal peptide is located at the N-terminus of the target protein (e.g., a first target protein, a second target protein, and / or a third target protein). In some embodiments, the signal peptide allows the transport of RNA-encoded polypeptides or proteins to defined cellular compartments, such as the cell surface, endoplasmic reticulum (ER), or endosome-lysosome compartments. In some embodiments, the signal peptide sequence includes, but is not limited to, signal peptide sequences derived from sequences encoding human MHC class I complexes (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3). In some embodiments, the signal peptide comprises an amino acid sequence as shown in SEQ ID NO:92 or having at least 80% sequence identity with it.

[0297] In some embodiments, the target protein (e.g., a first target protein, a second target protein, and / or a third target protein) further comprises a MITD domain. The MITD domain may correspond to the transmembrane and cytoplasmic domains of MHC class I molecules, also referred to as an MHC class I transport domain. In some embodiments, the MITD domain enhances the processing and presentation of the antigenic peptide. In some embodiments, the MITD domain comprises an amino acid sequence as shown in SEQ ID NO:93 or having at least 80% sequence identity with it.

[0298] In some embodiments, the composition comprises a first RNA, a second RNA, and an optional third RNA, the composition being selected from any group thereof:

[0299] (a) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it.

[0300] (b) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:50 or having at least 90% sequence identity with it.

[0301] (c) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:59 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it.

[0302] (d) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it, and the third RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:71 or having at least 90% sequence identity with it.

[0303] (e) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it.

[0304] (f) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:53 or having at least 90% sequence identity with it.

[0305] (g) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:59 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it.

[0306] (h) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it, and the third RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:71 or having at least 90% sequence identity with it.

[0307] (i) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:47 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it.

[0308] (j) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:62 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:68 or having at least 90% sequence identity with it.

[0309] (k) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:47 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it.

[0310] (l) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:62 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it.

[0311] In some embodiments, the RNA molecule comprises a nucleotide sequence as shown in or having at least 90% sequence identity with any of SEQ ID NO:46, 49, 58, 61, 64, 67 and 70.

[0312] In some embodiments, the RNA molecule (e.g., the first, second, and / or third RNA molecule) further comprises an untranslated region element (UTR). In some embodiments, the UTR is derived from the UTR of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. In some embodiments, the above-mentioned genes are human genes.

[0313] In some embodiments, the untranslated region element (UTR) includes a 5' UTR and a 3' UTR. In some embodiments, the 3' UTR and 5' UTR are of the same or different origins, for example, from the same or different genes. For example, the 3' UTR originates from the 3' UTR of gene ACTG1, and the 5' UTR originates from the 5' UTR of gene ACTG1. Another example is that the 3' UTR originates from the 3' UTR of gene CTSB, and the 5' UTR originates from the 5' UTR of gene CHCHD10. In some embodiments, the 5' UTR and 3' UTR originate from the same or different species.

[0314] In some embodiments, the 5'UTR is located upstream of the ORF. In some embodiments, the 5'UTR is located at the 5' end of the ORF. In some embodiments, the 5'UTR is selected from the 5'UTR or a derived sequence of any of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. In some embodiments, the 5'UTR is derived from or is a derived sequence of the 5'UTR of the gene ACTG1. In some embodiments, the 5'UTR contains the sequence shown in SEQ ID NO:33 or has at least 80% identity with it.

[0315] In some embodiments, the 3'UTR is located downstream of the ORF. In some embodiments, the 3'UTR is located at the 3' end of the ORF. In some embodiments, the 3'UTR is selected from the 3'UTR or a derived sequence of any of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9. In some embodiments, the 3'UTR is derived from or is a derived sequence of the 3'UTR of the gene CTSB. In some embodiments, the 3'UTR contains the sequence shown in SEQ ID NO:35 or has at least 80% identity with it.

[0316] In some embodiments, the RNA molecule (e.g., first, second, and / or third RNA molecules) contains a 5' UTR and a 3' UTR, wherein:

[0317] The 5'UTR is selected from the 5'UTR or a derivative sequence of any of the following genes: ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. The 3'UTR is selected from the 3'UTR or a derivative sequence of any of the following genes: ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9.

[0318] In some embodiments, the first, second, and / or third RNA contains a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any of the following:

[0319] The 5'UTR is derived from or is a 5'UTR of the ACTG1 gene or a derivative thereof, and the 3'UTR is derived from or is a 3'UTR of the CTSB gene or a derivative thereof.

[0320] In some embodiments, the RNA molecule (e.g., a first, second, and / or third RNA molecule) contains a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following:

[0321] The 5'UTR contains or is a nucleotide sequence as shown in SEQ ID NO:33 or a nucleotide sequence having at least 80% sequence identity with it, and / or the 3'UTR contains or is a nucleotide sequence as shown in SEQ ID NO:35 or a nucleotide sequence having at least 80% sequence identity with it;

[0322] In some embodiments, the RNA molecule (e.g., the first, second, and / or third RNA molecule) also includes a poly-A tail.

[0323] In some embodiments, the poly-A tail in the RNA molecule (e.g., the first, second, and / or third RNA molecule) is located downstream of the 3' UTR. In some embodiments, the poly-A tail in the RNA molecule (e.g., the first, second, and / or third RNA molecule) is located at the 3' end of the 3' UTR. In some embodiments, the poly-A tail is located at the 3' end of the RNA molecule. In some embodiments, the poly-A tail is at least about 50, 100, 150, 200, 300, 400, or 500 nucleotides in length.

[0324] In some implementations, the poly-A tail includes, but is not limited to, tails selected from 120A, Poly A-3070, HGH polyA, SV40polyA, BGH polyA, rbGlob polyA, or SV40late polyA.

[0325] In some embodiments, the poly-A tail is selected from 120A or Poly A-3070, which comprises or has at least 80% sequence identity with the sequence shown in SEQ ID NO:36.

[0326] In some embodiments, the RNA molecule (e.g., the first, second, and / or third RNA molecule) further comprises a 5' cap structure.

[0327] In some embodiments, the 5' cap structure in the RNA molecule (e.g., the first, second, and / or third RNA molecule) is located upstream of the 5' UTR. In some embodiments, the 5' cap structure in the RNA molecule is located at the 5' end of the 5' UTR. In some embodiments, the 5' cap structure is a cap structure known to those skilled in the art, such as Cap0 (methylation of the first base, e.g., m7GpppN), Cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN, e.g., m7G(5')ppp(5')(2'OMeA)pG), Cap2 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphate thioester modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deazo-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0328] In some implementations, 5'-cap structures (such as Cap0 or Cap1) are formed using chemical RNA synthesis or in vitro RNA transcription (co-transcriptional capping).

[0329] In some embodiments, a capping enzyme (e.g., a vaccinia virus capping enzyme and / or a cap-dependent 2'-O methyltransferase) is used to form a 5'-cap structure (such as Cap0 or Cap1) via enzymatic capping. In some embodiments, an immobilized capping enzyme is used to add a 5' cap structure (Cap0 or Cap1). The capping methods and means described in WO2016 / 193226 are incorporated herein by reference in their entirety.

[0330] In some implementations, the 5' cap structure includes, but is not limited to, ARCA, 3'OMe-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0331] In some embodiments, the composition is selected from any one of the following groups:

[0332] (a) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:46 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:40 or having at least 90% sequence identity with it;

[0333] (b) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:58 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:52 or having at least 90% sequence identity with it;

[0334] (c) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:61 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:67 or having at least 90% sequence identity with it.

[0335] (d) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:58 or having at least 90% sequence identity with it, the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:40 or having at least 90% sequence identity with it, and the third RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:73 or having at least 90% sequence identity with it.

[0336] (e) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:46 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:43 or having at least 90% sequence identity with it;

[0337] (f) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:58 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:55 or having at least 90% sequence identity with it;

[0338] (g) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:61 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:67 or having at least 90% sequence identity with it.

[0339] (h) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:58 or having at least 90% sequence identity with it, the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:43 or having at least 90% sequence identity with it, and the third RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:73 or having at least 90% sequence identity with it.

[0340] (i) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:49 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:43 or having at least 90% sequence identity with it;

[0341] (j) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:64 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:70 or having at least 90% sequence identity with it;

[0342] (k) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:49 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:40 or having at least 90% sequence identity with it;

[0343] (l) The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:64 or having at least 90% sequence identity with it, and the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:67 or having at least 90% sequence identity with it.

[0344] In some implementations, the RNA molecule may be mRNA.

[0345] In some embodiments, this disclosure provides a target protein encoded by any of the aforementioned RNA molecules. The target protein is produced in vitro or in vivo.

[0346] Polynucleotides, vectors

[0347] This disclosure also provides an isolated polynucleotide. Exemplarily, the polynucleotide includes, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threonucleic acid (TNA), gamma-hydroxyl nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), including LNA having a β-D-ribose configuration, α-LNA having an α-L-ribose configuration (diastereomers of LNA), 2′-amino-LNA having 2′-amino functionalization and 2′-amino-α-LNA having 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or chimeras or combinations thereof.

[0348] In some embodiments, the polynucleotide encodes the aforementioned target protein or polypeptide, for example, the polynucleotide is RNA (e.g., mRNA) containing an open reading frame sequence encoding the target protein or polypeptide. In some embodiments, the polynucleotide encodes the aforementioned RNA molecule, for example, the polynucleotide is template DNA (e.g., cDNA) containing the RNA molecule transcribed from the aforementioned RNA molecule.

[0349] In some embodiments, the polynucleotide is isolated DNA, which is transcribed to obtain the aforementioned RNA molecule. Exemplarily, in vitro transcription of RNA is known in the art and described in WO / 2014 / 152027, which is incorporated herein by reference in its entirety. For example, in some embodiments, an RNA transcript is produced by using a non-amplified, linearized DNA template in an in vitro transcription reaction. In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA transcript is purified by chromatographic methods, such as using an oligomeric dT substrate. Some embodiments do not include the use of DNases. In some embodiments, RNA transcripts are synthesized from a non-amplified, linear DNA template encoding the gene of interest via an enzymatic in vitro transcription reaction using a T7 phage RNA polymerase of desired chemical properties and a nucleotide triphosphate. Any number of RNA polymerases or variants can be used in the methods of this disclosure. The polymerase may be selected from (but is not limited to) phage RNA polymerases (e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNa polymerase), and / or mutant polymerases, such as (but not limited to) polymerases capable of combining modified nucleic acids and / or modified nucleotides (including chemically modified nucleic acids and / or nucleotides).

[0350] In some embodiments, non-amplified, linearized plasso DNA is used as template DNA for in vitro transcription. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of RNA polynucleotides. In some embodiments, cells (e.g., bacterial cells, such as E. coli, or DH-1 cells) are transfected with the plasso DNA template. In some embodiments, the transfected cells are cultured to replicate the plasso DNA, which is then isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, such as a 5' promoter located at the gene of interest and a T7 promoter operatively linked to the gene of interest.

[0351] In some implementations, the polynucleotide is codon-optimized. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some implementations, optimization algorithms are used to optimize open reading frame (ORF) sequences.

[0352] In some implementations, this disclosure provides nucleic acid constructs that encode the aforementioned RNA molecules.

[0353] In some embodiments, the nucleic acid construct includes an open reading frame (ORF) encoding at least one of the aforementioned target proteins or peptides.

[0354] In some embodiments, the target protein or polypeptide comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, OAS1, TERT, and / or survivin.

[0355] In some embodiments, the antigenic peptide derived from ECT2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:1 and / or SEQ ID NO:2. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TPX2 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:3. In some embodiments, the antigenic peptide derived from C12orf32 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:5. In some embodiments, the antigenic peptide derived from OAS1 comprises an amino acid sequence shown in or having at least 80% sequence identity with SEQ ID NO:6.

[0356] In some embodiments, the ECT2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, containing at least 9-31 consecutive amino acids. In some embodiments, the ECT2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the ECT2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:7 or 8, or having at least 80% sequence identity with it.

[0357] In some embodiments, the antigenic peptide derived from TOP2A comprises the amino acid sequence shown in SEQ ID NO:4, which contains at least 11-31 consecutive amino acids. In some embodiments, the antigenic peptide derived from TOP2A further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:4. In some embodiments, the antigenic peptide derived from TOP2A comprises an amino acid sequence as shown in SEQ ID NO:10 or having at least 80% sequence identity with it.

[0358] In some embodiments, the TPX2-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:3, which contains at least 11-31 consecutive amino acids. In some embodiments, the TPX2-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the TPX2-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:9 or having at least 80% sequence identity with it.

[0359] In some embodiments, the C12orf32-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:5, which contains at least 9-31 consecutive amino acids. In some embodiments, the C12orf32-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:5. In some embodiments, the C12orf32-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:11 or having at least 80% sequence identity with it.

[0360] In some embodiments, the OAS1-derived antigenic peptide comprises the amino acid sequence shown in SEQ ID NO:6, which contains at least 10-31 consecutive amino acids. In some embodiments, the OAS1-derived antigenic peptide further comprises an enzyme cleavage site at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the OAS1-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:12 or having at least 80% sequence identity with it.

[0361] In some embodiments, the target protein comprises an amino acid sequence as shown in any one of SEQ ID NO:1, 2, 3, 4, 5, 6 or having at least 80% sequence identity with it, or any combination thereof.

[0362] In some embodiments, the target protein comprises an amino acid sequence as shown in any one of SEQ ID NO:7, 8, 9, 10, 11, 12 or having at least 80% sequence identity with it, or any combination thereof.

[0363] In some implementations, the target protein further comprises one or more antigenic peptides derived from hTERT and / or survivin.

[0364] In some embodiments, the hTERT-derived antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or having at least 80% sequence identity with it, or any combination thereof. In some embodiments, the survivin-derived antigenic peptide comprises an amino acid sequence as shown in SEQ ID NO:21 or 22 or having at least 80% sequence identity with it, or any combination thereof.

[0365] In some embodiments, the target protein or polypeptide contains at least one antigenic peptide derived from KRAS.

[0366] In some embodiments, the KRAS-derived antigenic peptide comprises an amino acid sequence as shown in any one of SEQ ID NO:13-16 or having at least 80% sequence identity with it, or any combination thereof.

[0367] In some implementations, the antigenic peptides in the target protein are arbitrarily linked directly by peptide bonds or by linkers.

[0368] In some implementations, the linker is a peptide linker.

[0369] In some implementations, the connector is as follows: (GS)a(GGS)b(GGGS)c(GGGGS)d(GGGGG)e, where a, b, c, d, and e are independent integers greater than or equal to 0; or the connector is selected from: (EAAAK)3(SEQ ID NO: 94), (EAAAR)3(SEQ ID NO: 95), (EGGGK)3(SEQ ID NO: 96), (EGGGR)3(SEQ ID NO: 97), (DAAAR)3(SEQ ID NO: 98), (DAAAK)3(SEQ ID NO: 99), (DGGGR)3(SEQ ID NO: 100) or (DGGGK)3(SEQ ID NO: 101); or the connector is (GxS)y, where x is an integer selected from 1 to 5, and y is an integer selected from 1 to 6, including but not limited to GGGS(SEQ ID NO: 102) or GGSGGGGSGG(SEQ ID NO: 102). NO: 103); or the connector is (GxZy)i, where Z is selected from P, A, L or S, x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to GGPPG (SEQ ID NO: 104), GAGPG (SEQ ID NO: 105) or GPLS (SEQ ID NO: 106); or the connector is (AxYz)i, where x is selected from integers from 0 to 8, y is selected from integers from 0 to 8, and i is selected from integers from 1 to 5, including but not limited to AAY.

[0370] In some implementations, the connector is EAAAK (SEQ ID NO: 107), AAY, or GGPPG (SEQ ID NO: 104).

[0371] In some embodiments, the target protein or polypeptide further comprises a signal peptide. In some embodiments, the signal peptide is fused to the target protein or polypeptide of this disclosure directly or via a linker (e.g., a linker having the amino acid sequence GGSGGGGSGG (SEQ ID NO: 103)). In some embodiments, the signal peptide has a length of about 15 to 30 amino acids. In some embodiments, the signal peptide is located at the N-terminus of the target protein or polypeptide. In some embodiments, the signal peptide allows the transport of RNA-encoded polypeptides or proteins to defined cellular compartments, such as the cell surface, endoplasmic reticulum (ER), or endosome-lysosome compartments. In some embodiments, the signal peptide sequence includes, but is not limited to, signal peptide sequences derived from sequences encoding human MHC class I complexes (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3). In some embodiments, the signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 92 or having at least 80% sequence identity with it.

[0372] In some embodiments, the target protein or polypeptide further comprises a MITD domain. The MITD domain may correspond to the transmembrane and cytoplasmic domains of MHC class I molecules, also known as an MHC class I transport domain. In some embodiments, the MITD domain enhances the processing and presentation of the antigenic peptide. In some embodiments, the MITD domain comprises an amino acid sequence as shown in SEQ ID NO:93 or having at least 80% sequence identity with it.

[0373] In some embodiments, the target protein or polypeptide comprises an amino acid sequence as shown in or having at least 90% sequence identity with any of SEQ ID NO:44, 47, 56, 59, 62, 65, 68.

[0374] In some embodiments, the nucleic acid construct further comprises untranslated region elements (UTRs). In some embodiments, the UTR is derived from the UTR of genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. In some embodiments, the aforementioned genes are human genes.

[0375] In some embodiments, the untranslated region element (UTR) includes a 5' untranslated region element (5'UTR) and a 3' untranslated region element (3'UTR). In some embodiments, the 3'UTR and 5'UTR are of the same or different origins, for example, from the same or different genes. In some embodiments, the 5'UTR and 3'UTR are from the same or different species.

[0376] In some embodiments, the 5'UTR is located upstream of the ORF. In some embodiments, the 5'UTR is located at the 5' end of the ORF. In some embodiments, the 5'UTR is selected from the 5'UTR or a derived sequence of any of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP. In some embodiments, the 5'UTR is derived from or is a derived sequence of the 5'UTR of the gene ACTG1. In some embodiments, the 5'UTR contains the sequence shown in SEQ ID NO:33 or has at least 80% identity with it.

[0377] In some embodiments, the 3'UTR is located downstream of the ORF. In some embodiments, the 3'UTR is located at the 3' end of the ORF. In some embodiments, the 3'UTR is selected from the 3'UTR or a derived sequence of any of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9. In some embodiments, the 3'UTR is derived from or is a derived sequence of the 3'UTR of the gene CTSB. In some embodiments, the 3'UTR contains the sequence shown in SEQ ID NO:35 or has at least 80% identity with it.

[0378] In some embodiments, the RNA molecule comprises a 5'UTR and a 3'UTR, wherein: the 5'UTR is selected from the 5'UTR or a derivative sequence thereof derived from or from any of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, NDUFB9, CHCHD10, SLC38A2, NDUFA11, NDUFV3, PRDX5, GUK1, IAH1, ABHD16A, SLC25A39, ATPIF1, ANAPC11, CCDC12, MRPL14, or APOA1BP; and the 3'UTR is selected from the 3'UTR or a derivative sequence thereof derived from or from any of the genes ACTG1, ATP6V0B, ATP6V0E1, CFL1, COX4I1, CTSB, FAM166A, or NDUFB9.

[0379] In some embodiments, the RNA molecule contains a 5'UTR and a 3'UTR, wherein the 5'UTR and 3'UTR are selected from any one of the following:

[0380] The 5'UTR is derived from or is a 5'UTR of the ACTG1 gene or a derivative thereof, and the 3'UTR is derived from or is a 3'UTR of the CTSB gene or a derivative thereof.

[0381] In some implementations, the nucleic acid construct further includes a poly-A tail.

[0382] In some embodiments, the poly-A tail in the nucleic acid construct is located downstream of the 3' UTR. In some embodiments, the poly-A tail in the nucleic acid construct is located at the 3' end of the 3' UTR. In some embodiments, the poly-A tail is located at the 3' end of the nucleic acid construct. In some embodiments, the poly-A tail is at least about 50, 100, 150, 200, 300, 400, or 500 nucleotides long.

[0383] In some implementations, the poly-A tail includes, but is not limited to, tails selected from 120A, Poly A-3070, HGH polyA, SV40polyA, BGH polyA, rbGlob polyA, or SV40late polyA.

[0384] In some embodiments, the poly-A tail is selected from 120A or Poly A-3070, which comprises or has at least 80% sequence identity with the sequence shown in SEQ ID NO:36.

[0385] The nucleic acid constructs disclosed herein can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on the nucleotide sequence information disclosed herein, and / or can be isolated from suitable natural sources.

[0386] Chemical modification

[0387] In some embodiments, the polynucleotides (e.g., RNA) disclosed herein contain at least one chemical modification.

[0388] The terms “chemically modified” and “chemically modified” refer to modifications of at least one of the ribonucleotides or deoxyribonucleotides, namely adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C), in terms of their position, pattern, percentage, or population. Typically, these terms do not refer to modifications of the ribonucleotides in the naturally occurring 5′ end cap of mRNA.

[0389] Modifications to polynucleotides include, but are not limited to, those described herein, and include (but are not explicitly limited to) those involving chemical modifications. Polynucleotides (e.g., RNA, such as mRNA) may contain naturally occurring, non-naturally occurring modifications, or a combination of naturally occurring and non-naturally occurring modifications. Polynucleotides may include any suitable modifications to, for example, inter-sugar, base, or nucleoside linkages (e.g., linkages to phosphate esters, phosphodiester linkages, or the phosphodiester backbone).

[0390] For example, chemical modifications include, but are not limited to, the modification types disclosed in WO2017070601A and WO2017070623A, which are incorporated herein by reference.

[0391] In some embodiments, the polynucleotide (e.g., RNA, such as mRNA) contains multiple (more than one) different modifications. In some embodiments, specific regions of the polynucleotide contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified RNA (e.g., modified mRNA) introduced into cells or organisms exhibits reduced degradation relative to the unmodified polynucleotide in cells or organisms. In some embodiments, the modified RNA (e.g., modified mRNA) introduced into cells or organisms may exhibit reduced immunogenicity (e.g., reduced innate response) in cells or organisms.

[0392] In some implementations, the polynucleotide (e.g., RNA, such as mRNA) comprises a non-naturally modified nucleotide introduced during or after polynucleotide synthesis to achieve the desired function or property. Modifications can be present at internucleotide links, bases such as purines or pyrimidines, or sugars. Modifications can be introduced chemically or via polymerase at the chain terminus or any other location on the chain. Any region of the polynucleotide can be chemically modified.

[0393] This disclosure provides modified nucleosides and nucleotides of polynucleotides (e.g., RNA, such as mRNA). "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof, combined with a base (e.g., purine, pyrimidine, or a derivative thereof). "Nucleotide" refers to a nucleoside, including a phosphate ester group. Modified nucleotides can be synthesized by any suitable method, such as chemical, enzymatic, or recombinant methods, to include one or more modified or non-natural nucleosides. Polynucleotides may contain one or more regions of the linked nucleoside. Such regions may have variable backbone bonds. The bonds may be standard phosphodiester bonds, in which case the polynucleotide will contain the nucleotide region.

[0394] Modified nucleotide base pairings encompass 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 bonding between non-standard bases and standard bases or between two complementary non-standard base structures (for example, such as those polynucleotides having at least one chemical modification). An example of such non-standard base pairings is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of base / sugar or linker may be incorporated into the polynucleotides of this disclosure.

[0395] The polynucleotide (e.g., RNA, such as mRNA) modifications (including but not limited to chemical modifications) applicable to this disclosure include, but are not limited to, the following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine; 1-methyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine (phosphate ester); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; 2-methylthio-N6-hydroxyn-valinecarbamoyladenosine; 2′ -O-methyl adenosine; 2′-O-ribosyl adenosine (phosphate ester); isopentenyl adenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2′-O-dimethyl adenosine; N6,2′-O-dimethyl adenosine; N6,N6,2′-O-trimethyl adenosine; N6,N6-dimethyl adenosine; N6-acetyl adenosine; N6-hydroxyn-valinecarbamoyl adenosine; N6-methyl-N6-threonylcarbamoyl adenosine; 2-methyl adenosine; 2-methylthio-N6-isopentenyl adenosine; 7-deaza-adenosine; N1-methyl-adenosine; N6,N6(dimethyl)adenosine; N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2-(amino)adenosine; 2-(aminopropyl)adenosine Purine; 2-(methylthio)N6-(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halogenated)adenine; 2-(halogenated)adenine; 2-(propyl)adenine; 2′-amino-2′-deoxy-ATP; 2′-azido-2′-deoxy-ATP; 2′-deoxy-2′-a-aminoadenine TP; 2′-deoxy-2′-a-azido-adenine TP; 6-(alkyl)adenine; 6-(methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7-(deaza)adenine; 8-(alkenyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(thioalkyl)adenine; 8-(alkenyl ... 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halogenated)adenine; 8-(hydroxy)adenine; 8-(thioalkyl)adenine; 8-(thiolyl)adenine; 8-azido-adenine; azaadenine; deazaadenine; N6(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-azaadenine; 7-methyladenine; 1-deazaadenine TP; 2′-fluoro-N6-Bz-deoxyadenine TP; 2′-OMe-2-amino-ATP; 2′-O-methyl-N6-Bz-deoxyadenine TP; 2′-α-ethynyladenine TP; 2-aminoadenine; 2-aminoadenine TP; 2-amino-ATP;2′-a-trifluoromethyladenosine TP; 2-azidoadenosine TP; 2′-b-ethynyladenosine TP; 2-bromoadenosine TP; 2′-b-trifluoromethyladenosine TP; 2-chloroadenosine TP; 2′-deoxy-2′,2′-difluoroadenosine TP; 2′-deoxy-2′-a-mercaptoadenosine TP; 2′-deoxy-2′-a-thiomethoxyadenosine TP; 2′-deoxy-2′-b-aminoadenosine TP; 2′-deoxy-2′-b-azidoadenosine TP; 2′-deoxy-2′-b-bromoadenosine TP; 2′-deoxy-2′-b-chloroadenosine TP; 2′-deoxy-2′-b-fluoroadenosine TP; 2′-deoxy-2′-b-iodoadenosine TP; 2′-deoxy-2′-a-difluoro ... -b-mercaptoadenosine TP; 2′-deoxy-2′-b-thiomethoxyadenosine TP; 2-fluoroadenosine TP; 2-iodoadenosine TP; 2-mercaptoadenosine TP; 2-methoxy-adenosine; 2-methylthio-adenosine; 2-trifluoromethyladenosine TP; 3-deaza-3-bromoadenosine TP; 3-deaza-3-chloroadenosine TP; 3-deaza-3-fluoroadenosine TP; 3-deaza-3-iodoadenosine TP; 3-deaza-3-adenosine TP; 4′-azidoadenosine TP; 4′-carbocyclic adenosine TP; 4′-ethynyladenosine TP; 5′-homo-adenosine TP; 8-aza-ATP; 8-bromo-adenosine TP; 8-trifluoromethyladenosine TP; 9-deazaadenosine TP; 2-aminopurine; 7-deaza-2 6-Diaminopurine; 7-Deaza-8-aza-2,6-Diaminopurine; 7-Deaza-8-aza-2-aminopurine; 2,6-Diaminopurine; 7-Deaza-8-aza-adenine; 7-Deaza-2-aminopurine; 2-Thiocytidine; 3-Methylcytidine; 5-Formylcytidine; 5-Hydroxymethylcytidine; 5-Methylcytidine; N4-Acetylcytidine; 2′-O-Methylcytidine; 2′-O-Methylcytidine; 5,2′-O-Dimethylcytidine; 5-Formyl-2′-O-Methylcytidine; Lysidine; N4,2′-O-Dimethylcytidine; N4-Acetyl-2′-O-Methylcytidine; N4-Methylcytidine; N4,N4-Dimethyl-2′-OM e-cytidine TP; 4-methylcytidine; 5-aza-cytidine; pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine; 2-(thio)cytosine; 2′-amino-2′-deoxy-CTP; 2′-azido-2′-deoxy-CTP; 2′-deoxy-2′-a-aminocytidine TP; 2′-deoxy-2′-a-azidocytidine TP; 3(deaza)5(aza)cytidine; 3(methyl)cytidine; 3-(alkyl)cytidine; 3-(deaza)5(aza)cytidine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine; 5(halogenated)cytidine; 5(methyl)cytidine; 5(propynyl)cytidine; 5(trifluoromethyl)cytidine; 5-(alkyl)cytidine5-(Alynyl)cytosine; 5-(halogenated)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propynylcytosine; 6-(azo)cytosine; 6-aza-cytidine; aza-cytosine; deaza-cytosine; N4-(acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza- -Zebularine; 5-Methyl-Zebularine; Pyrrolo-Pseudoisocytidine; Zebularine; (E)-5-(2-bromo-vinyl)cytidine TP; 2,2′-Dehydro-cytidine TP hydrochloride; 2′-Fluoro-N4-Bz-cytidine TP; 2′-Fluoro-N4-acetyl-cytidine TP; 2′-O-methyl-N4-acetyl-cytidine TP; 2′-O-methyl-N4-Bz-cytidine TP; 2′-a-ethynylcytidine TP; 2′-a-trifluoromethylcytidine TP; 2′-b-ethynylcytidine TP; 2′-b-trifluoromethylcytidine TP; 2′-deoxy-2′,2′-difluorocytidine TP; 2′-deoxy-2′-a-mercaptocytidine TP; 2′-deoxy-2′ -a-thiomethoxycytidine TP; 2′-deoxy-2′-b-aminocytidine TP; 2′-deoxy-2′-b-azidocytidine TP; 2′-deoxy-2′-b-bromocytidine TP; 2′-deoxy-2′-b-chlorocytidine TP; 2′-deoxy-2′-b-fluorocytidine TP; 2′-deoxy-2′-b-iodocytidine TP; 2′-deoxy-2′-b-mercaptocytidine TP; 2′-deoxy-2′-b-thiomethoxycytidine TP; 2′-O-methyl-5-(1-propynyl)cytidine TP; 3′-ethynylcytidine TP; 4′-azidocytidine TP; 4′-carbocycliccytidine TP; 4′-ethynylcytidine TP; 5-(1-propynyl)arsylcytidine TP; 5 -(2-chloro-phenyl)-2-thiocytidine TP; 5-(4-amino-phenyl)-2-thiocytidine TP; 5-aminoallyl-CTP; 5-cyanocytidine TP; 5-ethynylarsyl-cytidine TP; 5-ethynylcytidine TP; 5′-homocytidine TP; 5-methoxycytidine TP; 5-trifluoromethyl-cytidine TP; N4-amino-cytidine TP; N4-benzylyl-cytidine TP; pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine; N2-methylguanosine; Wyorrhin; 1,2′-O-dimethylguanosine; 1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate ester); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate ester);7-Aminomethyl-7-deazaguanosine; 7-Cyano-7-deazaguanosine; Archapurin; Methylwyoside; N2,7-Dimethylguanosine; N2,N2,2′-O-Trimethylguanosine; N2,N2,7-Trimethylguanosine; N2,N2-Dimethylguanosine; N2,7,2′-O-Trimethylguanosine; 6-Thio-guanosine; 7-Deaza-guanosine; 8-O-guanosine; N1-Methyl-guanosine; α-Thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP; 2′-Azide-2′-deoxy-GTP; 2′-deoxy-2′-a-aminoguanosine TP; 2′-deoxy-2′-a-azido-guanosine TP; 6(methyl)guanosine Ale; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(halogenated)guanine; 8-(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halogenated)guanine; 8-(hydroxy)guanine; 8-(thioalkyl)guanine; 8-(thiolyl)guanine; azaguanine; deazaguanine; N-(methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio- Guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′-fluoro-N2-isobutyl-guanosine TP; 2′-O-methyl-N2-isobutyl-guanosine TP; 2′-a-ethynylguanosine TP; 2′-a-trifluoromethylguanosine TP; 2′-b-ethynylguanosine TP; 2′-b-trifluoromethylguanosine TP; 2′-deoxy-2′,2′-difluoroguanosine TP; 2′-deoxy-2′-a-mercaptoguanosine TP; 2′-deoxy-2′-a-thiomethoxyguanosine TP; 2′-deoxy-2′-b-aminoguanosine TP; 2′-deoxy-2′-b-azidoguanosine TP; 2′-deoxy-2′-b-bromoguanosine TP; 2′-deoxy-2′-b-chloroguanosine TP; 2′-deoxy-2′-b-fluoroguanosine TP; 2′-deoxy-2′-b-iodoguanosine TP; 2′-deoxy-2′-b-mercaptoguanosine TP; 2′-deoxy-2′-b-thiomethoxyguanosine TP; 4′-azidoguanosine TP; 4′-carbocyclic guanosine TP; 4′-ethynylguanosine TP; 5′-homo-guanosine TP; 8-bromo-guanosine TP; 9-deazaguanosine TP; N2-isobutyl-guanosine TP;1-Methylinosine; Inosine; 1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine; 2′-O-methylinosine; epoxy brassinoside; galactosyl brassinoside; mannosyl brassinoside; brassinoside; allylamino-thymidine; azathymidine; dezathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine; 3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurate methyl-2-thiouridine; 5-taurate methyluridine; dihydrouridine; pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; 1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseudouridine; 1-ethylpseudouridine; 2′-O-methyluridine Glycosides; 2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine; 3-methyl-pseudo-uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methyl ester; 5,2′-O-dimethyluridine; 5,6-dihydrouridine; 5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine; 5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2′-O-methyluridine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethyl Aminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5-carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine; 5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine; 5-methyluridine, 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-methyldihydrouridine; 5-oxyacetic acid-uridine TP; 5-oxyacetic acid-methyl ester-uridine TP; N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3- (3-Amino-3-carboxypropyl)-uridine TP; 5-(isopentenylaminomethyl)-2-thiouridine TP; 5-(isopentenylaminomethyl)-2′-O-methyluridine TP; 5-(isopentenylaminomethyl)uridine TP; 5-propynyluracil; α-thiouridine; 1-(aminoalkylamino-carbonylvinyl)-2-(thio)-pseudouracil; 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil; 1-(aminoalkylaminocarbonylvinyl)-4-(thio)pseudouracil; 1-(aminoalkylaminocarbonylvinyl)-pseudouracil; 1-(aminocarbonylvinyl)-2-(thio)-pseudouracil; 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil;1(aminocarbonylvinyl)-4(thio)pseudouracil; 1(aminocarbonylvinyl)-pseudouracil; 1-substituted 2(thio)-pseudouracil; 1-substituted 2,4-(dithio)pseudouracil; 1-substituted 4(thio)pseudouracil; 1-substituted pseudouracil; 1-(aminoalkylamino-carbonylvinyl)-2-(thio)-pseudouracil; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-methyl-pseudo-UTP; 1-ethyl-pseudo-UTP; 2(thio)pseudouracil; 2′deoxyuridine; 2′fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2′methyl,2′ Amino, 2′-azido, 2′-fluoro-guanosine; 2′-amino-2′-deoxy-UTP; 2′-azido-2′-deoxy-UTP; 2′-azido-deoxyuridine TP; 2′-O-methylpseudouridine; 2′-deoxyuridine; 2′-fluorouridine; 2′-deoxy-2′-a-aminouridine TP; 2′-deoxy-2′-a-azidouridine TP; 2-methylpseudouridine; 3(3amino-3-carboxypropyl)uracil; 4(thio)pseudouridine; 4-(thio)pseudouridine; 4-(thio)uracil; 4-thiouracil; 5(1,3-diazol-1-alkyl)uracil; 5(2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5(dimethylaminoalkyl)uracil 5-(guanidinyl)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl)-2-(thio)uracil; 5-(methyl)-2,4-(dithio)uracil; 5-(methyl)-4-(thio)uracil; 5-(methylaminomethyl)-2-(thio)uracil; 5-(methylaminomethyl)-2,4-(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4-(dithio)pseudouracil; 5-(alkyl)-4-(thio)pseudouracil; 5-(alkyl)pseudouracil Pyridine; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidinyl)uracil; 5-(halogenated)uracil; 5-(1,3-diazol-1-alkyl)uracil; 5-(methoxy)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl)2-(thio)uracil; 5-(methyl)2,4-(dithio)uracil; 5-(methyl)4-(thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4-(dithio)pseudouracil;5-(methyl)-4-(thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2-(thio)uracil; 5-(methylaminomethyl)-2,4-(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6-(azo)uracil; 6-(azo)uracil; 6-aza-uridine; allylamino-uridine; aza-uridine; deaza-uridine; N3(methyl)uracil; pseudo-UTP-1-2-acetic acid; pseudouracil; 4-thio-pseudo-UTP; 1-carboxymethyl-pseudouracil; 1-Methyl-1-deaza-pseudouridine; 1-propynyl-uridine; 1-Taurate methyl-1-methyl-uridine; 1-Taurate methyl-4-thio-uridine; 1-Taurate methyl-pseudouridine; 2-Methoxy-4-thio-pseudouridine; 2-Thio-1-methyl-1-deaza-pseudouridine; 2-Thio-1-methyl-pseudouridine; 2-Thio-5-aza-uridine; 2-Thio-dihydropseudouridine; 2-Thio-dihydrouridine; 2-Thio-pseudouridine; 4-Methoxy-2-thio-pseudouridine; 4-Methoxy-pseudouridine; 4-Thio-1-methyl-pseudouridine; 4-Thio-pseudouridine; 5-aza-uridine; dihydropseudouridine; (±)1-(2-hydroxypropyl)pseudouridine TP; (2R)-1- (2-Hydroxypropyl)pseudouridine TP; (2S)-1-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2-bromo-vinyl)arsyluridine TP; (E)-5-(2-bromo-vinyl)uridine TP; (Z)-5-(2-bromo-vinyl)arsyluridine TP; (Z)-5-(2-bromo-vinyl)uridine TP; 1-(2,2,2-trifluoroethyl)pseudo-UTP; 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine TP; 1-(2,2-diethoxyethyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudo-UTP; 1-(2,4,6-trimethylbenzyl)pseudo-U TP; 1-(2-amino-2-carboxyethyl) pseudo-UTP; 1-(2-amino-ethyl) pseudo-UTP; 1-(2-hydroxyethyl) pseudouridine TP; 1-(2-methoxyethyl) pseudouridine TP; 1-(3,4-bis-trifluoromethoxybenzyl) pseudouridine TP; 1-(3,4-dimethoxybenzyl) pseudouridine TP; 1-(3-amino-3-carboxypropyl) pseudo-UTP; 1-(3-amino-propyl) pseudo-UTP; 1-(3-cyclopropyl-prop-2-ynyl) pseudouridine TP; 1-(4-amino-4-carboxybutyl) pseudo-UTP; 1-(4-amino-benzyl) pseudo-UTP; 1-(4-amino-butyl) pseudo-UTP; 1-(4-amino-phenyl) pseudo-UTP;1-(4-azidobenzyl)pseudouridine TP; 1-(4-bromobenzyl)pseudouridine TP; 1-(4-chlorobenzyl)pseudouridine TP; 1-(4-fluorobenzyl)pseudouridine TP; 1-(4-iodobenzyl)pseudouridine TP; 1-(4-methanesulfonylbenzyl)pseudouridine TP; 1-(4-methoxybenzyl)pseudo-UTP; 1-(4-methoxy-phenyl)pseudo-UTP; 1-(4-methylbenzyl)pseudouridine TP; 1-(4-methyl-benzyl)pseudo-UTP; 1-(4-nitrobenzyl)pseudouridine TP; 1-(4-nitro-benzyl)pseudo-UTP; 1-(4-nitro-phenyl)pseudo-UTP; 1-(4-thiomethoxybenzyl)pseudouridine TP; 1-(4-trifluoromethoxybenzyl)pseudouridine TP; 1-(4-trifluoromethylbenzyl)pseudouridine TP; 1-(5-amino-pentyl)pseudo-UTP; 1-(6-amino-hexyl)pseudo-UTP; 1,6-dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudouridine TP; 1-acetylpseudouridine TP; 1-alkyl-6-(1-propynyl)-pseudo-UTP; 1-alkyl-6-(2-propynyl)-pseudo-UTP; 1-alkyl-6-allyl-pseudo-UTP; 1-alkyl-6 -Ethynyl-pseudo-UTP; 1-alkyl-6-homallyl-pseudo-UTP; 1-alkyl-6-vinyl-pseudo-UTP; 1-allyl-pseudo-uridine TP; 1-aminomethyl-pseudo-UTP; 1-benzylyl-pseudo-uridine TP; 1-benzyloxymethyl-pseudo-uridine TP; 1-benzyl-pseudo-UTP; 1-biotinyl-PEG2-pseudo-uridine TP; 1-biotinyl-pseudo-uridine TP; 1-butyl-pseudo-UTP; 1-cyanomethyl-pseudo-uridine TP; 1-cyclobutylmethyl-pseudo-UTP; 1-cyclobutyl-pseudo-UTP; 1-cycloheptylmethyl-pseudo-UTP; 1-cycloheptyl-pseudo-UTP; 1-cyclohexylmethyl-pseudo-UTP; 1-cyclohexyl-pseudo-UTP; 1-cyclooctylmethyl-pseudo- UTP; 1-Cyclooctyl-pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallyl-pseudouridine TP; 1-Hydroxymethyl-pseudo-uridine TP; 1-Iso-propyl-pseudo-UTP; 1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP; 1-Me-α-thio-pseudo-UTP; 1-Methanesulfonylmethyl-pseudouridine TP; 1-Meoxymethyl-pseudouridine TP; 1-Methyl-6-(2,2,2-trifluoroethyl)pseudo-UTP; 1-Methyl-6-(4-morpholinyl)pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substituted phenyl)-pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP; 1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP; 1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP; 1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP; 1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-carboxylic acid ethyl ester-pseudo-UTP; 1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP; 1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6 -hydroxyamino-pseudo-UTP; 1-methyl-6-hydroxy-pseudo-UTP; 1-methyl-6-iodo-pseudo-UTP; 1-methyl-6-iso-propyl-pseudo-UTP; 1-methyl-6-methoxy-pseudo-UTP; 1-methyl-6-methylamino-pseudo-UTP; 1-methyl-6-phenyl-pseudo-UTP; 1-methyl-6-propyl-pseudo-UTP; 1-methyl-6-tert-butyl-pseudo-UTP; 1-methyl-6-trifluoromethoxy-pseudo-UTP; 1-methyl-6-trifluoromethyl-pseudo-UTP; 1-morpholinylmethylpseudouridine TP; 1-pentyl-pseudo-UTP; 1-phenyl-pseudo-UTP; 1-trimethylacetylpseudouridine TP; 1-propynylpseudouridine TP; 1-propyl- Pseudo-UTP; 1-Propyno-pseudouridine; 1-p-Tolyl-pseudo-UTP; 1-Tertiaryl-pseudo-UTP; 1-Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2′-Dehydr-uridine TP; 2′-Bromo-deoxyuridine TP; 2′-F-5-methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP; 2′-a-ethynyluridine TP; 2′-a-trifluoromethyluridine TP; 2′-b-ethynyluridine TP; 2′-b-trifluoromethyluridine TP; 2′-deoxy-2′ , 2′-difluorouridine TP; 2′-deoxy-2′-a-mercaptouridine TP; 2′-deoxy-2′-a-thiomethoxyuridine TP; 2′-deoxy-2′-b-aminouridine TP; 2′-deoxy-2′-b-azidouridine TP; 2′-deoxy-2′-b-bromouridine TP; 2′-deoxy-2′-b-chlorouridine TP; 2′-deoxy-2′-b-fluorouridine TP; 2′-deoxy-2′-b-iodouridine TP; 2′-deoxy-2′-b-mercaptouridine TP; 2′-deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2′-O-methyl-5-(1-propynyl)uridine TP;3-alkyl-pseudo-UTP; 4′-azidouridine TP; 4′-carbocyclic uridine TP; 4′-ethynyluridine TP; 5-(1-propynyl)arsyluridine TP; 5-(2-furanyl)uridine TP; 5-cyanouridine TP; 5-dimethylaminouridine TP; 5′-homouridine TP; 5-iodo-2′-fluoro-deoxyuridine TP; 5-phenylethynyluridine TP; 5-trideuterylmethyl-6-deuteryluridine TP; 5-tri Fluoromethyl-uridine TP; 5-vinylaradaurin uridine TP; 6-(2,2,2-trifluoroethyl)-pseudo-UTP; 6-(4-morpholino)-pseudo-UTP; 6-(4-thiomorpholino)-pseudo-UTP; 6-(substituted phenyl)-pseudo-UTP; 6-amino-pseudo-UTP; 6-azido-pseudo-UTP; 6-bromo-pseudo-UTP; 6-butyl-pseudo-UTP; 6-chloro-pseudo-UTP; 6-cyano- False-UTP; 6-Dimethylamino-False-UTP; 6-Ethoxy-False-UTP; Ethyl 6-carboxylate-False-UTP; 6-Ethyl-False-UTP; 6-Fluoro-False-UTP; 6-Formyl-False-UTP; 6-Hydroxyamino-False-UTP; 6-Hydroxy-False-UTP; 6-Iodo-False-UTP; 6-Iso-propyl-False-UTP; 6-Methoxy-False-UTP; 6-Methylamino-False-UTP; 6- Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-Tertiary-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP; α-Thio-pseudo-UTP; Pseudouridine 1-(4-methylbenzenesulfonic acid)TP; Pseudouridine 1-(4-methylbenzoic acid)TP; Pseudouridine TP1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; pseudouridine TP 1-methylphosphonic acid; pseudouridine TP Diethyl 1-methylphosphonate; pseudo-UTP-N1-3-propionic acid; pseudo-UTP-N1-4-butyric acid; pseudo-UTP-N1-5-valeric acid; pseudo-UTP-N1-6-hexanoic acid; pseudo-UTP-N1-7-heptanoic acid; pseudo-UTP-N1-methyl-p-benzoic acid; pseudo-UTP-N1-p-benzoic acid; huaitoside; hydroxyhuaitoside; isowaitoside; peroxyhuaitoside; incompletely modified hydroxyhuaitoside; 4-demethylhuaitoside; 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 1,3,5-(triza)-2,6-(dioxa)-naphthalene; 2-(amino)purine; 2,4,5-(trimethyl)phenyl; 2′methyl, 2′amino, 2′azido, 2′fluoro-cytidine; 2′methyl, 2′amino, 2′azido, 2′fluoro-adenine; 2′methyl, 2′amino, 2′azido, 2′fluoro-uridine; 2′-amino-2′-deoxyribose; 2-amino-6-chloro-purine; 2-aza-inosine; 2′-azido-2′-deoxyribose; 2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′- O-Methyl-ribose; 2-oxo-7-aminopyridinidin-3-yl; 2-oxo-pyridinidin-3-yl; 2-pyridinone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)isoquinolone; 3-(methyl)isoquinolone; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindol; 5-substituted pyrimidine; 5-(methyl)isoquinolone; 5-nitroindol; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; 7-(aminoalkylhydroxy)-1-( 7-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-yl; 7-(guanidinylhydroxy)-1-(aza)-2-(thio) -3-(aza)-phenoxazine-1-yl; 7-(guanidinium alkyl hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl; 7-(guanidinium alkyl hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl; 7-(guanidinium alkyl hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl Azine-1-yl; 7-(guanidinyl alkyl hydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl; 7-(propynyl)isoquinolone; 7-(propynyl)isoquinolone, propynyl-7-(aza)indolyl; 7-deaza-inosine; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazine-1-yl;7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidazopyridyl; aminoindolyl; anthracene; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolopyrimidin-2-one-3-yl; bis-ortho-substituted 6-phenyl-pyrrolopyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridyl; inosine; isoquinolone; isoguanosine; N2-substituted purine; N6 -Methyl-2-amino-purine; N6-substituted purine; N-alkylated derivative; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nubularine; O6-substituted purine; O-alkylated derivative; o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; ortho-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxo-meta-mold TP; p-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; para-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; pentaphenyl; benzanthyl; phenyl; propynyl-7-(aza)indolyl; pyrene; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolopyrimidyl; pyrrolopyrimidyl Azine; stilbene; substituted 1,2,4-triazole; tetraphenyl; tubercidine; xanthine; xanthine nucleotide-5′-TP; 2-thio-zebraline; 5-aza-2-thio-zebraline; 7-deaza-2-amino-purine; pyridine-4-ketoribonucleoside; 2-amino-riboside-TP; mestyromycin ATP; mestyromycin BTP; pyrrolosine TP; 2′-OH-arabinoside-adenosine TP; 2′-OH-arabinoside-cytidine TP; 2′-OH-arabinoside-uridine TP; 2′-OH-arabinoside-guanosine TP; 5-(2-methoxycarbonylvinyl)uridine TP; and N6-(19-amino-pentaenoyl)adenosine TP.

[0396] In some implementations, the modified bases in the RNA (such as mRNA) are selected from the group consisting of: pseudouridine (ψ), 2-thiouridine (s2U), 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2′-O-methyl 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4′-thiouridine, 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6-diaminopurine, (I), 1-methyl-inosine (m1I), wyoside (imG), methyl wyoside (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza hetero-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine, 2-geranylthiouridine, 2-lysicryl, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2′-O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2-thiouridine Urate, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-demethylwyoside, 7-aminocarboxypropylwyoside, 7-aminocarboxypropylwyoside methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-quinoline, incompletely methylated hydroxywaibutidine, N4,N4,2′-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQ0base, preQ1base, and combinations of two or more thereof. In some embodiments, at least one chemically modified nucleoside is selected from the group consisting of: pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and combinations thereof. In some embodiments, RNA (such as mRNA) comprises at least two (e.g., 2, 3, 4, or more) combinations of the aforementioned modified bases. In some embodiments, RNA (such as mRNA) comprises at least two (e.g., 2, 3, 4, or more) combinations of the aforementioned modified bases.

[0397] In some embodiments, the modified bases in the polynucleotide (e.g., RNA, such as mRNA) are selected from the group consisting of: 1-methyl-pseuuridine (m1ψ), 1-ethyl-pseuuridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine, and α-thio-adenosine. In some embodiments, the RNA comprises a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified bases, including but not limited to chemical modifications.

[0398] In some embodiments, the polynucleotide (e.g., RNA, such as mRNA) comprises pseudouridine (ψ) and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA (such as mRNA) comprises 1-ethyl-pseudouridine (e1ψ). In some embodiments, the RNA (such as mRNA) comprises 1-methyl-pseudouridine (m1ψ) and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises 1-ethyl-pseudouridine (e1ψ) and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises 2-thiouridine (s2U). In some embodiments, the RNA (such as mRNA) comprises 2-thiouridine and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises methoxyuridine (mo5U). In some embodiments, the RNA (such as mRNA) comprises 5-methoxyuridine (mo5U) and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises 2′-O-methyluridine. In some embodiments, the RNA (such as mRNA) comprises 2′-O-methyluridine and 5-methylcytidine (m5C). In some embodiments, the RNA (such as mRNA) comprises N6-methyladenosine (m6A). In some embodiments, the RNA (such as mRNA) comprises N6-methyladenosine (m6A) and 5-methylcytidine (m5C).

[0399] In some implementations, polynucleotides (e.g., RNA, such as mRNA) are uniformly modified (e.g., completely modified, modified throughout the sequence) to have specific modifications. For example, a polynucleotide may be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a polynucleotide may be uniformly modified to any similar nucleoside residues present in the sequence by replacing modified residues such as those described above.

[0400] host cells

[0401] This disclosure also provides a host cell comprising any of the nucleic acid constructs, nucleic acid molecules, RNA, polynucleotides, or vectors described in the preceding embodiments. In some embodiments, the cell is capable of expressing one or more polypeptides of the nucleic acid constructs, nucleic acid molecules, RNA, polynucleotides, or vectors disclosed herein. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0402] Bacterial cells include, for example, cells of Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0403] Fungal cells include, for example, cells of species from the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells of species from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichia pastoris* and *Pichia methanolica*), and *Hansenula*.

[0404] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0405] However, this disclosure may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0406] Production or preparation method

[0407] This disclosure provides a method for preparing tumor-associated antigen peptides, polynucleotides, vectors, nucleic acid molecules, or RNA.

[0408] In some embodiments, the methods used to prepare the polypeptides, polynucleotides, nucleic acid molecules, or RNA are known in the art, such as specific suitable vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions. Similarly, protein isolation and purification techniques suitable for the methods of producing the polypeptides encoded by this disclosure are well known to those skilled in the art.

[0409] In some embodiments, the method for preparing tumor-associated antigen peptides includes: culturing host cells containing polynucleotides, vectors, nucleic acid molecules, or RNA, and recovering the resulting peptides from the culture.

[0410] In some embodiments, the method for preparing RNA includes: generating the RNA encoding the target protein in an in vitro transcription reaction using a linearized DNA template. In some embodiments, the RNA is generated in an in vitro transcription reaction using a non-amplified linearized DNA template. In some specific embodiments, the method further includes adding a 5' cap to the 5' end of the RNA molecule.

[0411] In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of a polynucleotide (RNA, such as, but not limited to, respiratory viral mRNA). In some embodiments, cells, such as bacterial cells, such as *E. coli*, such as DH-1 cells, are transfected with the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA, and then the plasmid DNA is isolated and purified. In some embodiments, the DNA template includes an RNA polymerase promoter, such as the T7 promoter located at the 5' of the target gene and operatively linked thereto.

[0412] In some embodiments, the polynucleotides, vectors, RNAs, or peptides of this disclosure may also be obtained by other production methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0413] lipid nanoparticles

[0414] This disclosure also provides a lipid nanoparticle comprising the aforementioned nucleic acid molecule, RNA molecule, polynucleotide, or carrier. In some embodiments, the lipid nanoparticle comprises the aforementioned RNA molecule.

[0415] In some embodiments, the RNA molecules of this disclosure can be delivered into cells and / or in vivo using nanolipid nanoparticles of any type in the art. Exemplarily, the lipid nanoparticles may comprise ionizable cationic lipids, non-cationic lipids, sterols and / or PEG lipid components, and a target nucleic acid. Exemplarily, the nanolipid nanoparticles include, but are not limited to, the lipid particles disclosed in WO2017075531, WO2018081480A1, WO2017049245A2, WO2017099823A1, WO2022245888A1, WO2022150717A1, CN101291653A, CN102119217A, WO2011000107A1, and CN107028886A, all of which are incorporated herein by reference in their entirety.

[0416] In some embodiments, at least one RNA molecule (e.g., first, second, and / or third RNA molecules) of this disclosure is formulated in the same or different lipid nanoparticles.

[0417] Pharmaceutical Composition

[0418] This disclosure also provides a pharmaceutical composition comprising any of the preceding polypeptides, polynucleotides, carriers, nucleic acid molecules and / or lipid nanoparticles, as well as pharmaceutically acceptable excipients, diluents or excipients.

[0419] In some embodiments, the pharmaceutical composition is a tumor vaccine. In some embodiments, the tumor vaccine comprises an open reading frame encoding the aforementioned target protein.

[0420] Tumor vaccines

[0421] This disclosure also provides a tumor vaccine comprising any of the preceding polypeptides, nucleic acid molecules, polynucleotides, carriers, lipid nanoparticles, and / or pharmaceutical compositions.

[0422] Methods of treating and / or preventing diseases and pharmaceutical uses

[0423] This disclosure provides the use of ECT2, TOP2A, TPX2, C12orf32, and / or OAS1 proteins, or immunogenic fragments thereof, in the preparation of pharmaceutical compositions for treating or preventing cancer. In some embodiments, the cancer is a KRAS-mutated malignant tumor. In some embodiments, the cancer is selected from pancreatic cancer, colorectal cancer, and / or lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer.

[0424] In some embodiments, the immunogenic fragment contains a high-affinity HLA sequence, exhibits enhanced in vivo immune response levels after immunization, and / or has antitumor activity.

[0425] In some embodiments, the pharmaceutical composition further comprises at least one antigenic peptide derived from KRAS, hTERT, and / or survivin.

[0426] In some embodiments, the immunogenic fragment comprises: an antigenic peptide derived from ECT2 comprising the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7 or SEQ ID NO:8; an antigenic peptide derived from TOP2A comprising the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10; an antigenic peptide derived from TPX2 comprising the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:9; an antigenic peptide derived from C12orf32 comprising the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:11; and an antigenic peptide derived from OAS1 comprising the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:12.

[0427] In some implementations, the ECT2, TOP2A, TPX2, C12orf32, and OAS1 proteins contain the amino acid sequences shown in SEQ ID NO:126, 129, 132, 135, and 138, respectively.

[0428] In some embodiments, the antigenic peptide derived from hTERT comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof.

[0429] In some implementations, the antigenic peptide derived from survivin contains an amino acid sequence as shown in SEQ ID NO:21 and / or 22.

[0430] In some embodiments, at least one KRAS-derived antigenic peptide has a G12 and / or G13 mutation. In some embodiments, the KRAS-derived antigenic peptide has a G12A, G12C, G12D, G12R, G12S, G12V, and / or G13D mutation. In some embodiments, the KRAS-derived antigenic peptide comprises at least one of the sequences shown in SEQ ID NO:13-16.

[0431] In some embodiments, the pharmaceutical composition is a tumor vaccine. In some embodiments, the tumor vaccine is an mRNA vaccine or a recombinant protein vaccine.

[0432] In some embodiments, this disclosure provides methods for inducing an immune response against an antigen of interest in a subject (e.g., a human subject). In one embodiment, the method includes administering to a subject any of the foregoing peptides, RNA molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and / or tumor vaccines. In one embodiment, inducing an immune response includes stimulating cytokine production. In another embodiment, inducing an immune response includes inducing cellular immunity (T cell response), such as stimulating antigen-specific CD8+ T cell activity, stimulating antigen-specific CD4+ T cell activity, or increasing the percentage of "effect memory" CD62L10T cells. In another embodiment, inducing an immune response includes inducing humoral immunity (B cell response), such as stimulating antigen-specific antibody production. The induction of an immune response against one or more antigens of interest in a subject by the peptides, RNA molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and / or tumor vaccines of this disclosure can be assessed using a variety of methods established in the art for evaluating immune responses, including but not limited to the methods described in the examples.

[0433] In some embodiments, the antigen of interest is a tumor antigen. In some embodiments, the tumor antigen includes those antigens disclosed herein (e.g., ECT2, TOP2A, TPX2, C12orf32, OAS1, KRAS, hTERT-1, and / or survivin).

[0434] In some embodiments, this disclosure provides the use of any of the foregoing peptides, nucleic acid molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and / or tumor vaccines in the preparation of medicaments for the prevention and / or treatment of diseases in subjects in need.

[0435] In some embodiments, the aforementioned subjects include humans as well as non-human primates and other animals, such as cattle, mice, and monkeys. In some embodiments, the subjects in need have been diagnosed with cancer or are considered to be at risk of developing cancer. In some embodiments, the cancer is a KRAS-mutated malignancy. In some embodiments, the cancer is liver cancer, colorectal cancer, melanoma, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), cervical cancer, or head and neck cancer. In some embodiments, the cancer is a hematopoietic cancer. In some embodiments, the cancer is acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, myelodystrophy syndrome (including refractory anemia and refractory cytopenia), or myeloproliferative vegetations or diseases (including polycythemia vera, essential thrombocythemia, and primary myelofibrosis). In other embodiments, the cancer is a blood-based cancer or a hematopoietic cancer. In some embodiments, the cancer is a KRAS-mutated malignancy. In some embodiments, the cancer is a refractory or relapsed cancer.

[0436] Based on the information disclosed herein, administer to subjects in need a preventive or therapeutically effective amount of any of the aforementioned peptides, nucleic acid molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and / or tumor vaccines.

[0437] In some embodiments, this disclosure provides methods for preventing and / or treating diseases, including administering to a subject in need a preventive and / or therapeutically effective amount of any of the foregoing peptides, nucleic acid molecules, polynucleotides, carriers, lipid nanoparticles, pharmaceutical compositions, and / or tumor vaccines.

[0438] In some embodiments, this disclosure provides the use of molecules targeting ECT2, TOP2A, TPX2, C12orf32, and / or OAS1 in the preparation of medicaments for the prevention and / or treatment of cancer. In some embodiments, the molecules regulate the activity of ECT2, TOP2A, TPX2, C12orf32, and / or OAS1. In some embodiments, the molecules downregulate the activity of ECT2, TOP2A, TPX2, C12orf32, and / or OAS1. In some embodiments, the cancer is a KRAS-mutated malignant tumor. In some embodiments, the cancer is pancreatic cancer, colorectal cancer, or lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer.

[0439] In this disclosure, "malignant tumor" refers to a tumor that is carcinogenic. Attached Figure Description

[0440] Figure 1: Differentially Expressed Genes. Figure 1A shows a tumor sample from colorectal cancer TCGA and adjacent normal tissue of TCGA; Figure 1B shows a tumor sample from colorectal cancer TCGA and normal GTEx sample; Figure 1C shows a tumor sample from pancreatic cancer TCGA and adjacent normal tissue of TCGA; Figure 1D shows a tumor sample from pancreatic cancer TCGA and normal GTEx sample; Figure 1E shows a tumor sample from non-small cell lung cancer TCGA and adjacent normal tissue of TCGA; Figure 1F shows a tumor sample from non-small cell lung cancer TCGA and normal GTEx sample.

[0441] Figures 2A to 2C: Bioinformatics prediction of in vivo immunogenicity screening of TAA-related antigens. Figure 2A shows the ELISpot results of immunogenicity evaluation after immunizing HLA-A*1101 mice with pancreatic cancer-associated antigen-encoded mRNA (SEQ ID NO:25), Figure 2B shows the ELISpot results of immunogenicity evaluation after immunizing HLA-A*1101 mice with lung cancer-associated antigen-encoded mRNA (SEQ ID NO:31), and Figure 2C shows the ELISpot results of immunogenicity evaluation after immunizing HLA-A*1101 mice with colorectal cancer-associated antigen-encoded mRNA (SEQ ID NO:28).

[0442] Figure 3: ELISpot results of immunogenicity evaluation after immunizing HLA-A*1101 mice with mRNA encoding each TAA antigen peptide individually.

[0443] Figure 4: Evaluation of the in vitro killing activity of specific T cells induced and activated after immunizing HLA-A*1101 mice with individual TAA-encoded mRNAs.

[0444] Figure 5: ELISpot results of immunogenicity evaluation of HLA-A*1101 mice after immunization with the full-length gene-encoded mRNAs of ECT2, TOP2A, TPX2, C12orf32, and OAS1.

[0445] Figure 6: Evaluation of the in vitro killing activity of specific T cells generated by HLA-A*1101 mice after immunization with the full-length gene-encoded mRNAs of ECT2, TOP2A, TPX2, C12orf32, and OAS1.

[0446] Figure 7: Detection of recombinant fusion protein expression levels. Each recombinant polypeptide was labeled with the His tag molecule, encoding mRNA, and then transfected into HEK293 cells using a transfection reagent. The expression level of the target protein was detected by Western blotting.

[0447] Figures 8A to 8C: Immunogenicity evaluation of mRNA vaccines Comb.1, Comb.2, and Comb.3 after immunization in animals with different antigen combinations. Figure 8A: ELISpot detection of KRAS-related antigen immune response levels after 3 immunizations. Figure 8B: ELISpot detection of TAA-related antigen immune response levels after 3 immunizations via bioinformatics screening. Figure 8C: ELISpot detection of hTERT Survivin-related antigen immune response levels after 3 immunizations.

[0448] Figures 9A to 9C: Immunogenicity evaluation of mRNA vaccines Comb.1 and Comb.4 after immunization in animals with different antigen combinations. Figure 9A: ELISpot detection of KRAS-related antigen immune response levels after 3 immunizations. Figure 9B: ELISpot detection of TAA-related antigen immune response levels after 3 immunizations via bioinformatics screening. Figure 9C: ELISpot detection of hTERT Survivin-related antigen immune response levels after 3 immunizations.

[0449] Figures 10A and 10B: Immunogenicity evaluation of the combined mRNA vaccine Comb.1 and the single KRAS mRNA vaccine under multiple antigen co-stimulation. Here, KRAS mixture represents stimulation with a mixture of four peptide libraries: G12C, G12D, G12V, and G13D; hTERT and Survivin mixture represents stimulation with a mixture of hTERT-1, hTERT-2, hTERT-3, hTERT-4, Survivin-1, and Survivin-2; TAA mixture represents stimulation with a mixture of six peptides: ECT2, TPX2, TOP2A, C12orf32, and OAS1; and KRAS+TAA+hTERT+Survivin mixture represents stimulation with a mixture of the above three peptide libraries. Figure 10A shows the immune response levels induced by the combined vaccine under different antigen stimulations. Figure 10B shows the immune response levels induced by the combined vaccine and the single KRAS-encoding mRNA vaccine under combined antigen stimulation. Statistical differences between the single-stimulatory peptide and the combined-stimulatory peptide in the Comb.1 group were determined by one-way repeated measures ANOVA and Dunnett's multiple comparisons assay. *p≤0.05.

[0450] Figure 11: Evaluation of the in vitro cytotoxic activity of specific T cells after immunization with the Comb.1 mRNA vaccine and the KRAS mRNA vaccine alone (the horizontal axis corresponds to the peptide stimulation that amplifies effector cells; only G12V cytotoxicity was evaluated in the KRAS immunization group). Statistical differences between the single-stimulatory peptide and the combined-stimulatory peptide in the Comb.1 group were determined by one-way repeated measures ANOVA and Dunnett's multiple comparison assay. **p≤0.01, ***p≤0.001, ****p≤0.0001.

[0451] Figure 12: Evaluation of in vivo killing activity of the combined mRNA vaccine Comb.1 and the KRAS mRNA vaccine alone in the presence of all antigens. Detailed Implementation

[0452] To facilitate understanding of this disclosure, certain techniques and scientific methods are specifically defined below. Unless otherwise expressly defined in this disclosure, all other techniques and scientific methods used in this disclosure have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0453] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0454] The term "vaccine" refers to a composition that induces an immune response upon administration to a subject. In some embodiments, the induced immune response provides therapeutic immunity.

[0455] The term “immune response” refers to any detectable response of one or more cells of the host mammal’s immune system to a stimulus (such as an immunogen), including but not limited to innate immune responses (e.g., activation of the Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells of the immune system, such as antigen-specific T cells, and non-specific cells), and humoral immune responses (e.g., B cell-mediated reactions, such as the production and secretion of antibodies into plasma, lymph, and / or tissue fluid). Examples of immune responses include the following alterations (e.g., increases): activation of Toll-like receptors; expression or secretion of lymphokines (e.g., cytokines (e.g., Th1, Th2, or Th17 type cytokines) or chemokines); activation of macrophages; activation of dendritic cells; activation of T cells (e.g., CD4+ or CD8+ T cells); activation of NK cells; activation of B cells (e.g., antibody production and / or secretion); binding of immunogens (e.g., antigens (e.g., immunogenic peptides)) to MHC molecules; induction of cytotoxic T lymphocyte (“CTL”) responses; induction of B cell responses (e.g., antibody production); and expansion of immune system cells (e.g., T cells and B cells) (e.g., cell population growth); and increased antigen processing and presentation by antigen-presenting cells. The term “immune response” also covers any detectable response of one or more components of the vertebrate immune system to a specific substance (such as an antigen or immunogen) in vitro.

[0456] "Nucleic acid molecule" refers to a polymeric form of nucleotides, which may include the sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers described above. A nucleotide refers to a modified form of ribonucleotide, deoxynucleotide, or any type of nucleotide. As used herein, the term "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide." The term includes DNA in both single-stranded and double-stranded forms. Polynucleotides may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. "cDNA" refers to DNA in single-stranded or double-stranded form that is complementary to or identical to mRNA. "Encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide (e.g., gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological characteristics.

[0457] “Vector” or “expression vector” refers to a replicon, such as a plasmid, rod-like particle, bacteriophage, virus, virion, or granule, that can link another DNA segment, or “insertion,” to enable replication of the linked segment within the cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral in origin and / or final form, such as the PUC57 DNA vector used herein. The term “vector” encompasses any genetic element that, when bound to a suitable control element, is capable of replication and can transfer a gene sequence into a cell. In some embodiments, a vector can be an expression vector or a recombinant vector.

[0458] "Nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule, such as a DNA fragment, that is modified or synthesized to contain nucleic acid segments in a manner not naturally present, said nucleic acid molecule containing one or more control sequences or regulatory elements. In the context of this disclosure, nucleic acid constructs contain recombinant nucleotide sequences that are substantially composed of, optionally, one, two, three, or more separate nucleotide sequences, including a 5' UTR, an open reading frame (ORF), and a 3' UTR. In embodiments involving constructs comprising two or more sequences, the sequences are operatively linked to each other within the construct.

[0459] An "open reading frame" (ORF) is a segment or region of an mRNA molecule that encodes a polypeptide. An ORF consists of consecutive, non-overlapping in-frame codons, starting with a start codon and ending with a stop codon, and is translated by the ribosome.

[0460] The term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into peptides or proteins. Transcription includes "in vitro transcription," where the term "in vitro transcription" refers to methods for synthesizing RNA, particularly mRNA, in vitro in a cell-free system.

[0461] The terms “expression” or “translation” refer to a process within the cell’s ribosomes through which the mRNA chain directs the assembly of amino acid sequences to produce peptides or proteins.

[0462] As is known in the art, the terms "identity" or "homology" refer to the relationship between sequences of two or more polypeptides or polynucleotides, as determined by sequence comparison. In the art, identity also means the degree of sequence correlation, as determined by the number of matches between two or more amino acid residues or nucleic acid residue strings. Identity measures the percentage of consistent matches between the smaller of two or more sequences having gap alignments (if present) proposed by a specific mathematical model or computer program (e.g., an "algorithm"). "Identity %" when applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical to residues in the amino acid or nucleic acid sequence of the second sequence after sequence alignment and, where necessary, the introduction of gaps to achieve the maximum identity percentage. Methods and computer programs used for alignment are well known in the art. It should be understood that identity depends on the calculation of the identity percentage, but its value can vary due to gaps and penalties introduced in the calculation. Typically, as determined by sequence alignment procedures and parameters described herein and known to those skilled in the art, a variant of a particular polynucleotide or polypeptide has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with a particular reference polynucleotide or polypeptide. Such tools used for alignment include those in BLAST kits (Stephen F. Altschul, et al. (1997), “GappedBLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25: 3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, TF and Waterman, MS (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147: 195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB and Wunsch, CD (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48: 443-453).A new Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has recently been developed, which is claimed to generate global alignments of nucleotide and protein sequences faster than other optimized global alignment methods (including the Needleman-Wunsch algorithm). Other tools are described in this paper, especially in the definition of “identity” below.

[0463] "Codon optimization" refers to replacing codons in a target sequence that are generally rare in genes highly expressed in a given species with codons that are common in genes highly expressed in the same species, while the original and unreplaced codons encode the same amino acid. Different species exhibit specific preferences for certain codons of particular amino acids. Codon preferences (differences in codon use between organisms) are generally related to the translation efficiency of messenger RNA (mRNA), which is considered to be particularly dependent on the characteristics of the codons translated and the utilization of specific transfer RNA (tRNA) molecules. The dominance of the chosen tRNA in the cell is generally a reflection of the most frequently used codons in peptide synthesis. Therefore, based on codon optimization, genes can be modified to target optimal gene expression in a given organism. Thus, the selection of the optimal codon depends on the codon use preferences of the host genome.

[0464] "Cell" or "host cell" includes any cell type that is readily transformed, transfected, transduced, etc., by the nucleic acid constructs or vectors of this disclosure. As a non-limiting example, the host cell can be any of isolated primary cells, pluripotent stem cells, CD34+ cells, induced pluripotent stem cells, or many immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell can be an in situ or in vivo cell in a tissue, organ, or organism.

[0465] An "effective amount" or "pharmacologically effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medically diagnosed disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0466] "Treatment or prevention of disease" refers to, for example, suppressing the complete development of a disease or condition in a subject at risk of disease, such as cancer. "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. Regarding a disease or pathological condition, the term "improvement" refers to any observable beneficial effect of treatment. For example, a beneficial effect can be demonstrated by delaying the onset of clinical symptoms of the disease in a susceptible subject, reducing the severity of some or all of the clinical symptoms of the disease, slowing the progression of the disease, improving the overall health or well-being of the subject, or by parameters known in the art that are specific to a particular disease. "Preventive" treatment is treatment applied to subjects who do not exhibit signs of disease or only exhibit early signs, with the aim of reducing the risk of pathological development.

[0467] The term "mammal" includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as non-human primates. Therefore, administration to a subject can include administration to human subjects. Non-limiting examples of veterinary subjects include domestic animals (e.g., cats and dogs), livestock (e.g., cattle, horses, pigs, sheep, and goats), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, and non-human primates).

[0468] The term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The term "protein" refers to a large peptide, particularly a peptide having at least about 151 amino acids, but the terms "peptide" and "protein" are generally used synonymously herein.

[0469] The term "antigen" refers to a reagent that includes an epitope against which an immune response can be generated. The term "antigen" specifically includes proteins and peptides. In one embodiment, the antigen is presented by cells of the immune system, such as antigen-presenting cells, like dendritic cells or macrophages. In one embodiment, the antigen or its processed product, such as a T-cell epitope, binds to T-cell or B-cell receptors or to immunoglobulin protein molecules, such as antibodies. Therefore, the antigen or its processed product can specifically react with antibodies or T lymphocytes (T cells). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, and the epitope is derived from such an antigen.

[0470] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to produce a disease-specific cellular immune response and / or humoral antibody response. Therefore, disease-associated antigens or their epitopes can be used for therapeutic purposes. Disease-associated antigens can be associated with cancer (usually tumors).

[0471] The term "tumor-associated antigen" or "TAA" refers to components of cancer cells that can originate from the cytoplasm, cell surface, and nucleus. Specifically, it refers to antigens produced intracellularly or as surface antigens on tumor cells. In some implementations, tumor-associated antigens refer to antigens expressed at significantly higher levels in cancer cells than in normal cells, such as carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA). In some implementations, the tumor antigens disclosed herein may be ECT2 (Epithelial cell transforming 2, Ensembl Gene ID: ENSG00000114346), TOP2A (DNA topoisomerase II alpha, Ensembl Gene ID: ENSG00000131747), TPX2 (TPX2 microtubule nucleation factor, Ensembl Gene ID: ENSG00000088325), C12orf32 (RAD9-HUS1-RAD1 interacting nuclear orphan 1, Ensembl Gene ID: ENSG00000171792), OAS1 (2'-5'-oligoadenylate synthetase 1, Ensembl Gene ID: ENSG00000089127), KRAS (KRAS proto-oncogene, Ensembl Gene ID: ENSG00000089127), or KRAS (KRAS proto-oncogene, Ensembl Gene ID: ENSG00000089127). One or more of the following: ID: ENSG00000133703, hTERT (Telomerase reverse transcriptase, Ensembl Gene ID: ENSG00000164362), and Survivin (Baculoviral IAP repeat containing 5, Ensembl Gene ID: ENSG00000089685).

[0472] The term "epitope" refers to a portion or fragment of a molecule (such as an antigen) that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen may include continuous or discontinuous portions of the antigen and may be between about 5 and 100 amino acids in length. In one embodiment, the epitope is about 10 to about 25 amino acids long. The term "epitope" includes T-cell epitopes.

[0473] The term "T-cell epitope" refers to a portion or fragment of a protein that is recognized by T cells in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" encompasses MHC class I and MHC class II molecules and refers to gene complexes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in immune responses, where MHC proteins or molecules bind peptide epitopes and present them for recognition by T-cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and present T cells with self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments from invading microorganisms). In the case of class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, while longer and shorter peptides may be effective.

[0474] Example

[0475] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in the Cell Culture Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents whose specific source is not specified are commercially available, conventional reagents.

[0476] The following are examples of standard experimental procedures 1) to 5):

[0477] 1) In vitro transcription of mRNA

[0478] To generate in vitro transcribed mRNA, the plasmid was linearized downstream of the polyadenylated tail using XbaI I (Vazyme, DD4304, China) and purified using a PCR purification kit (QIAGEN, 28106, Germany). Using the purified linearized plasmid as a template, in vitro transcription was performed to synthesize mRNA according to the following procedure. A 100 μL mRNA reaction system was synthesized; the reaction system is shown in Table 1. In the reaction system, 2 μg of linearized template was added to 100 μL of nuclease-free water, and the reaction was carried out at 37°C for 4 hours, followed by DNase I digestion for 30 min. The synthesized mRNA showed low levels of the byproduct dsRNA and excellent yield. The capping process was achieved chemically during in vitro transcription synthesis, with the cap structure being m7G(5')ppp(5')(2'OMeA)pG·NH4 (Hongene, ON-134). The mRNA was processed using MEGAclear. TM The kit (Invitrogen, AM1908, USA) was used to purify the mRNA to a relatively high purity for subsequent in vitro cell transfection and other experiments.

[0479] Table 1. mRNA reaction system

[0480] 2) Preparation of liposome nanoparticles encapsulating mRNA

[0481] Lipid solutions were prepared by dissolving ionizable lipids, DSPC (Avitar), cholesterol (Avitar), and DMG-PEG (Avitar) in ethanol at a molar ratio of 48:10:40.5:1.5. An aqueous solution of mRNA was prepared by dissolving mRNA in pH 5 acetate buffer. The ethanol-lipid solution and the mRNA-water solution were mixed via microfluidic mixing, with a total lipid to mRNA weight ratio of approximately 20:1, to prepare lipid nanoparticles. Ethanol was removed by dialyzing in 20 mM Tris pH 7.5 solution, and the nanoparticles were then frozen in 20 mM Tris pH 7.5, 8% sucrose solution to obtain mRNA-encapsulated liposome nanoparticles.

[0482] 3) Extraction of whole spleen lymphocytes from mice

[0483] The spleen was removed from the mouse by cervical dislocation. After grinding the cells using a 70µm cell sieve, erythrocytes were lysed with ACK lysis buffer (Gibco, A10492-01). The cells were then resuspended in complete culture medium (RPMI 1640 Medium + 10% FBS + 1% Penicillin Streptomycin) to obtain a cell suspension, which was then filtered through a 70µm filter membrane for counting.

[0484] 4) ELISpot detection

[0485] Antibody coating was performed the day before the test. The specific method was as follows: the plate was moistened with dampening solution (35% ethanol aqueous solution, dampening time upper limit is 1 min), washed 5 times with sterile PBS, and then coated with antibody using Capture mAb AN18 (Mabtech, 3321-2A) and incubated overnight at 4°C.

[0486] On the day of testing, remove the well plates coated the previous day, block them at room temperature for 30 min, wash them, add the prepared peptide and cell suspension mixture, and incubate at 37℃ with 5% CO2 for 24 h. Remove the well plates, wash them, add Detection mAb R4-6A2 biotin (Mabtech, 3321-2A), and incubate at room temperature for 1.5 h; wash them again, add Streptavidin-ALP (Mabtech, 3321-2A), and incubate at room temperature for 1 h; wash them again, add BCIP / NBT-plus substrate for ELISpot chromogenic solution (Mabtech, 3650-10), and react at room temperature in the dark (5-30 min); wash them again, invert the plates to air dry in the dark, and then examine and count the spots using an ELISPOT reader or analytical microscope.

[0487] 5) FACS testing

[0488] Add the prepared peptide-cell suspension mixture to the well plate, mix well by pipetting, and incubate at 37°C with 5% CO2 for 6 hours. Mix the cells by pipetting and transfer to a U-shaped plate, wash twice with washing buffer (2% FBS + PBS buffer); add 5 μg / ml Anti-mouse CD16 / 32 Antibody, block at 4°C for 15 min; wash twice, then add 4 μg / ml FITC anti-mouse CD3 Antibody, 1 μg / ml PerCP cy5.5 Rat Anti-Mouse CD8a Antibody, 0.5 μg / ml BV605 Rat Anti-Mouse CD4 Antibody, and 0.5 μL Zombie NIR. TMIncubate with Fixable Viability Kit at 4°C for 20 min, shaking every 10 min. After washing twice, add 4% paraformaldehyde to the U-plate and incubate at room temperature for 15 min. After washing twice, add stain buffer to the U-plate and incubate overnight at 4°C. Remove the U-plate with fixed cells and centrifuge (4°C, 300g, 5 min), discarding the supernatant. Add 1×BD Perm / Wash to the U-plate and centrifuge (4°C, 300g, 5 min), discarding the supernatant. Add 2.4ug / ml APC Rat Anti-Mouse IFN-γ Antibody to the U-plate and incubate at 4°C for 40 min, shaking every 20 min. Centrifuge (4°C, 300g, 5 min) and discard the supernatant. Wash twice with 1×BD Perm / Wash. Add stain buffer to the U-plate and analyze.

[0489] Example 1: TAA Antigen Screening Based on AI Model

[0490] Computer algorithms were used to predict potential TAA antigens for pancreatic cancer, colorectal cancer, and non-small cell lung cancer. The databases used were: (1) HLA Ligand Atlas database, which covers 51 MHC I subtypes and 86 MHC II subtypes, containing 90,428 MHC type I peptides and 142,625 MHC type II peptides, involving a total of 15,262 proteins. HLA Ligand Atlas is a benign HLA ligand library, ensuring the safety of TAA antigens. (2) UCSC Xena database, which downloads genomic, transcriptomic, and clinical data of pancreatic cancer, colorectal cancer, and non-small cell lung cancer from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases.

[0491] The method was as follows: (1) The R software DESeq2 was used to screen differentially expressed genes (DEGs) in pancreatic cancer, colorectal cancer, and non-small cell lung cancer tumor samples and adjacent normal tissues of TCGA, as well as between TCGA tumor samples and normal samples of GTEx (as shown in Figure 1). The screening criteria for DEGs were |log2foldchange|≥1 and false discovery rate (FDR)<0.05. The number of differentially expressed genes between TCGA tumor samples and adjacent normal tissues in pancreatic cancer, colon cancer, and non-small cell lung cancer, and between TCGA tumor samples and GTEx normal samples, were as follows: 794 genes upregulated, 863 genes downregulated, 4080 genes upregulated, 2966 genes downregulated, 2908 genes upregulated, 2814 genes downregulated, 2863 genes upregulated, 4867 genes downregulated, 3045 genes upregulated, 2313 genes downregulated, 3154 genes upregulated, and 3434 genes downregulated.

[0492] (2) The AI ​​mass spectrometry model was used to predict the presentation of peptides in A11:01-regulated genes. The union of upregulated genes for each cancer type was obtained, resulting in 4274, 4323, and 4286 genes for pancreatic cancer, colon cancer, and non-small cell lung cancer, respectively. The corresponding uniprot protein sequences of the upregulated genes were extracted and truncated into peptides of 8-11 bp in length. The expression of the corresponding gene was represented by the median expression value of the gene in the corresponding cancer type. For A11:01 genotyping, a cutoff of 0.79994 with recall = 40% was used to screen the final peptides, and HLA Ligand Atlas peptides were used for filtering. The final identified peptides are shown in Tables 2, 3, and 4.

[0493] Table 2. TAA peptides for pancreatic cancer prediction

[0494] Table 3. TAA peptides for colorectal cancer prediction

[0495] Table 4. Predicted TAA peptides for non-small cell lung cancer

[0496] Example 2: Screening of TAA antigens

[0497] Immunogenicity screening was performed on TAA antigens highly expressed in pancreatic cancer, colorectal cancer, and non-small cell lung cancer as predicted by AI models to obtain antigens or gene combinations for use in combination with KRAS.

[0498] The TAA peptides predicted by the bioinformatics algorithm were linked to form fusion peptides corresponding to pancreatic cancer, colorectal cancer, and non-small cell lung cancer, respectively (SEQ ID NO: 23, 26, and 29). KRAS G12C, KRAS G12V, KRAS G12D, and KRAS G13D were linked to form fusion peptide A (SEQ ID NO: 38). The DNA templates corresponding to the above four fusion peptides were inserted into the MCS region of the vector pD32 (SEQ ID NO: 37), and four mRNAs (SEQ ID NO: 25, 28, 31, and 40) were obtained by in vitro transcription via IVT.

[0499] Three mRNAs (SEQ ID NO: 25, 28, and 31) encoding the fusion peptides of pancreatic cancer, colorectal cancer, and non-small cell lung cancer, respectively, were mixed with mRNA encoding fusion protein A (SEQ ID NO: 40) at a mass ratio of 7:3 and encapsulated in LNPs. HLA-A*11:01Tg mice (Taconic, 9660F strain, n=3) were administered the mixture at a dose of 5 μg per 20 g of animal on days 1, 14, and 28. On day 35, the spleens of the mice were harvested, and whole spleen lymphocytes were extracted and restimulated in vitro with the corresponding group of antigen peptides (10 μg / ml for each peptide) for 24 hours. The immune response level of the TAA gene was detected by ELISPOT.

[0500] Table 5. Sequences related to each fusion protein used for TAA screening.

[0501] Table 6. Vector nucleotide sequences

[0502] The results are shown in Figure 2A. For pancreatic cancer, MXRA5, ECT2-KSI, and LAMA3 showed strong responses, and the positive rate of secondary activation of in vitro peptides by lymphocytes in immunized mice was 100%. For lung cancer, ECT2-KSI, C12orf32, OAS1, and TOP2A-RVI showed strong responses, and the positive rate of secondary activation of in vitro peptides by lymphocytes in immunized mice was 100%. GOLGA5 showed a weak response, and the positive rate of secondary activation of in vitro peptides by lymphocytes in immunized mice was 50% (see Figure 2B). For colorectal cancer, ECT2-KSS and TOP2A showed strong responses. The positive rate of secondary activation of in vitro peptides by lymphocytes in ECT2-KSS-immunized mice was 100%, and the positive rate of TOP2A was 75%. TPX2 showed a weak response, and the positive rate of secondary activation of in vitro peptides by lymphocytes in immunized mice was 75% (see Figure 2C).

[0503] Based on the above immunogenicity results, the final candidate genes or peptides were screened. The screening principles were based on the immunogenicity of the peptides, cross-reactivity across different cancer types, and patient coverage. The final selected genes were ECT2 (ECT2-KSI, ECT2-KSS), TOP2A, TPX2, C12orf32, and OAS1. The final selected TAA antigen peptides are shown in the table below.

[0504] Table 7. Screened TAA antigen peptides

[0505] Example 3: Evaluation of the immunogenicity and antitumor properties of various TAA antigen peptides

[0506] Animals were immunized with mRNA encoding TAA antigen peptides to evaluate the immunogenicity and antitumor activity of each TAA antigen peptide listed in Table 7. Specifically, 8-9 amino acids (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12) were extended at the N-terminus and C-terminus of each TAA antigen peptide in Table 7, and a signal peptide and MITD domain were ligated to obtain the constructed TAA antigen peptides (SEQ ID NO:74, 77, 80, 83, 86, and 89). The DNA template of this peptide was inserted into the MCS region of the vector pD32, and in vitro transcription via IVT was used to obtain the individual mRNA sequences encoding each TAA antigen peptide (SEQ ID NO:76, 79, 82, 85, 88, and 91). HLA-A*1101 mice were immunized with 5 μg / mouse mRNA-LNP vaccine on days 0, 14 and 28. Spleens were harvested 7 days after immunization. The level of immune response induced by the vaccine was detected by ELISpot (stimulating antigen peptides are shown in Table 7), and the antitumor performance was detected by in vitro killing assay.

[0507] The immunogenicity evaluation results of ELISpot are shown in Figure 3, where anti-CD3 antibody was used as a positive control (PC) and spleen cell suspension without stimulating peptide was used as a negative control (NC). Figure 3 shows that animals immunized with the above mRNA vaccines all exhibited a certain degree of immune response.

[0508] In the in vitro killing experiment, specific T cells from immunized mice were used as effector cells, and spleen cells from unimmunized animals were used as target cells. Specifically, spleen cells from immunized animals were incubated at 50 U / ml IL-2 with 20 μg / ml of the corresponding stimulating amplification peptide (stimulating antigen peptides are shown in Table 7), and T cells were isolated after 5 days of incubation at 37°C. Spleen cells from unimmunized animals were incubated at 10 μg / ml of the corresponding antigen peptide (stimulating antigen peptides are shown in Table 7) for 2 hours. Target cells were labeled with CFSE and mixed with amplified T cells at a 30:1 effector-to-target ratio, and incubated at 37°C for 20 hours. Target cell viability was detected by flow cytometry after labeling with a live / dead dye. The in vitro killing results are shown in Figure 4. The specific T cells generated in mice immunized with the above mRNA vaccine exhibited good target cell killing activity.

[0509] Example 4: Verification of the immunogenicity and antitumor properties of full-length TAA protein

[0510] Five full-length TAA proteins encoded by mRNA were selected, and the immunogenicity and antitumor activity of each gene were evaluated. Specifically, the DNA sequences corresponding to ECT2, TOP2A, TPX2, C12orf32, and OAS1 were inserted into the MCS region of vector pD32 (SEQ ID NO:37) to obtain the corresponding DNA templates. In vitro transcription (IVT) was then used to obtain mRNA (SEQ ID NO:128, 131, 134, 137, 140). After encapsulation in liposomes, each TAA vaccine was administered to HLA-A*1101 transgenic mice at a dose of 5 μg / mouse on days 0, 14, and 28. Seven days post-immunization, the spleens of the mice were harvested. The level of the immune response induced by the vaccines was detected using ELISApot (a stimulating peptide library was constructed using 9-10 aa HLA high-affinity peptides), and the antitumor activity was assessed by in vitro killing assay.

[0511] The results of the ELISpot immunogenicity evaluation are shown in Figure 5. After immunization with ECT2, TOP2A, TPX2, C12orf32, and OAS1, HLA-A*1101 transgenic mice all showed a certain degree of immune response, and the positive rate of response was 100%.

[0512] In vitro killing experiments used specific T cells from immunized mice as effector cells and spleen cells from unimmunized animals as target cells. Specifically, spleen cells from immunized animals were incubated at 50 U / ml IL-2 with corresponding stimulatory amplification peptides (using a stimulatory peptide library constructed from 9-10 aa HLA high-affinity peptides) for 5 days at 37°C, followed by T cell isolation. Spleen cells from unimmunized animals were incubated with corresponding antigenic peptides (using a stimulatory peptide library constructed from 9-10 aa HLA high-affinity peptides) for 2 hours. After CFSE labeling, the cells were mixed with the expanded T cells from immunized animals at a 30:1 effector-to-target ratio and incubated at 37°C for 20 hours. Target cell viability was detected by flow cytometry after labeling with live / dead dyes. The in vitro killing results are shown in Figure 6. Different genes exhibited varying degrees of killing activity, with specific T cells activated by TPX2, C12orf32, and OAS1 immunization showing excellent target cell lysis ability.

[0513] Example 5: Sequence Combination Form Screening

[0514] By adjusting the antigen linking sequence and introducing linkers with different hydrophilicities, the N-terminus and C-terminus of the six TAA antigen peptides screened in Example 2 were extended by 8-9 amino acids (SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12), and the four KRAS antigen peptides (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16), four hTERT antigen peptides (SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20), and two Survivin antigen peptides (SEQ ID NO:21, SEQ ID NO:22) were arranged and distributed in two fusion proteins in different combinations. The antigen peptides in the fusion proteins are distributed as shown in Table 8. The principles include KRAS antigen encoding a single mRNA, or bioinformatics predicting that TAA antigen encodes a single mRNA, or antigens being allocated according to immunogenicity. Fusion proteins A through K (SEQ ID NO: 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, and 68) were obtained. The DNA template of the fusion protein was inserted into the MCS region of vector pD32 (SEQ ID NO: 37) to obtain the corresponding vaccine DNA template, which was then transcribed in vitro via IVT to obtain the mRNA sequence. To evaluate the expression levels of each combination, His-tagged fusion proteins AT through KT were obtained.

[0515] Table 8. Antigenic peptides used to form fusion proteins

[0516] The expression of fusion proteins was detected using protein blotting. Specifically, the mRNAs obtained from each construction were transfected into cells, and cell lysates were collected from the wells. The lysates were then subjected to polyacrylamide gel electrophoresis, membrane transfer, antibody incubation, and color development to determine the in vitro expression level of each mRNA. The results are shown in Figure 7. Based on the in vitro expression level of the mRNAs, the fusion proteins shown in Table 9 were selected for subsequent experiments.

[0517] Table 9. Structure of the fusion protein (Note: Linker 1:EAAAK; Linker 2:AAY; Linker 3:GPGPG)

[0518] Example 6: Evaluation of the immunogenicity of different tumor vaccine combinations

[0519] After arranging the mRNAs encoding each antigenic peptide shown in Table 9 in different ways, they were assigned to different mRNA sequences, and the resulting mRNA sequences were combined as follows:

[0520] Comb.1: mRNA encoding fusion protein B (SEQ ID NO: 43) and mRNA encoding fusion protein C (SEQ ID NO: 46), with masses of 2.5 μg and 2.5 μg respectively;

[0521] Comb. 2: mRNA encoding fusion protein F (SEQ ID NO: 55) and mRNA encoding fusion protein G (SEQ ID NO: 58), with masses of 3 μg and 2 μg respectively; and

[0522] Comb.3: mRNA encoding fusion protein I (SEQ ID NO: 64) and mRNA encoding fusion protein K (SEQ ID NO: 70), with masses of 3 μg and 2 μg, respectively.

[0523] HLA-A*1101 mice were immunized with the three combined mRNA vaccines on days 0, 14, and 28. Spleens were harvested 7 days post-immunization, and the level of immune response induced by the vaccines was evaluated using ELISpot. The results are shown in Figures 8A to 8C. The three combined mRNA vaccines induced varying degrees of immune response, with comparable TAA response levels. Surprisingly, Comb.1 showed a higher level of KRAS activation.

[0524] A new combination, Comb.4, was formed by allocating a single mRNA from each of the KRAS (SEQ ID NO: 43), TAA (SEQ ID NO: 58), and hTERT / survivin (SEQ ID NO: 73) related antigens, with the three components weighing 4 μg, 3 μg, and 3 μg, respectively. The immune response levels of Comb.4 and Comb.1 were compared using the method described above, with the two components of Comb.1 being changed to 6 μg and 4 μg, respectively. The results are shown in Figures 9A to 9C. Both combinations elicited broad-spectrum immune responses; both KRAS and TAA showed good immune responses. Compared to the two, the Comb.1 combination exhibited a stronger immune response.

[0525] Example 7: Combination antigens enhance immune response

[0526] The immunogenicity enhancement of the KRAS, TAA, TRET, and Survivin antigen combination vaccine Comb.1 (SEQ ID NO: 43 and SEQ ID NO: 46, with two components weighing 6 μg and 4 μg, respectively) was evaluated compared with that of the KRAS mRNA alone (SEQ ID NO: 43). Specifically, HLA-A*1101 mice were immunized with the same KRAS dose (6 μg) of both the KRAS mRNA alone and the universal KRAS mRNA combination vaccine Comb.1 on days 0, 14, and 28. Spleens were harvested 7 days post-immunization, and the level of the immune response induced by the vaccine was detected by ELISPOT. The combined efficacy of the combination vaccine was assessed by in vitro and in vivo killing assays.

[0527] Immunogenicity evaluation results showed that, under multiple antigen stimulation, the combination vaccine immunization group exhibited a higher level of immune response compared to the KRAS-only immunization group (Figure 10B); at the same time, the combination vaccine showed higher immunogenicity under all antigen stimulation compared to single antigen stimulation with KRAS, TAA, hTRET, and Survivin (Figure 10A).

[0528] In the in vitro killing assay, specific T cells from immunized mice were used as effector cells, and spleen cells from unimmunized animals were used as target cells. Specifically, spleen cells from immunized animals were incubated at 50 U / ml IL-2 with 20 μg / ml of the corresponding stimulatory amplification peptide, and cultured at 37°C for 5 days before T cells were isolated. Spleen cells from unimmunized animals were incubated with 10 μg / ml of the corresponding antigenic peptide for 2 hours. After CFSE labeling, the cells were mixed with expanded T cells from immunized animals at different effector-to-target ratios (E / T) (30:1, 10:1) and cultured at 37°C for 20 hours. Target cell viability was then detected by flow cytometry after labeling with live / dead dyes. In vitro killing experiments were conducted to verify the antitumor performance of the combination vaccine. The antigens with the strongest immunogenicity, KRAS, TAA, and hTERT / survivin, were selected as representatives for the in vitro killing experiment. The results are shown in Figure 11. Against the KRAS antigen, the killing effect of the combination vaccine was comparable to that of the KRAS vaccine alone. At the same time, the killing effect of the combination vaccine stimulated by multiple antigens was significantly better than that of KRAS, TAA, and hTRET single antigen stimulation.

[0529] In vivo killing experiments used spleen cells from unimmunized animals as target cells, which were then reinfused into immunized animals to evaluate antitumor performance. Specifically, spleen cells from unimmunized animals were incubated at 37°C for 2 hours with 10 μg / ml of antigenic peptide. The experimental group was incubated with 1 μM CFSE-labeled peptide, while the control group was labeled with 0.1 μM CFSE. The experimental and control groups were mixed 1:1 and reinfused into immunized mice. Sixteen hours later, the spleens of the mice were harvested, and the killing effect was evaluated by flow cytometry detection of CFSE signals. The results, shown in Figure 12, indicate that the in vivo antitumor performance of the combined vaccine was significantly superior to that of the KRAS vaccine alone when all antigens were present.

[0530] The sequence of this disclosure is shown below:

[0531] >ECT2-KSI antigen-1

[0532] >ECT2-KSS antigen-1

[0533] TPX2 antigen-1

[0534] >TOP2A-RVI antigen-1

[0535] >C12orf32 antigen-1

[0536] OAS1 antigen-1

[0537] >ECT2-KSI antigen-2

[0538] >ECT2-KSS antigen-2

[0539] TPX2 antigen-2

[0540] >TOP2A-RVI antigen-2

[0541] >C12orf32 antigen-2

[0542] OAS1 antigen-2

[0543] KRAS G12D antigen

[0544] KRAS G12V antigen

[0545] KRAS G13D antigen

[0546] KRAS G12C antigen

[0547] hTERT-1 antigen

[0548] hTERT-2 antigen

[0549] hTERT-3 antigen

[0550] hTERT-4 antigen

[0551] Survivin-1 antigen

[0552] Survivin-2 antigen

[0553] >Pancreatic cancer-associated TAA antigen amino acid sequence

[0554] >Pancreatic cancer-associated TAA antigen nucleotide sequence

[0555] >Constructed pancreatic cancer-associated TAA antigen mRNA sequence

[0556] >Amino acid sequence of TAA antigen associated with colorectal cancer

[0557] > Nucleotide sequence of TAA antigen associated with colorectal cancer

[0558] >Constructed colorectal cancer-associated TAA antigen mRNA sequence

[0559] Amino acid sequence of TAA antigen associated with non-small cell lung cancer

[0560] Nucleotide sequence of TAA antigen associated with non-small cell lung cancer

[0561] >Constructed non-small cell lung cancer-associated TAA antigen mRNA sequence

[0562] T7 Promotor

[0563] >5'UTR

[0564] MCS

[0565] >3'UTR

[0566] >poly(A)

[0567] >vector-pD32

[0568] >Amino acid sequence of fusion protein A

[0569] >nucleotide sequence of fusion protein A

[0570] >Post-construction fusion protein A mRNA sequence

[0571] >Amino acid sequence of fusion protein B

[0572] >Fusion protein B nucleotide sequence

[0573] >Post-construction fusion protein B mRNA sequence

[0574] >C amino acid sequence of the fusion protein

[0575] >C nucleotide sequence of the fusion protein

[0576] >Post-construction fusion protein C mRNA sequence

[0577] >Methylamine sequence of fusion protein D

[0578] >Fusion protein D nucleotide sequence

[0579] >Constructed fusion protein D mRNA sequence

[0580] >Amino acid sequence of fusion protein E

[0581] >Fusion protein E nucleotide sequence

[0582] >Constructed fusion protein E mRNA sequence

[0583] >F amino acid sequence of fusion protein

[0584] >F nucleotide sequence of fusion protein

[0585] >Post-construction fusion protein F mRNA sequence

[0586] >Fuse protein G amino acid sequence

[0587] >Fusion protein G nucleotide sequence

[0588] >Post-construction fusion protein G mRNA sequence

[0589] >H amino acid sequence of the fusion protein

[0590] >H nucleotide sequence of the fusion protein

[0591] >Post-construction fusion protein H mRNA sequence

[0592] >Amino acid sequence of fusion protein I

[0593] >Fusion protein I nucleotide sequence

[0594] >Post-construction fusion protein I mRNA sequence

[0595] >Fuse protein J amino acid sequence

[0596] >Fusion protein J nucleotide sequence

[0597] >Post-construction fusion protein J mRNA sequence

[0598] >Fuse protein K amino acid sequence

[0599] >Fusion protein K nucleotide sequence

[0600] >Constructed fusion protein K mRNA sequence

[0601] >Fusion protein L amino acid sequence

[0602] >Fusion protein L nucleotide sequence

[0603] >Post-construction fusion protein L mRNA sequence

[0604] >Constructed C12orf32 amino acid sequence

[0605] >Constructed C12orf32 nucleotide sequence

[0606] >Constructed C12orf32 mRNA sequence

[0607] >Constructed ECT2-KSI amino acid sequence

[0608] >Constructed ECT2-KSI nucleotide sequence

[0609] >Constructed ECT2-KSI mRNA sequence

[0610] >Constructed ECT2-KSS amino acid sequence

[0611] >Constructed ECT2-KSS nucleotide sequence

[0612] >Constructed ECT2-KSS mRNA sequence

[0613] >Constructed OAS1 amino acid sequence

[0614] >Constructed OAS1 nucleotide sequence

[0615] >Constructed OAS1 mRNA sequence

[0616] >Constructed TOP2A amino acid sequence

[0617] >Constructed TOP2A nucleotide sequence

[0618] >Constructed TOP2A mRNA sequence

[0619] >Constructed TPX2 amino acid sequence

[0620] >Constructed TPX2 nucleotide sequence

[0621] >Constructed TPX2 mRNA sequence

[0622] SP signal peptide

[0623] MITD domain

[0624] >C12ORF32 amino acid sequence

[0625] >C12ORF32 nucleotide sequence

[0626] >C12ORF32 mRNA sequence

[0627] >ECT2 amino acid sequence

[0628] >ECT2 nucleotide sequence

[0629] >ECT mRNA sequence

[0630] >OAS1 amino acid sequence

[0631] >OAS1 nucleotide sequence

[0632] >OAS1 mRNA sequence

[0633] >TOP2A amino acid sequence

[0634] >TOP2A nucleotide sequence

[0635] >TOP2A mRNA sequence

[0636] >TPX2 amino acid sequence

[0637] >TPX2 nucleotide sequence

[0638] >TPX2 mRNA sequence

Claims

1. A composition comprising at least one RNA molecule, wherein the RNA molecule encodes at least one antigenic peptide derived from a tumor-associated antigen (TAA) and at least one antigenic peptide derived from KRAS.

2. The composition according to claim 1, wherein the composition comprises 1-10 RNA molecules, preferably, the composition comprises 1, 2, 3, 4, or 5 RNA molecules.

3. The composition according to claim 1 or 2, wherein the RNA molecule comprises an open reading frame (ORF) encoding a protein of interest, and wherein the ORF encoding the at least one antigenic peptide derived from a TAA and the ORF encoding the at least one antigenic peptide derived from KRAS are located on the same RNA molecule, or wherein the ORF encoding the at least one antigenic peptide derived from a TAA and the ORF encoding the at least one antigenic peptide derived from KRAS are located on different RNA molecules.

4. The composition according to any one of claims 1-3, wherein the antigenic peptide derived from a TAA, or the antigenic peptide derived from KRAS has a length of 9-50 amino acids, preferably 9-35 amino acids.

5. The composition according to any one of claims 1-4, wherein the at least one antigenic peptide derived from a TAA comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, OAS1, hTERT, and / or survivin.

6. The composition according to any one of claims 1-5, wherein the antigenic peptide derived from ECT2 comprises an amino acid sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 7, or SEQ ID NO: 8; the antigenic peptide derived from TOP2A comprises an amino acid sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 10; the antigenic peptide derived from TPX2 comprises an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 9; the antigenic peptide derived from C12orf32 comprises an amino acid sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 11; the antigenic peptide derived from OAS1 comprises an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 12; the antigenic peptide derived from hTERT comprises an amino acid sequence as set forth in any one of SEQ ID NO: 17-20 or any combination thereof; and / or the antigenic peptide derived from survivin comprises an amino acid sequence as set forth in SEQ ID NO: 21 and / or 22.

7. The composition according to any one of claims 1-6, wherein the at least one antigenic peptide derived from KRAS has a G12 and / or G13 mutation; preferably, the antigenic peptide derived from KRAS has a G12A, G12C, G12D, G12R, G12S, G12V, and / or G13D mutation; preferably, the antigenic peptide derived from KRAS comprises at least one of the sequences set forth in SEQ ID NO: 13-16. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 8. The composition according to any one of claims 1-7, wherein the KRAS-derived antigenic peptide synergistically enhances the level of in vivo immune response and / or antitumor activity with the TAA-derived antigenic peptide.

9. The composition according to any one of claims 1-8, comprising a first RNA molecule and a second RNA molecule, wherein the first RNA molecule and the second RNA molecule each comprise an open reading frame (ORF) encoding a target protein, wherein: A. The target protein encoded by the first RNA molecule includes: (1) One or more antigenic peptides derived from ECT2; preferably, wherein the antigenic peptides derived from ECT2 comprise the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, wherein the antigenic peptides derived from ECT2 comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; (2) One or more antigenic peptides derived from TOP2A; preferably, wherein the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, wherein the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10; (3) One or more antigenic peptides derived from TPX2; preferably, wherein the antigenic peptides derived from TPX2 comprise the amino acid sequence shown in SEQ ID NO:3; more preferably, wherein the antigenic peptides derived from TPX2 comprise the amino acid sequence shown in SEQ ID NO:9; (4) One or more antigenic peptides derived from C12orf32; preferably, wherein the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, wherein the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11; (5) One or more antigenic peptides derived from OAS1; preferably, wherein the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, wherein the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:

12. (6) One or more antigenic peptides derived from hTERT; preferably, wherein the antigenic peptides derived from hTERT comprise an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; and (7) One or more antigenic peptides derived from survivin, preferably wherein the antigenic peptides derived from survivin contain an amino acid sequence as shown in SEQ ID NO:21 and / or 22; The target protein encoded by the second RNA molecule includes: (1) One or more antigenic peptides derived from KRAS and having a G12D mutation; preferably, wherein the antigenic peptides derived from KRAS contain an amino acid sequence as shown in SEQ ID NO:13; (2) One or more antigenic peptides derived from KRAS and having a G12V mutation; preferably, wherein the antigenic peptides derived from KRAS contain an amino acid sequence as shown in SEQ ID NO:14; (3) One or more antigenic peptides derived from KRAS and having a G13D mutation; preferably, wherein the KRAS-derived antigenic peptides comprise the amino acid sequence shown in SEQ ID NO:15; and (4) One or more antigenic peptides derived from KRAS and having a G12C mutation; preferably, wherein the KRAS-derived antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:16; Preferably, the target protein encoded by the first RNA molecule comprises the sequence shown in SEQ ID NO:44 or an amino acid sequence having at least 90% sequence identity with it; and / or the target protein encoded by the second RNA molecule comprises the sequence shown in SEQ ID NO:41 or an amino acid sequence having at least 90% sequence identity with it; or B. The target protein encoded by the first RNA molecule includes: (1) One or more antigenic peptides derived from ECT2; preferably, wherein the antigenic peptides derived from ECT2 comprise the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, wherein the antigenic peptides derived from ECT2 comprise the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; (2) One or more antigenic peptides derived from TOP2A; preferably, wherein the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, wherein the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10; (3) One or more antigenic peptides derived from TPX2; preferably, wherein the antigenic peptides derived from TPX2 comprise the amino acid sequence shown in SEQ ID NO:3; more preferably, wherein the antigenic peptides derived from TPX2 comprise the amino acid sequence shown in SEQ ID NO:9; (4) One or more antigenic peptides derived from C12orf32; preferably, wherein the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, wherein the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11; and (5) One or more antigenic peptides derived from OAS1; preferably, wherein the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, wherein the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:

12. The target protein encoded by the second RNA molecule includes: (1) One or more antigenic peptides derived from KRAS and having a G12D mutation; preferably, wherein the KRAS-derived antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:13; (2) One or more antigenic peptides derived from KRAS and having a G12V mutation; preferably, wherein the KRAS-derived antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:14; (3) One or more antigenic peptides derived from KRAS and having a G13D mutation; preferably, wherein the KRAS-derived antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:15; (4) One or more antigenic peptides derived from KRAS and having a G12C mutation; preferably, wherein the KRAS-derived antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:16; (5) One or more antigenic peptides derived from hTERT; preferably, wherein the antigenic peptides derived from hTERT comprise an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; and (6) One or more antigenic peptides derived from survivin; preferably, wherein the antigenic peptides derived from survivin contain amino acid sequences as shown in SEQ ID NO:21 and / or 22; Preferably, the target protein encoded by the first RNA molecule comprises the sequence shown in SEQ ID NO:56 or an amino acid sequence having at least 90% sequence identity with it; and / or the target protein encoded by the second RNA molecule comprises the sequence shown in SEQ ID NO:53 or an amino acid sequence having at least 90% sequence identity with it.

10. The composition according to any one of claims 1-9, wherein the antigenic peptides encoded by the RNA molecules are directly linked by peptide bonds or linked by linkers; Preferably, the linker is selected from (GxS)y, EAAAK, AAY or GPGPG, wherein, x is an integer from 1 to 5, and y is an integer from 1 to 6.

11. The composition according to claim 9 or 10, wherein: A. The first RNA molecule contains the nucleotide sequence shown in SEQ ID NO:46 or a nucleotide sequence having at least 90% sequence identity with it; the second RNA molecule contains the nucleotide sequence shown in SEQ ID NO:43 or a nucleotide sequence having at least 90% sequence identity with it; or B. The first RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:58 or a nucleotide sequence having at least 90% sequence identity with it; the second RNA molecule contains a nucleotide sequence as shown in SEQ ID NO:55 or a nucleotide sequence having at least 90% sequence identity with it.

12. The composition according to any one of claims 1-11, wherein the RNA molecule is mRNA.

13. A composition comprising a first RNA molecule and a second RNA molecule, optionally, the composition further comprises a third RNA molecule, wherein, The first, second, and third RNA molecules each contain an open reading frame (ORF) encoding the target protein; The composition is selected from any one of the following groups: (a) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it; (b) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:50 or having at least 90% sequence identity with it; (c) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:59 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it; (d) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it; the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it; and the third RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:71 or having at least 90% sequence identity with it. (e) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it. (f) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:53 or having at least 90% sequence identity with it; (g) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:59 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it. (h) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it, and the third RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:71 or having at least 90% sequence identity with it. (i) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:47 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it; (j) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:62 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:68 or having at least 90% sequence identity with it; (k) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:47 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it; (l) The first RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:62 or having at least 90% sequence identity with it, and the second RNA molecule encodes an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it.

14. The composition according to any one of claims 1-13, wherein the RNA molecule comprises an untranslated region element (UTR); preferably, wherein the UTR comprises a 5' untranslated region element (5'UTR) and / or a 3' untranslated region element (3'UTR); Preferably, the 5'UTR contains the sequence shown in or having at least 90% identity with SEQ ID NO:33, and / or the 3'UTR contains the sequence shown in or having at least 90% identity with SEQ ID NO:

35.

15. The composition according to any one of claims 1-14, wherein the RNA molecule comprises a poly-A tail; Preferably, the poly-A tail is selected from 120A, Poly A-3070, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA, or SV40 late polyA.

16. The composition according to any one of claims 1-15, wherein the RNA molecule comprises a 5' cap structure; Preferably, 5'Cap is selected from Cap0, Cap1, Cap2, Cap3, Cap4, ARCA, modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. More preferably, 5'Cap is selected from ARCA, 3'OMe-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5')( 2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

17. The composition according to any one of claims 1-16, wherein the RNA molecule comprises at least one chemical modification; preferably, the chemical modification is uracil modification.

18. The composition according to any one of claims 1-17, wherein at least one RNA molecule is formulated in the same or different lipid nanoparticles.

19. A composition comprising at least one polypeptide, wherein the polypeptide comprises at least one antigenic peptide derived from a tumor-associated antigen (TAA) and at least one antigenic peptide derived from KRAS.

20. The composition according to claim 19, wherein the composition comprises 1-10 polypeptides, preferably, the composition comprises 1, 2, 3, 4, or 5 polypeptides.

21. The composition according to claim 19 or 20, wherein at least one antigenic peptide derived from TAA and at least one antigenic peptide derived from KRAS are located on the same polypeptide, or At least one antigenic peptide derived from TAA and at least one antigenic peptide derived from KRAS are located on different polypeptides.

22. The composition according to any one of claims 19-21, wherein the antigenic peptide derived from TAA and / or the antigenic peptide derived from KRAS has a length of 9-50 amino acids, preferably 9-35 amino acids.

23. The composition according to any one of claims 19-22, wherein at least one antigenic peptide derived from TAA comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, OAS1, hTERT and / or survivin.

24. The composition according to claim 23, wherein, The antigenic peptide derived from ECT2 contains the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7 or SEQ ID NO:8; The antigenic peptide derived from TOP2A contains the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10; The antigenic peptide derived from TPX2 contains the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:9; The antigenic peptide derived from C12orf32 contains the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:11; The antigenic peptide derived from OAS1 contains the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:12; The antigenic peptide derived from hTERT comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; and / or The antigenic peptide derived from survivin contains an amino acid sequence as shown in SEQ ID NO:21 and / or 22.

25. The composition according to any one of claims 19-24, wherein the at least one KRAS-derived antigenic peptide has a G12 and / or G13 mutation; Preferably, the antigenic peptide derived from KRAS has G12A, G12C, G12D, G12R, G12S, G12V and / or G13D mutations; Preferably, the antigenic peptide derived from KRAS comprises at least one of the sequences shown in SEQ ID NO:13-16.

26. The composition according to any one of claims 19-25, wherein the KRAS-derived antigenic peptide synergistically enhances the level of in vivo immune response and / or antitumor activity with the TAA-derived antigenic peptide.

27. The composition according to any one of claims 19-26, wherein the composition comprises a first polypeptide and a second polypeptide, characterized in that: A. The first polypeptide contains: (1) One or more antigenic peptides derived from ECT2; preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:

8. (2) One or more antigenic peptides derived from TOP2A; preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10; (3) One or more antigenic peptides derived from TPX2; preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:3; more preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:9; (4) One or more antigenic peptides derived from C12orf32; preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11; (5) One or more antigenic peptides derived from OAS1; preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:

12. (6) One or more antigenic peptides derived from hTERT; preferably, the antigenic peptides derived from hTERT comprise an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; and (7) One or more antigenic peptides derived from survivin, preferably, the antigenic peptides derived from survivin contain amino acid sequences as shown in SEQ ID NO:21 and / or 22; The second polypeptide contains: (1) One or more antigenic peptides derived from KRAS and having a G12D mutation; preferably, the antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:13; (2) One or more antigenic peptides derived from KRAS and having a G12V mutation; preferably, the antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:14; (3) One or more antigenic peptides derived from KRAS and having a G13D mutation; preferably, the antigenic peptides comprise the amino acid sequence shown in SEQ ID NO:15; and (4) One or more antigenic peptides derived from KRAS and having a G12C mutation; preferably, the antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:16; Preferably, the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it; and / or the second polypeptide comprises a sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it; or B. The first polypeptide contains: (1) One or more antigenic peptides derived from ECT2; preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:

8. (2) One or more antigenic peptides derived from TOP2A; preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10; (3) One or more antigenic peptides derived from TPX2; preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:3; more preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:9; (4) One or more antigenic peptides derived from C12orf32; preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11; and (5) One or more antigenic peptides derived from OAS1; preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:

12. The second polypeptide contains: (1) One or more antigenic peptides derived from KRAS and having a G12D mutation; preferably, the antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:13; (2) One or more antigenic peptides derived from KRAS and having a G12V mutation; preferably, the antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:14; (3) One or more antigenic peptides derived from KRAS and having a G13D mutation; preferably, the antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:15; (4) One or more antigenic peptides derived from KRAS and having a G12C mutation; preferably, the antigenic peptides comprise an amino acid sequence as shown in SEQ ID NO:16; (5) One or more antigenic peptides derived from hTERT; preferably, the antigenic peptides derived from hTERT comprise an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof; and (6) One or more antigenic peptides derived from survivin; preferably, the antigenic peptides derived from survivin contain amino acid sequences as shown in SEQ ID NO:21 and / or 22; Preferably, the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it; and / or the second polypeptide comprises a sequence as shown in SEQ ID NO:53 or having at least 90% sequence identity with it.

28. The composition of any one of claims 19-27, wherein, The antigenic peptides in the polypeptide are directly linked by peptide bonds or linked by linkers. Preferably, the connector is selected from (GxS)y, EAAAK, AAY or GGPPG, where x is selected from an integer from 1 to 5 and y is selected from an integer from 1 to 6.

29. A composition comprising a first polypeptide and a second polypeptide, preferably, the composition further comprising a third polypeptide. The composition is selected from any one of the following groups: (a) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it. (b) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:50 or having at least 90% sequence identity with it. (c) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:59 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it. (d) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it, and the third polypeptide comprises an amino acid sequence as shown in SEQ ID NO:71 or having at least 90% sequence identity with it. (e) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it. (f) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:53 or having at least 90% sequence identity with it. (g) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:59 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it. (h) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:56 or having at least 90% sequence identity with it, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it, and the third polypeptide comprises an amino acid sequence as shown in SEQ ID NO:71 or having at least 90% sequence identity with it. (i) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:47 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:41 or having at least 90% sequence identity with it. (j) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:62 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:68 or having at least 90% sequence identity with it. (k) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:47 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:38 or having at least 90% sequence identity with it. (l) The first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:62 or having at least 90% sequence identity with it, and the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:65 or having at least 90% sequence identity with it.

30. A nucleic acid molecule comprising (a) an open reading frame (ORF) encoding a protein of interest; wherein, The target protein comprises one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32, and OAS1; Preferably, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; The antigenic peptide derived from TOP2A contains the amino acid sequence shown in SEQ ID NO:4; The antigenic peptide derived from TPX2 contains the amino acid sequence shown in SEQ ID NO:3; The antigenic peptide derived from C12orf32 contains the amino acid sequence shown in SEQ ID NO:5; The antigenic peptide derived from OAS1 contains the amino acid sequence shown in SEQ ID NO:

6.

31. The nucleic acid molecule according to claim 30, wherein the length of the antigenic peptide is 9-50 amino acids, preferably 9-35 amino acids.

32. The nucleic acid molecule according to claim 30 or 31, characterized in that, The antigenic peptide derived from ECT2 contains the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; The antigenic peptide derived from TOP2A contains the amino acid sequence shown in SEQ ID NO:10; The antigenic peptide derived from TPX2 contains the amino acid sequence shown in SEQ ID NO:9; The antigenic peptide derived from C12orf32 contains the amino acid sequence shown in SEQ ID NO:11; The antigenic peptide derived from OAS1 contains the amino acid sequence shown in SEQ ID NO:

12.

33. The nucleic acid molecule according to any one of claims 30-32, wherein the target protein comprises: (1) One or more antigenic peptides derived from ECT2; preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:

8. (2) One or more antigenic peptides derived from TOP2A; preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10; (3) One or more antigenic peptides derived from TPX2; preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:3; more preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:9; (4) One or more antigenic peptides derived from C12orf32; preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11; and (5) One or more antigenic peptides derived from OAS1; preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:

12.

34. The nucleic acid molecule according to any one of claims 30-33, wherein the target protein further comprises one or more antigenic peptides derived from hTERT and / or survivin; Preferably, the antigenic peptide derived from hTERT comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof. The antigenic peptide derived from survivin contains an amino acid sequence as shown in SEQ ID NO:21 or 22.

35. The nucleic acid molecule of any one of claims 30-34, wherein, The antigenic peptides are directly linked by peptide bonds or linked by linkers. Preferably, the connector is selected from (GxS)y, EAAAK, AAY or GGPPG, where x is selected from an integer from 1 to 5 and y is selected from an integer from 1 to 6.

36. The nucleic acid molecule of any one of claims 30-35, wherein, The target protein comprises an amino acid sequence as shown in any one of SEQ ID NO:44, 47, 56, 59, 62, 65, 68 or having at least 90% sequence identity with it.

37. The nucleic acid molecule according to any one of claims 30-36, further comprising (b) an untranslated region element (UTR); preferably, the untranslated region element (UTR) comprises a 5' untranslated region element (5'UTR) and / or a 3' untranslated region element (3'UTR); Preferably, the 5'UTR contains the sequence shown in or having at least 90% identity with SEQ ID NO:33, and / or the 3'UTR contains the sequence shown in or having at least 90% identity with SEQ ID NO:

35.

38. The nucleic acid molecule according to any one of claims 30-37, further comprising: (c) Poly-A tail; Preferably, the poly-A tail is selected from 120A, Poly A-3070, HGH polyA, SV40 polyA, BGH polyA, rbGlob polyA, or SV40 late polyA.

39. The nucleic acid molecule according to any one of claims 30-38, comprising: (d) 5' Cap structure; Preferably, the 5'Cap is selected from Cap0, Cap1, Cap2, Cap3, Cap4, ARCA, modified ARCA, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-denitro-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. More preferably, the 5'Cap is selected from ARCA, 3'OMe-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pU, m7Gppp(A2'O-MOE)pG, m7G(5')ppp(5') (2'OMeA)pG, m7G(5')ppp(5')(2'OMeG)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

40. The nucleic acid molecule according to any one of claims 30-39, comprising at least one chemical modification; preferably, the chemical modification is uracil modification.

41. The nucleic acid molecule according to any one of claims 30-40, wherein the nucleic acid molecule is DNA or RNA, preferably RNA, and more preferably mRNA.

42. The nucleic acid molecule of any one of claims 30-41, wherein, The nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NO:46, 49, 58, 61, 64, 67, 70 or has at least 90% sequence identity with it.

43. A polypeptide comprising one or more antigenic peptides derived from ECT2, TOP2A, TPX2, C12orf32 and OAS1; Preferably, the antigenic peptide derived from ECT2 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; The antigenic peptide derived from TOP2A contains the amino acid sequence shown in SEQ ID NO:4; The antigenic peptide derived from TPX2 contains the amino acid sequence shown in SEQ ID NO:3; The antigenic peptide derived from C12orf32 contains the amino acid sequence shown in SEQ ID NO:5; The antigenic peptide derived from OAS1 contains the amino acid sequence shown in SEQ ID NO:

6.

44. The polypeptide according to claim 43, wherein the length of the antigenic peptide is 9-35 amino acids.

45. The polypeptide according to claim 43 or 44, characterized in that, The antigenic peptide derived from ECT2 contains the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:8; The antigenic peptide derived from TOP2A contains the amino acid sequence shown in SEQ ID NO:10; The antigenic peptide derived from TPX2 contains the amino acid sequence shown in SEQ ID NO:9; The antigenic peptide derived from C12orf32 contains the amino acid sequence shown in SEQ ID NO:11; The antigenic peptide derived from OAS1 contains the amino acid sequence shown in SEQ ID NO:

12.

46. ​​The polypeptide according to any one of claims 43-45, comprising: (1) One or more antigenic peptides derived from ECT2; preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; more preferably, the antigenic peptides derived from ECT2 contain the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:

8. (2) One or more antigenic peptides derived from TOP2A; preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:4; more preferably, the antigenic peptides derived from TOP2A contain the amino acid sequence shown in SEQ ID NO:10; (3) One or more antigenic peptides derived from TPX2; preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:3; more preferably, the antigenic peptides derived from TPX2 contain the amino acid sequence shown in SEQ ID NO:9; (4) One or more antigenic peptides derived from C12orf32; preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:5; more preferably, the antigenic peptides derived from C12orf32 comprise the amino acid sequence shown in SEQ ID NO:11; and (5) One or more antigenic peptides derived from OAS1; preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:6; more preferably, the antigenic peptides derived from OAS1 contain the amino acid sequence shown in SEQ ID NO:

12.

47. The polypeptide according to any one of claims 43-46, further comprising one or more antigenic peptides derived from hTERT and / or survivin; Preferably, the antigenic peptide derived from hTERT comprises an amino acid sequence as shown in any one of SEQ ID NO:17-20 or any combination thereof. The antigenic peptide derived from survivin contains an amino acid sequence as shown in SEQ ID NO:21 and / or 22.

48. The polypeptide of any of claims 43-47, wherein, The antigenic peptides are directly linked by peptide bonds or linked by linkers. Preferably, the connector is selected from (GxS)y, EAAAK, AAY or GGPPG, where x is selected from an integer from 1 to 5 and y is selected from an integer from 1 to 6.

49. The polypeptide according to any one of claims 43-48, comprising an amino acid sequence as shown in or having at least 90% sequence identity with any one of SEQ ID NO: 44, 47, 56, 59, 62, 65, 68.

50. Use of ECT2, TOP2A, TPX2, C12orf32 and / or OAS1 protein or their immunogenic fragments in the preparation of pharmaceutical compositions for the treatment or prevention of cancer; Preferably, the cancer is a KRAS-mutated malignant tumor; more preferably, the cancer is selected from pancreatic cancer, colorectal cancer, and / or lung cancer; preferably, the lung cancer is non-small cell lung cancer.

51. The application according to claim 50, wherein the immunogenic fragment comprises a high-affinity HLA sequence, exhibits an enhanced immune response level and / or has antitumor activity after immunization.

52. The application according to claim 50 or 51, wherein the pharmaceutical composition further comprises at least one antigenic peptide derived from KRAS, hTERT and / or survivin.

53. The application according to any one of claims 50-52, wherein the immunogenic fragment comprises: The antigenic peptide derived from ECT2 contains the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7 or SEQ ID NO:8; The antigenic peptide derived from TOP2A contains the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:10; The antigenic peptide derived from TPX2 contains the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:9; The antigenic peptide derived from C12orf32 contains the amino acid sequence shown in SEQ ID NO:5 or SEQ ID NO:11; The antigenic peptide derived from OAS1 contains the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:

12.

54. The use according to any one of claims 50-53, wherein, The proteins ECT2, TOP2A, TPX2, C12orf32, and OAS1 contain the amino acid sequences shown in SEQ ID NO:126, 129, 132, 135, and 138, respectively.

55. The use according to any one of claims 50-54, wherein, The antigenic peptides derived from hTERT contain amino acid sequences as shown in any one of SEQ ID NO:17-20 or any combination thereof; The antigenic peptides derived from survivin contain amino acid sequences as shown in SEQ ID NO:21 and / or 22; and / or, at least one antigenic peptide derived from KRAS has a G12 and / or G13 mutation; Preferably, the antigenic peptide derived from KRAS has G12A, G12C, G12D, G12R, G12S, G12V and / or G13D mutations; Preferably, the antigenic peptide derived from KRAS contains at least one of the sequences shown in SEQ ID NO:13-16.

56. The application according to any one of claims 50-55, wherein the pharmaceutical composition is a tumor vaccine, preferably an mRNA vaccine or a recombinant protein vaccine.

57. A polypeptide comprising an amino acid sequence as shown in or having at least 90% sequence identity with any one of SEQ ID NO:44, 47, 56, 59, 62, 65 and 68.

58. An RNA molecule comprising or having at least 90% sequence identity with any one of SEQ ID NO:46, 49, 58, 61, 64, 67 and 70.

59. A DNA molecule comprising a nucleotide sequence as shown in or having at least 90% sequence identity with any one of SEQ ID NO:45, 48, 57, 60, 63, 66 and 69.

60. A polynucleotide encoding a polypeptide as described in any one of claims 43-49 or 57, or a polypeptide in a composition as described in any one of claims 19-29.

61. A vector comprising a DNA molecule as claimed in claim 59 or a polynucleotide as claimed in claim 60.

62. A host cell comprising the polynucleotide of claim 60 or the vector of claim 61.

63. Lipid nanoparticles comprising a nucleic acid molecule as described in any one of claims 30-42, an RNA molecule as described in claim 58, a composition as described in any one of claims 1-18, a DNA molecule as described in claim 59, a polynucleotide as described in claim 60, or a carrier as described in claim 61.

64. A pharmaceutical composition comprising any one or any combination of the following: The nucleic acid molecule as described in any one of claims 30-42, the RNA molecule as described in claim 58, the composition as described in any one of claims 1-18, the polypeptide as described in any one of claims 43-49 or 57, the composition as described in any one of claims 19-29, the DNA molecule as described in claim 59, the polynucleotide as described in claim 60, the carrier as described in claim 61, or the lipid nanoparticle as described in claim 63; Optionally, it contains pharmaceutically acceptable excipients, diluents, or excipients.

65. A tumor vaccine comprising the pharmaceutical composition as described in claim 64; Preferably, the tumor vaccine is a universal pancreatic cancer, colorectal cancer, and / or non-small cell lung cancer vaccine.

66. The use of any nucleic acid molecule as claimed in any one of claims 30-42, the RNA molecule as claimed in claim 58, the composition as claimed in any one of claims 1-18, the polypeptide as claimed in any one of claims 43-49 or 57, the composition as claimed in any one of claims 19-29, the DNA molecule as claimed in claim 59, the polynucleotide as claimed in claim 60, the carrier as claimed in claim 61, or the lipid nanoparticle as claimed in claim 63, the pharmaceutical composition as claimed in claim 64, or the tumor vaccine as claimed in claim 65 in (1) the preparation of a medicament for inducing an immune response in a subject, or (2) the preparation of a medicament for the prevention and / or treatment of cancer; Preferably, the cancer is a KRAS-mutated malignant tumor; more preferably, the cancer is selected from pancreatic cancer, colorectal cancer, and / or lung cancer; preferably, the lung cancer is non-small cell lung cancer.

67. Methods for preparing antigenic peptides include: The antigenic peptide is produced by expressing the polynucleotide as described in claim 60 or the vector as described in claim 61 in the host cell.

68. Methods for preparing RNA molecules include: RNA molecules are obtained by transcription using the polynucleotide of claim 60 or the vector of claim 61 as templates. Preferably, the method further includes adding a 5'Cap to the 5' end of the RNA molecule.

69. Use of molecules targeting ECT2, TOP2A, TPX2, C12orf32 and / or OAS1 in the preparation of medicaments for the prevention and / or treatment of cancer; Preferably, the molecule regulates the activity of ECT2, TOP2A, TPX2, C12orf32 and / or OAS1; Preferably, the cancer is a KRAS-mutated malignant tumor; more preferably, the cancer is selected from pancreatic cancer, colorectal cancer, and / or lung cancer; preferably, the lung cancer is non-small cell lung cancer.

Images