Immunomodulatory therapeutic mRNA compositions encoding activated EGFR mutant peptides

Abstract

The present disclosure relates generally to immunomodulatory therapeutic compositions of mRNA encoding an activated EGFR mutant peptide and mRNA encoding a polypeptide that enhances an immune response to the activated EGFR mutant peptide. The disclosure also features methods of using the immunomodulatory therapeutic compositions, for example, to treat a cancer expressing a mutated EGFR or to delay the progression of a cancer in a subject.

Description

Technical Field

[0001] The present disclosure generally relates to immunomodulatory therapeutic compositions of mRNA encoding activated EGFR mutant peptides and mRNA encoding polypeptides that enhance immune responses to activated EGFR mutant peptides. The present disclosure also features methods of using the immunomodulatory therapeutic compositions, such as treating a subject's cancer expressing mutant EGFR or delaying cancer progression. Background Art

[0002] In China, mutations in the epidermal growth factor receptor (EGFR) cause almost 40-50% of non-small cell lung cancer (NSCLC), and NSCLC is one of the most common malignant tumors with a high mortality rate [1]. EGFR is a member of the erbB family of receptor tyrosine kinase proteins, which contains an extracellular binding domain, a transmembrane segment, and a cytoplasmic tyrosine kinase domain. When the ligand of EGFR engages the extracellular binding domain, EGFR activates, dimerizes, and autophosphorylates the tyrosine kinase domain. This phosphorylation triggers downstream signaling pathways involved in cell growth and proliferation. When EGFR gain-of-function mutations occur, they lead to constitutive activation of the tyrosine kinase domain, which promotes uncontrolled cell growth and proliferation and ultimately mediates tumorigenesis [2].

[0003] EGFR mutations are common in exons 18 to 21, which encode the ATP binding site of the tyrosine kinase. Among these EGFR mutations, exon 19 deletions account for 47% (E746_A750 del accounts for 65% of exon 19 deletions), followed by L858R mutations on exon 21, which account for 41%, and other rare mutations, including exon 20 insertions, p.G719X, and p.L861Q, account for 12% [3,4]. Patients carrying the common mutations of L858R and 19del show significant responses to EGFR-tyrosine kinase inhibitors (TKIs), while those positive for uncommon EGFR mutations are considered to be less sensitive to these drugs [5]. T790M exists as a minor allele before TKI therapy and accounts for approximately half of cases of acquired resistance [6].

[0004] Currently, targeted drugs called tyrosine kinase inhibitors (TKIs) are the standard of care for patients with NSCLC who harbor EGFR mutations [2]. The first generation of TKIs (including gefitinib, erlotinib, and icotinib) bind reversibly to EGFR (primarily targeting EGFR L858R and exon 19 deletions) and prevent ATP from binding to its TK domain, subsequently impeding cell proliferation and ultimately leading to cell death. Gefitinib and erlotinib have been approved worldwide for the treatment of patients with NSCLC who harbor EGFR mutations, while icotinib has been approved in China. The second generation of EGFR TKIs include afatinib and dacomitinib, which bind irreversibly to EGFR. Like gefitinib and erlotinib, afatinib is approved worldwide for the first-line treatment of patients with EGFR mutations, and dacomitinib is also expected to be approved. Although the first and second generation EGFR TKIs have advanced the objective response rate (ORR) to as high as approximately 60-70% and significantly prolonged the progression-free survival (PFS) of NSCLC patients with EGFR mutations, all patients will sooner or later develop acquired resistance through a variety of mechanisms after 9-15 months of treatment with first or second generation TKIs [2,7]. The most common mechanism of acquired resistance is the T790M mutation on EGFR exon 20, which accounts for approximately 55% of all EGFR TKI resistance in NSCLC patients [7]. Although the third generation EGFR TKIs (osimertinib and almonertinib, which are designed to mainly target EGFR sensitizing mutations and T790M mutations) can partially overcome the resistance caused by the T790M mutation, patients treated with third generation EGFR-TKIs still develop acquired resistance (mainly the on-target C797S mutation) after approximately 10 months, and the resistance mechanisms are diverse and complex. Recently, a new generation of EGFR TKIs (BLU-701, BBT-176, and HS-10375) has been developed to target acquired osimertinib resistance, especially the C797S mutation. However, other resistance mechanisms will eventually emerge [8,9]. Currently, there is no effective treatment for patients with multi-TKI resistance, so there is an urgent need to develop new effective drugs targeting EGFR mutations to eventually overcome the endless TKI resistance.

[0005] Among EGFR mutations, L858R substitution and E746_A750 deletion are the fundamental mutations of EGFR, which mainly lead to the development of lung cancer, and T790M point mutation is the main mechanism of resistance to first- and second-generation TKIs. In addition, C797S mutation is the main resistance mechanism of third-generation TKIs. At present, although targeted drugs (TKIs) have significantly improved the ORR (objective response rate) of NSCLC treatment, the median PFS (progression-free survival) and OS (overall survival) are still unsatisfactory due to the heterogeneity of cancer escape mechanisms. However, fundamental mutations including L858R and E746_A750del always exist, and T790M and C797S also persist, and even produce new escape mechanisms, so it is very attractive to develop multivalent therapeutic mRNA cancer vaccines targeting EGFR L858R, E746_A750del, T790M and C797S. The therapeutic EGFR cancer vaccine mentioned above can theoretically enhance T cell activation and intentionally expand T cells expressing EGFR mutation-specific TCRs to effectively eradicate xenogeneic cancer cells and ultimately cure cancer. Due to the mode of action, a successfully developed vaccine can potentially be applied in a) neoadjuvant / adjuvant therapy, b) monotherapy or combination with TKI or immunotherapy for patients with advanced EGFRmut NSCLC.

[0006] In addition, mRNA vaccines as an innovative model have shown obvious advantages, including, a) the "self-adjuvant effect" of mRNA and LNP, which contributes to better immunogenicity of antigen-specific cytotoxic T cell responses; b) there is no risk of host genome integration, and mRNA is rapidly degraded after efficient antigen expression; c) mRNA-encoded antigens are internally processed and presented in antigen-presenting cells, and HLA restrictions are minimal to cover a larger HLA population.

[0007] In general, mRNA vaccines targeting EGFR mutations are promising therapeutic approaches for NSCLC treatment to improve overall survival and even lead to tumor cure. Therefore, there is a need in the art to develop new and effective mRNA vaccines targeting EGFR mutations for the treatment of cancer, including NSCLC. Summary of the invention

[0008] In one aspect, provided herein are polypeptides comprising an EGFR domain, wherein the EGFR domain comprises at least one mutated fragment of human EGFR.

[0009] In some embodiments, the polypeptide further comprises a degradation domain configured to mark the EGFR domain for proteasomal degradation.

[0010] In some embodiments, the degradation domain comprises a ubiquitin peptide.

[0011] In some embodiments, the ubiquitin peptide is a ubiquitin monomer, a ubiquitin polymer, a ubiquitin-like domain (ULD), or a functional derivative thereof.

[0012] In some embodiments, the polypeptide comprises an EGFR domain fused directly or indirectly to the C-terminus of a degradation domain, and wherein the EGFR domain is at least about 10 amino acids in length.

[0013] In some embodiments, the EGFR domain is located in the C-terminal portion of the polypeptide.

[0014] In some embodiments, the EGFR domain comprises two or more mutated fragments of human EGFR.

[0015] In some embodiments, the EGFR domain is fused directly to the C-terminus of the degradation domain, and wherein the EGFR domain is at least about 10 amino acids in length.

[0016] In some embodiments, the spacer peptide is located between the degradation domain and the EGFR domain; optionally wherein the spacer peptide is at least about 15 amino acids in length; optionally wherein the spacer peptide is about 15-30 amino acids in length; optionally wherein the spacer peptide is about 20 amino acids, about 25 amino acids, or about 30 amino acids in length.

[0017] In some embodiments, the degradation domain consists of, consists essentially of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:1.

[0018] In some embodiments, the degradation domain further comprises a spacer positioned between the ubiquitin peptide and the EGFR domain, and wherein the spacer peptide consists of, consists essentially of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:2.

[0019] In some embodiments, the polypeptide further comprises one or more linker sequences.

[0020] In some embodiments, the EGFR domain comprises at least one mutant fragment of human EGFR, wherein each mutant fragment comprises one or more EGFR mutations selected from T790M, C797S, L858R and E746_A750 del; optionally wherein the mutant fragment comprises one or more EGFR mutations selected from any one of (a) to (k):

[0021] (a) L858R;

[0022] (b) T790M;

[0023] (c) E746_A750 del;

[0024] (d) C797S;

[0025] (e) T790M and C797S;

[0026] (f) L858R and T790M;

[0027] (g) L858R and E746_A750 del;

[0028] (h) E746_A750 del;

[0029] (i) E746_A750 del and T790M;

[0030] (j) L858R, E746_A750 del and T790M; and

[0031] (k) L858R, E746_A750 del, T790M, and C797S.

[0032] In some embodiments, the length of at least one mutant fragment of human EGFR is at least about 10 amino acids; optionally wherein the length of the human EGFR mutant fragment is about 10 amino acids, 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids or about 35 amino acids.

[0033] In some embodiments, the at least one mutated fragment of human EGFR includes two or more mutated fragments of human EGFR, wherein the two or more mutated fragments are the same or different.

[0034] In some embodiments, the EGFR domain comprises two or more mutant fragments of human EGFR, wherein at least two of the two or more mutant fragments are fused directly or through a linker peptide.

[0035] In some embodiments, the at least one mutant fragment of human EGFR comprises a L858R fragment, wherein the L858R fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the L858R mutation.

[0036] In some embodiments, the EGFR domain comprises two or more identical copies of the L858R fragment.

[0037] In some embodiments, the L858R fragment has the amino acid sequence of SEQ ID NO:3.

[0038] In some embodiments, the at least one mutant fragment of human EGFR includes a T790M fragment, wherein the T790M fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the T790M mutation.

[0039] In some embodiments, the EGFR domain comprises two or more identical copies of the T790M fragment.

[0040] In some embodiments, the T790M fragment has the amino acid sequence of SEQ ID NO:4.

[0041] In some embodiments, the at least one mutant fragment of human EGFR includes an E746_A750 del fragment, wherein the E746_A750 del fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the E746_A750 del mutation.

[0042] In some embodiments, the EGFR domain comprises two or more identical copies of the E746 A750 del fragment.

[0043] In some embodiments, the E746_A750 del fragment has the amino acid sequence of SEQ ID NO:5.

[0044] In some embodiments, the at least one mutant fragment of human EGFR comprises a C797S fragment, wherein the C797S fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the C797S mutation.

[0045] In some embodiments, the EGFR domain comprises two or more identical copies of the C797S fragment.

[0046] In some embodiments, the C797S fragment has the amino acid sequence of SEQ ID NO:6.

[0047] In some embodiments, the EGFR domain comprises:

[0048] (a) six copies of the L858R fragment, wherein each L858R fragment has the amino acid sequence of SEQ ID NO: 3;

[0049] (b) three copies of an L858R fragment and three T790M fragments, wherein each L858R fragment has the amino acid sequence of SEQ ID NO: 3, and wherein each T790M fragment has the amino acid sequence of SEQ ID NO: 4;

[0050] (c) three L858R fragments and three E746_A750 del fragments, wherein each L858R fragment has an amino acid sequence of SEQ ID NO: 3, and wherein each E746_A750 del fragment has an amino acid sequence of SEQ ID NO: 5;

[0051] (d) six E746_A750 fragments, wherein each E746_A750 fragment has an amino acid sequence of SEQ ID NO: 5;

[0052] (e) three E746_A750 del fragments and three T790M fragments, wherein each T790M fragment has the amino acid sequence of SEQ ID NO: 4, and wherein each E746_A750 fragment has the amino acid sequence of SEQ ID NO: 5;

[0053] (f) two L858R fragments, two E746_A750 del fragments, and two T790M fragments, wherein each L858R fragment has the amino acid sequence of SEQ ID NO:3, wherein each T790M fragment has the amino acid sequence of SEQ ID NO:4, and wherein each E746_A750 fragment has the amino acid sequence of SEQ ID NO:5; or

[0054] (g) two L858R fragments, two E746_A750 del fragments, one T790M fragment and one C797S fragment, wherein each L858R fragment has the amino acid sequence of SEQ ID NO:3, wherein each T790M fragment has the amino acid sequence of SEQ ID NO:4, wherein each E746_A750 fragment has the amino acid sequence of SEQ ID NO:5, and wherein each C797S fragment has the amino acid sequence of SEQ ID NO:6.

[0055] In one aspect, provided herein is an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28 and 31.

[0056] In one aspect, provided herein is a composition comprising a plurality of polypeptides, wherein each polypeptide is selected from the polypeptides described above, wherein at least two of the plurality of polypeptides are different.

[0057] In one aspect, provided herein is a nucleic acid comprising an open reading frame encoding a polypeptide as described above.

[0058] In some embodiments, the nucleic acid further comprises a 5' untranslated region (UTR); optionally wherein the 5'UTR comprises the sequence of SEQ ID NO:34 or comprises a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:34.

[0059] In some embodiments, the nucleic acid further comprises a 3'UTR; optionally wherein the 3'UTR comprises the sequence of SEQ ID NO:35 or comprises a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:35.

[0060] In some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule.

[0061] In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs selected from pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

[0062] In one aspect, provided herein is a vector comprising a nucleic acid as described above.

[0063] In one aspect, the present invention provides a composition comprising a nucleic acid as described above and at least one lipid; optionally, the lipid comprises a first lipid according to Formula 01-I or Formula 01-II; or a compound listed in Table 01-1; or a compound according to Formula 02-I; or a compound listed in Table 02-1; or a compound according to Formula 03-I; or a compound listed in Table 03-1; or a compound according to Formula 04-I; or a compound listed in Table 04-1; optionally, the lipid also comprises a second lipid according to Formula 05-I.

[0064] In one aspect, the present invention provides a composition comprising multiple species of nucleic acid molecules, each nucleic acid molecule encoding an EGFR domain, wherein each encoded EGFR domain comprises at least one mutant fragment of human EGFR, wherein the mutant fragment comprises one or more EGFR mutations selected from T790M, C797S, L858R and E746_A750 del, and wherein the EGFR domains encoded by at least two of the multiple species of nucleic acid molecules are different.

[0065] In some embodiments, the EGFR domain encoded by at least one of the plurality of species of nucleic acid molecules comprises two or more mutant fragments of human EGFR, wherein the two or more mutant fragments are the same or different.

[0066] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules comprises an EGFR mutation selected from any one of (a) to (K):

[0067] (a) L858R;

[0068] (b) T790M;

[0069] (c) E746_A750 del;

[0070] (d) C797S;

[0071] (e) T790M and C797S;

[0072] (f) L858R and T790M;

[0073] (g) L858R and E746_A750 del;

[0074] (h) E746_A750 del;

[0075] (i) E746_A750 del and T790M;

[0076] (j) L858R, E746_A750 del and T790M; and

[0077] (k) L858R, E746_A750 del, T790M, and C797S.

[0078] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is a L858R fragment, wherein the L858R fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the L858R mutation.

[0079] In some embodiments, each of the plurality of species of nucleic acid molecules encoding the L858R fragment comprises a coding sequence selected from SEQ ID NO: 9, 12, 15, 18, 21, 30 and 33, a homologous sequence thereof, or a degenerate sequence thereof.

[0080] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules include a T790M fragment, wherein the T790M fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the T790M mutation.

[0081] In some embodiments, each of the plurality of species of nucleic acid molecules encoding the T790M fragment comprises a coding sequence selected from SEQ ID NO: 12, 18, 21, 27, 30 and 33, a homologous sequence thereof, or a degenerate sequence thereof.

[0082] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is an E746_A750 del fragment, wherein the E746_A750 del fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the E746_A750 del mutation.

[0083] In some embodiments, each of the multiple species of nucleic acid molecules encoding the E746_A750 del fragment comprises a coding sequence selected from SEQ ID NO: 15, 18, 21, 24, 27, 30 and 33, a homologous sequence thereof, or a degenerate sequence thereof.

[0084] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is a C797S fragment, wherein the C797S fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the C797S mutation.

[0085] In some embodiments, each of the plurality of species of nucleic acid molecules encoding the C797S fragment comprises a coding sequence selected from SEQ ID NOs: 30 and 33, a homologous sequence thereof, or a degenerate sequence thereof.

[0086] In some embodiments, the multiple species of nucleic acid molecules also include at least one first species of nucleic acid molecules encoding an EGFR domain longer than about 10 amino acids, and wherein at least one first species of nucleic acid molecules also encodes a degradation domain, wherein the degradation domain is configured to mark the EGFR domain for proteasomal degradation.

[0087] In some embodiments, the degradation domain comprises a ubiquitin peptide.

[0088] In some embodiments, the ubiquitin peptide is a ubiquitin monomer, a ubiquitin polymer, a ubiquitin-like domain (ULD), or a functional derivative thereof.

[0089] In some embodiments, the spacer peptide is located between the degradation domain and the EGFR domain; optionally wherein the spacer peptide is at least about 15 amino acids in length; optionally wherein the spacer peptide is about 15-30 amino acids in length; optionally wherein the spacer peptide is about 20 amino acids, about 25 amino acids, or about 30 amino acids in length.

[0090] In some embodiments, at least one species of nucleic acid molecule comprises a coding sequence for a ubiquitin peptide selected from SEQ ID NOs: 37-45.

[0091] In some embodiments, the plurality of species of nucleic acid molecules further comprises at least one second species of nucleic acid molecule encoding an EGFR domain no longer than about 10 amino acids, and wherein the at least one second species of nucleic acid molecule lacks a coding sequence for a degradation domain.

[0092] In some embodiments, the plurality of species of nucleic acid molecules include mRNA molecules.

[0093] In some embodiments, at least one of the plurality of species of nucleic acid molecules further comprises a 3'-UTR and / or a 5'-UTR; optionally wherein the 5'-UTR comprises the sequence of SEQ ID NO:34, or comprises a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:34; optionally wherein the 3'-UTR comprises the sequence of SEQ ID NO:35, or comprises a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:35.

[0094] In some embodiments, the plurality of species of nucleic acid molecules include RNA molecules comprising a sequence selected from SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, 32, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, and 32.

[0095] In some embodiments, the plurality of species of nucleic acid molecules include DNA molecules comprising a sequence selected from SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, 33, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30 and 33.

[0096] In some embodiments, at least one of the plurality of species of nucleic acid molecules comprises one or more functional nucleotide analogs selected from pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

[0097] In some embodiments, the composition further comprises at least one lipid; optionally, the lipid comprises a first lipid according to Formula 01-I or Formula 01-II; or a compound listed in Table 01-1; or a compound according to Formula 02-I; or a compound listed in Table 02-1; or a compound according to Formula 03-I; or a compound listed in Table 03-1; or a compound according to Formula 04-I; or a compound listed in Table 04-1; optionally, the lipid further comprises a second lipid according to Formula 05-I.

[0098] In some embodiments, the composition is formulated as a lipid nanoparticle encapsulating the nucleic acid.

[0099] In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0100] In some embodiments, the composition is a vaccine.

[0101] In some embodiments, the composition is administered in combination with a second therapeutic agent; optionally, the second therapeutic agent comprises EGFR-TKI, PD1 antibody; optionally, the EGFR-TKI is erlotinib, gefitinib or dacomitinib.

[0102] In one aspect, the present invention provides a method for producing EGFR mutant-specific immune effector cells, comprising providing a starting population of immune effector cells, and contacting the starting population of immune effector cells with a polypeptide as described above, a nucleic acid as described above, or a composition as described above under suitable conditions, wherein upon contact, EGFR mutant-specific immune effector cells are produced.

[0103] In some embodiments, the contacting is performed in the presence of a proteasome system, and wherein the proteasome system degrades the EGFR domain into one or more peptides of about 9 to 15 amino acids in length.

[0104] In some embodiments, contacting is performed in the presence of antigen presenting cells (APCs), wherein the APCs present the EGFR domain or a degraded portion thereof on their surface.

[0105] In some embodiments, the APC is selected from the group consisting of a dendritic cell, a macrophage, a Langerhans cell, and a B cell.

[0106] In some embodiments, the APC expresses HLA class I and / or HLA class II molecules; optionally wherein the HLA class I molecule is of the HLA-A*0101 subtype or the HLA-A*0201 subtype.

[0107] In some embodiments, the immune effector cells are selected from T cells, peripheral blood lymphocytes and natural killer cells; optionally wherein the T cells include CD4+T cells, CD8+T cells, antigen-specific T cells, tumor-infiltrating T cells, γδT cells, cytotoxic T cells, natural killer T cells and mucosa-associated invariant T cells (MAIT).

[0108] In one aspect, provided herein is a method for forming a proinflammatory environment in tissue surrounding a population of diseased cells expressing mutant EGFR, comprising contacting the tissue with an effective amount of a polypeptide as described above, a nucleic acid as described above, or a composition as described above under suitable conditions.

[0109] In some embodiments, the infiltration of activated B cells, CD4+ T cells, CD8+ T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils and / or neutrophils in the tissue is increased.

[0110] In some embodiments, the concentration of a pro-inflammatory cytokine in a tissue is increased.

[0111] In some embodiments, the proinflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, IP-10, CXCL1, MCP-1, or any combination thereof.

[0112] In some embodiments, presentation of an antigen originating or derived from a diseased cell by antigen presenting cells in the tissue is increased.

[0113] In some embodiments, phagocytosis of diseased cells in a tissue is increased.

[0114] In some embodiments, apoptosis of diseased cells induced by cell-mediated cytotoxicity is increased in a tissue.

[0115] In some embodiments, apoptosis of diseased cells induced by antibody-dependent cellular cytotoxicity is increased in a tissue.

[0116] In some embodiments, the population of diseased cells in a tissue is reduced.

[0117] In some embodiments, the population of diseased cells in a tissue is reduced by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0118] In some embodiments, the methods are performed in vitro or in vivo.

[0119] In one aspect, provided herein is a method for inducing an immune response against a target cell expressing a mutant EGFR in a subject, wherein the method comprises administering to the subject a polypeptide as described above, a nucleic acid as described above, or a composition as described above, wherein upon said administration,

[0120] (a) the EGFR domain encoded by the nucleic acid molecule is expressed;

[0121] (b) EGFR domain degradation into fragments of approximately 9 to 15 amino acids;

[0122] (c) the EGFR domain or its degradation fragments associate with the HLA class I or HLA class II complex;

[0123] (d) increased innate immune responses against target cells;

[0124] (e) increased secretion of pro-inflammatory cytokines;

[0125] (f) an increase in the percentage of phagocytic macrophages in the macrophage population;

[0126] (g) the percentage of dendritic cells presenting the EGFR domain or its degradation fragments in the dendritic cell population is increased;

[0127] (h) an increase in the percentage of antigen-specific T cells specific for a domain of EGFR or an epitope thereof; or

[0128] (i) The percentage of B cells producing antibodies specific to the EGFR domain or an epitope thereof in the B cell population is increased.

[0129] In some embodiments, the innate immune response is measured by

[0130] (a) activation of the NF-kB signaling pathway; or

[0131] (b) increased secretion of cytokines selected from IFN-α, IP-10, TNF-α, CXCL1, MCP-1, IL-2, or any combination thereof.

[0132] In some embodiments, the proinflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof.

[0133] In some embodiments, the target cell is a diseased cell expressing a mutant EGFR having a mutation selected from L858R, T790M, E746_A750 del, and C797S; optionally wherein the diseased cell expresses a mutant EGFR having a double mutation of L858R and T790M.

[0134] In one aspect, provided herein is a method of eliminating diseased cells expressing mutant EGFR in a subject, comprising administering an effective amount of a polypeptide as described above, a nucleic acid as described above, or a composition as described above.

[0135] In some embodiments, the subject is a human subject having the HLA subtype HLA-A*0101 or HLA-A*0201.

[0136] In some embodiments, the diseased cell is a cancer cell.

[0137] In one aspect, provided herein is a method of treating or delaying progression of a cancer expressing mutant EGFR in a subject, wherein the method comprises administering to the subject a polypeptide as described above, a nucleic acid as described above, or a composition as described above.

[0138] In some embodiments, upon administration,

[0139] (a) the EGFR domain encoded by the nucleic acid molecule is expressed;

[0140] (b) EGFR domain degradation into fragments of approximately 9 to 15 amino acids;

[0141] (c) the EGFR domain or its degradation fragments associate with the HLA class I or HLA class II complex;

[0142] (d) increased innate immune response against cancer;

[0143] (e) increased secretion of pro-inflammatory cytokines;

[0144] (f) an increase in the percentage of phagocytic macrophages in the macrophage population;

[0145] (g) the percentage of dendritic cells presenting the EGFR domain or its degradation fragments in the dendritic cell population is increased;

[0146] (h) an increase in the percentage of antigen-specific T cells specific for a domain of EGFR or an epitope thereof; or

[0147] (i) The percentage of B cells producing antibodies specific to the EGFR domain or an epitope thereof in the B cell population is increased.

[0148] In some embodiments, the innate immune response is measured by

[0149] (a) activation of the NF-kB signaling pathway; or

[0150] (b) increased secretion of cytokines selected from IFN-α, IP-10, TNF-α, CXCL1, MCP-1, IL-2, or any combination thereof.

[0151] In some embodiments, the proinflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof.

[0152] In some embodiments, the mutant EGFR contains a mutation selected from L858R, T790M, E746_A750 del, and C797S; optionally wherein the mutant EGFR contains a L858R and T790M double mutation.

[0153] In some embodiments, the method further comprises administering a second therapeutic agent, either simultaneously or sequentially.

[0154] In some embodiments, the second therapeutic agent comprises EGFR-TKI, PD1 antibody; optionally, the EGFR-TKI is erlotinib, gefitinib or dacomitinib.

[0155] In some embodiments, the cancer expressing mutant EGFR is NSCLC.

[0156] In one aspect, the present disclosure relates to the use of a polypeptide as described above, a nucleic acid as described above, or a composition as described above in the manufacture of a medicament or a kit for treating a cancer expressing mutant EGFR in a subject or delaying the progression of the cancer.

[0157] In some embodiments, the mutant EGFR contains a mutation selected from L858R, T790M, E746_A750 del, and C797S; optionally wherein the mutant EGFR contains a L858R and T790M double mutation.

[0158] In some embodiments, the cancer expressing mutant EGFR is NSCLC. BRIEF DESCRIPTION OF THE DRAWINGS

[0159] Figure 1 is a scheme showing the basic structural elements of an illustrative mRNA construct according to the present disclosure.

[0160] Figure 2A-2C The specific IFN-γ secretion detected in Balb / c mice by ELISPOT according to Example 1 is shown, wherein the numbers in the upper left corner indicate the number of spots, representing the secretion of specific IFN-γ.

[0161] Figure 3 The levels of cytokines IFN-α, IP-10, TNF-α, CXCL1, MCP-1, and IL-6 measured by flow cytometry according to Example 2 are shown.

[0162] Figure 4A-4B The levels of IFN-γ secretion induced by EGFR L858R, E746_A750del and T790M antigen-specific T cells as determined by ELISPOT according to Example 3 are shown.

[0163] Figure 5 Evaluation of antigen-specific T cell killing induced by mRNA constructs in HLA-A*0101 transgenic mice according to Example 4 is shown.

[0164] Figure 6 Evaluation of antigen-specific T cell killing induced by mRNA constructs in HLA-A*1101 transgenic mice according to Example 5 is shown.

[0165] Figure 7 This is a graph evaluating the effect of EGFR antigen-specific T cells induced by mRNA constructs on EGFR mutant tumors according to Example 6, wherein tumor size was measured and tumor growth inhibition rate (TGI) was calculated.

[0166] Figure 8 is a graph showing the synergistic effect between the mRNA constructs of the present disclosure and α-PD-1 in the adoptive transfer assay according to Example 7.

[0167] Fig. 9 It is a graph showing that according to Example 8, EGFR (L858R+E746_A750+T790M+C797S) induces strong C797S and T790M antigen-specific T cell activation in HLA-A*0201 transgenic mice.

[0168] Fig.10is a graph showing that according to Example 9, C797S did not reduce the killing ability of L858R and T790M-specific T cells, and EGFR (L858R+E746_A750+T790M+C797S) exhibited the anti-tumor efficacy of antigen-specific T cells in vitro. DETAILED DESCRIPTION

[0169] The disclosed compositions and methods may be more readily understood by reference to the following detailed description in conjunction with the accompanying drawings which form a part of the present disclosure. It is to be understood that the disclosed compositions and methods are not limited to the specific compositions and methods described and / or shown herein, and that the terminology used herein is only for the purpose of describing specific embodiments by way of example and is not intended to limit the claimed compositions and methods.

[0170] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if fully set forth.

[0171] Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, exemplary materials and methods are described. In describing and claiming the present disclosure, the following terminology will be used.

[0172] Unless specifically stated otherwise, any description of a possible mechanism or mode of action or cause of improvement is meant to be illustrative only, and the disclosed compositions and methods are not limited by the correctness or otherwise of any such proposed mechanism or mode of action or cause of improvement.

[0173] Throughout this article, description refers to compositions and methods of using the compositions. Where the disclosure describes or claims a feature or embodiment related to a composition, such feature or embodiment also applies to methods of using the composition. Likewise, where the disclosure describes or claims a feature or embodiment related to a method of using a composition, such feature or embodiment also applies to the composition.

[0174] It will be appreciated that, for clarity, certain features of the disclosed compositions and methods described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed compositions and methods described in the context of a single embodiment may also be provided separately or in any sub-combination.

[0175] Unless otherwise described, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following description of the terms will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall prevail.

[0176] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0177] In some embodiments, the numerical parameters shown in the specification (claims are fully incorporated therein) are approximate values, which can vary according to the desired characteristics to be obtained by a specific embodiment. In some embodiments, the numerical parameters should be interpreted according to the number of significant figures reported and using common rounding techniques. Although the numerical ranges and parameters stating the broad range of some embodiments of the present disclosure are approximate values, the numerical values ​​stated in the specific examples are reported as accurately as possible. The numerical values ​​presented in some embodiments of the present disclosure may include certain errors, which are necessarily caused by the standard deviation in their respective test measurements. The reference to the value range herein is intended to serve as a simple method for individually relating to each different value falling within the range. Unless otherwise noted herein, each single value is incorporated into the specification as if it were individually quoted herein.

[0178] The terms "include", "have" and "comprise" are open conjunctions. Any form or tense of one or more of these verbs, such as "include", "comprise", "have", "have", "comprises" and "comprises", is also open. For example, any method that "includes", "has" or "comprises" one or more steps is not limited to having only these one or more steps, and can also cover other unlisted steps. Similarly, any composition that "includes", "has" or "comprises" one or more features is not limited to having only these one or more features, and can cover other unlisted features. The use of any and all examples or exemplary language (e.g., "such as") provided herein for certain embodiments is intended only to better illustrate the present disclosure, and is not intended to limit the scope of protection claimed by the present disclosure. The language in this specification should not be interpreted as indicating any non-required element as necessary for practicing the present disclosure.

[0179] The term “consisting essentially of” permits the presence of additional materials or steps that “do not materially affect the basic and novel characteristics of the claimed invention.”

[0180] The term "consisting of" refers to the compositions, methods, and respective components described herein, excluding any elements not enumerated in the description of that embodiment.

[0181] I. Peptides

[0182] Provided herein are polypeptides comprising an EGFR domain, wherein the EGFR domain comprises at least one mutated fragment of human EGFR.

[0183] In some embodiments, the polypeptide further comprises a degradation domain configured to mark the EGFR domain for proteasomal degradation.

[0184] In some embodiments, the EGFR domain comprises at least one mutant fragment of human EGFR, wherein each mutant fragment comprises one or more EGFR mutations selected from T790M, C797S, L858R and E746_A750 del; optionally wherein the mutant fragment comprises one or more EGFR mutations selected from any one of (a) to (k):

[0185] L858R;

[0186] T790M;

[0187] E746_A750 del;

[0188] C797S;

[0189] T790M and C797S;

[0190] L858R and T790M;

[0191] L858R and E746_A750 del;

[0192] E746_A750 del;

[0193] E746_A750 del and T790M;

[0194] L858R, E746_A750 del, and T790M; and

[0195] L858R, E746_A750 del, T790M and C797S.

[0196] In some embodiments, the at least one mutated fragment of human EGFR comprises a L858R fragment.

[0197] In some embodiments, the at least one mutated fragment of human EGFR includes a T790M fragment.

[0198] In some embodiments, at least one mutant fragment of human EGFR includes an E746_A750 del fragment.

[0199] In some embodiments, the at least one mutated fragment of human EGFR comprises a C797S fragment.

[0200] In some embodiments, the length of at least one mutant fragment of human EGFR is at least about 10 amino acids; optionally wherein the length of the human EGFR mutant fragment is about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, about 25 amino acids, about 26 amino acids, about 27 amino acids, about 28 amino acids, about 29 amino acids, about 30 amino acids, about 31 amino acids, about 32 amino acids, about 33 amino acids, about 34 amino acids or about 35 amino acids.

[0201] In some embodiments, the at least one mutated fragment of human EGFR includes two or more mutated fragments of human EGFR, wherein the two or more mutated fragments are the same or different.

[0202] In some embodiments, the EGFR domain comprises two or more mutant fragments of human EGFR, wherein at least two of the two or more mutant fragments are fused directly or through a linker peptide.

[0203] In some embodiments, the at least one mutant fragment of human EGFR comprises a L858R fragment, wherein the L858R fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the L858R mutation.

[0204] In some embodiments, the EGFR domain comprises two or more identical copies of the L858R fragment.

[0205] In certain embodiments, the L858R fragment comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO:3.

[0206] In some embodiments, the L858R fragment has the amino acid sequence of SEQ ID NO:3.

[0207] In some embodiments, the at least one mutant fragment of human EGFR includes a T790M fragment, wherein the T790M fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the T790M mutation.

[0208] In some embodiments, the EGFR domain comprises two or more identical copies of the T790M fragment.

[0209] In certain embodiments, the T790M fragment comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO:4.

[0210] In some embodiments, the T790M fragment has the amino acid sequence of SEQ ID NO:4.

[0211] In some embodiments, the at least one mutant fragment of human EGFR includes an E746_A750 del fragment, wherein the E746_A750 del fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the E746_A750 del mutation.

[0212] In some embodiments, the EGFR domain comprises two or more identical copies of the E746_A750 del fragment.

[0213] In certain embodiments, the E746_A750 del fragment comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO:5.

[0214] In some embodiments, the E746_A750 del fragment has the amino acid sequence of SEQ ID NO:5.

[0215] In some embodiments, the at least one mutant fragment of human EGFR comprises a C797S fragment, wherein the C797S fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the C797S mutation.

[0216] In some embodiments, the EGFR domain comprises two or more identical copies of the C797S fragment.

[0217] In certain embodiments, the C797S fragment comprises an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO:6.

[0218] In some embodiments, the C797S fragment has the amino acid sequence of SEQ ID NO:6.

[0219] In some embodiments, the EGFR domain comprises:

[0220] six copies of the L858R fragment, wherein each L858R fragment has an amino acid sequence of SEQ ID NO: 3;

[0221] three copies of an L858R fragment and three T790M fragments, wherein each L858R fragment has the amino acid sequence of SEQ ID NO:3, and wherein each T790M fragment has the amino acid sequence of SEQ ID NO:4;

[0222] three L858R fragments and three E746_A750 del fragments, wherein each L858R fragment has an amino acid sequence of SEQ ID NO: 3, and wherein each E746_A750 del fragment has an amino acid sequence of SEQ ID NO: 5;

[0223] six E746_A750 fragments, wherein each E746_A750 fragment has an amino acid sequence of SEQ ID NO: 5;

[0224] three E746_A750 del fragments and three T790M fragments, wherein each T790M fragment has the amino acid sequence of SEQ ID NO: 4, and wherein each E746_A750 fragment has the amino acid sequence of SEQ ID NO: 5;

[0225] two L858R fragments, two E746_A750 del fragments, and two T790M fragments, wherein each L858R fragment has the amino acid sequence of SEQ ID NO:3, wherein each T790M fragment has the amino acid sequence of SEQ ID NO:4, and wherein each E746_A750 fragment has the amino acid sequence of SEQ ID NO:5; or

[0226] Two L858R fragments, two E746_A750 del fragments, one T790M fragment and one C797S fragment, wherein each L858R fragment has an amino acid sequence of SEQ ID NO:3, wherein each T790M fragment has an amino acid sequence of SEQ ID NO:4, wherein each E746_A750 fragment has an amino acid sequence of SEQ ID NO:5, and wherein each C797S fragment has an amino acid sequence of SEQ ID NO:6.

[0227] In some embodiments, provided herein is an isolated polypeptide comprising an amino acid sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25, 28, and 31.

[0228] In some embodiments, provided herein is an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28 and 31.

[0229] Unless otherwise indicated, the term "epidermal growth factor receptor" or "EGFR" as used herein refers to any native EGFR from any vertebrate source, including mammals such as primates (e.g., humans, macaques (cynomolgus)) and rodents (e.g., mice and rats). The term encompasses unprocessed EGFR as well as any form of EGFR produced by intracellular processing. The term also encompasses naturally occurring variants of EGFR, such as splice variants or allelic variants. The amino acid sequence of an exemplary human EGFR is ELREA TSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLI T QLMPFGC LLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFG L AKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSPSTSRTPLLS SLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA(SEQ ID NO:36; T790, C797, L858, the five amino acids spanning positions E746-A750 are underlined and shown in bold. "Full-length" EGFR as used herein refers to a mature, native-length EGFR molecule. For example, full-length human EGFR refers to a molecule having 1210 amino acids (see, e.g., SEQ ID NO:36).

[0230] When used in connection with a polypeptide, a "fragment" refers to a contiguous amino acid sequence of the polypeptide that is less than the full length of the polypeptide.

[0231] The term "EGFR mutant" or "mutant EGFR" as used herein refers to an EGFR molecule comprising one or more modified wild-type amino acid residues that are normally located at that position. The term "wild-type" as used herein refers to an organism, cell, gene, protein, oligonucleotide, etc. that is found in nature and has no changes relative to these components found in nature (in the wild state).

[0232] "Modification" of an amino acid residue / position refers to a change in the primary amino acid sequence as compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue / position. For example, typical modifications include replacing a residue with another amino acid (e.g., conservative or substantial substitution), inserting one or more (e.g., usually less than 5, 4 or 3) amino acids adjacent to the residue / position, and / or deleting the residue / position. For example, a human EGFR mutant comprises one or more modifications in its amino acid sequence relative to the sequence shown in SEQ ID NO: 34, and such modifications are sometimes referred to as "EGFR mutations" in this application. Various names may be used herein to represent the same modification. For example, the modification of leucine at position 858 of human EGFR to arginine may be represented as L858R or Leu858Arg. The modification of threonine at position 790 of human EGFR to methionine may be represented as T790M or Thr790Met. The modification of cysteine ​​at position 797 of human EGFR to serine may be represented as C797S or Cys797Ser. The modification of deleting five consecutive amino acid residues spanning the E746_A750 position from the human EGFR sequence can be represented as E746_A750 del. The EGFR mutant fragment containing the E746_A750 del mutation refers to the EGFR continuous amino acid sequence obtained after removing 5 residues spanning the E746_A750 position from the EGFR sequence. In some embodiments, the mutant EGFR shares at least 75%, 80%, 85%, 90%, 95% or 97% sequence identity with the wild-type EGFR protein.

[0233] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues linked by covalent peptide bonds. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). As used herein, the term encompasses amino acid chains of any length, including full-length proteins.

[0234] In the context of polypeptides, the term "derivative" as used herein refers to an altered form of a polypeptide having a modification in the amino acid sequence of the starting polypeptide. A "functional derivative" of a polypeptide refers to a derivative that retains one or more functions or activities of the starting polypeptide from which the derivative is derived. For example, a functional derivative of a proteasome substrate may retain the ability to recruit the proteasome complex to degrade the polypeptide sequence to which it is attached.

[0235] In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising an EGFR domain and a degradation domain, which is configured to mark the EGFR domain for proteasome degradation. Without being bound by any theory, the degraded EGFR domain peptide is subsequently processed by antigen presenting cells (e.g., dendritic cells) and presented in combination with major histocompatibility complex I or II (MHC) proteins to train lymphocytes (e.g., B cells and T cells) to recognize and eliminate any cells expressing target antigens (e.g., cancer cells expressing mutated EGFR proteins). Antigen processing can be carried out through two different approaches: proteasome degradation and phagolysosomal degradation. Antigens found in the dendritic cell cytoplasm can be degraded by targeting proteasomes with chaperone proteins. Antigens that enter the phagosome through phagocytosis can be partially degraded by targeted processing through the fusion of phagosomes and lysosomes.

[0236] The ubiquitin pathway has been well described as targeting proteins to the proteasome. Ubiquitin molecules come in a variety of peptide forms, such as branched or linear polyubiquitin chains and heterotypic or homotypic chains. For example, homotypic K48-linked ubiquitin drives proteasomal degradation, while branched ubiquitin can act to promote a variety of cellular functions (see, e.g., Kolla et al., (2022), Trends Biochem Sci; 47(9): 759-771, hereby incorporated by reference in its entirety). Polyubiquitin chains attached to target proteins play different functions depending on the lysine residue of the ubiquitin to which they are attached: Lys-6-linked polyubiquitin chains may be involved in DNA repair; Lys-11-linked polyubiquitin chains are involved in ERAD (endoplasmic reticulum-associated degradation) and cell cycle regulation; Lys-29-linked polyubiquitin chains are involved in proteotoxic stress response and cell cycle; Lys-33-linked polyubiquitin chains are involved in kinase modification; Lys-48-linked polyubiquitin chains are involved in protein degradation by the proteasome; Lys-63-linked polyubiquitin chains are involved in endocytosis, DNA damage response, and signaling processes leading to the activation of the transcription factor NF-κ-B. Cell signaling is initiated by the linear polymer chain formed by the attachment of the initiator methionine. Ubiquitin is usually conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed.

[0237] The ubiquitin peptide is encoded by one of four genes. The UBA52 and RPS27A genes encode single copies of ubiquitin fused to ribosomal proteins L40 and S27a, respectively. The UBB and UBC genes encode polyubiquitin precursors with precise head-to-tail repeats, the number of which varies from species to species.

[0238] In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising an EGFR domain and a degradation domain, wherein the degradation domain is configured to mark the EGFR domain for proteasome degradation. In some embodiments, the degradation domain is fused to the EGFR domain. In some embodiments, the polypeptide includes an N-terminal portion comprising the degradation domain and a C-terminal portion comprising the EGFR domain. In some embodiments, the polypeptide includes an N-terminal portion comprising the EGFR domain and a C-terminal portion comprising the degradation domain.

[0239] In some embodiments, the degradation domain comprises a ubiquitin peptide. In some embodiments, the degradation domain comprises a ubiquitin monomer or a functional derivative thereof. In some embodiments, the degradation domain comprises a ubiquitin polymer or a functional derivative thereof. In some embodiments, the degradation domain comprises a ubiquitin-like domain (ULD) or a functional derivative thereof.

[0240] In some embodiments, the ubiquitin peptide comprises an amino acid sequence encoded by any one of the UBA52, RPS27A, UBB or UBC genes. In some embodiments, the ubiquitin peptide comprises an amino acid sequence having about 80% (such as any one of 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to an amino acid sequence encoded by any one of the UBA52, RPS27A, UBB or UBC genes.

[0241] In some embodiments described in this paragraph, the degradation domain is located at the N-terminal portion of the polypeptide, and the EGFR domain is located at the C-terminal portion of the polypeptide. In some embodiments, the EGFR domain is located at the C-terminal portion of the polypeptide and is fused to the C-terminal portion of the degradation domain. In some embodiments, the length of the C-terminal portion of the polypeptide is at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids. In some embodiments, the EGFR domain is directly fused to the degradation domain, wherein the length of the EGFR domain is at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids.

[0242] Alternative transcripts of ubiquitin peptides encoded by the UBB gene have been identified, wherein the peptide may also include a 19 amino acid C-terminal extension that inhibits proteasomal degradation activity, thereby stabilizing the protein to which the ubiquitin peptide is attached. However, when extended to 25 amino acids, alternative ubiquitin peptides attached to the N-terminus of larger proteins can effectively target larger proteins for proteasomal degradation (see Vergoef et al. (2009), FASEB J; 23 (1): 123-133, which is incorporated herein by reference in its entirety). In the context of eliciting a vaccine immune response, effective proteasomal processing and degradation of the target antigen increases the likelihood of vaccine efficacy. Therefore, enhancements to vaccine constructs, such as fusion of alternative ubiquitin peptides to vaccine constructs, can improve overall efficacy and the likelihood of successful vaccination and / or treatment.

[0243] Therefore, in some embodiments, the spacer peptide is located between the degradation domain and the EGFR domain. In some embodiments, the length of the spacer peptide is at least about 15 amino acids. In some embodiments, the length of the spacer peptide is about 15-30 amino acids. In some embodiments, the length of the spacer peptide is about 20 amino acids, about 25 amino acids, or about 30 amino acids. In some embodiments, the spacer peptide is a 25 amino acid peptide of any sequence.

[0244] The term "ubiquitin" is a technical term. Ubiquitin is an evolutionarily conserved, ubiquitously expressed eukaryotic protein. When comparing the amino acid sequences of ubiquitin from plants, humans, and yeast, there were only two amino acid differences between plant and yeast ubiquitin, while only three amino acid differences were observed when plant ubiquitin was compared to human ubiquitin (Sharp and Li, 1987; Callis and Vierstra, 1989). The amino acid sequence of ubiquitin is identical between plant species (Callis and Vierstra, 1989; Callis et al., 1995). The ubiquitin gene translation product is a polyubiquitin precursor protein containing tandemly fused ubiquitin molecules. Ubiquitin is also produced in the form of fusions to unrelated ribosomal proteins. The fusion protein is processed by a deubiquitinase (DUB) to produce a mature free ubiquitin monomer. (Amerik and Hochstrasser, 2004; Reyes-Turcu et al., 2009). In some species, ubiquitin monomers are small, consisting of 76 amino acids that fold into a compact, globular, tightly hydrogen-bonded structure with a terminal diglycine sequence (Vijay-Kumar et al., 1987). In some embodiments, the "ubiquitin monomer" has the amino acids of SEQ ID NO: 1 disclosed herein (MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRL IFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGV). As used herein, "ubiquitin multimers" refer to polypeptides comprising multiple copies of ubiquitin monomers that are directly fused or fused in series through a linker. In some embodiments, the ubiquitin complex comprises 2, 3, 4 or more than 4 copies of ubiquitin monomers.

[0245] The term "ubiquitin-like domain" or "UbL" is also a technical term and refers to the domain of certain proteins that are homologous to ubiquitin. Various ubiquitin-like domain proteins (UDP) and their UbL domains are known in the art (Yu et al., The EMBO Journal (2016) 35:1522-1536 https: / / doi.org / 10.15252 / embj.201593147). For example, Rad23, Dsk2, and Ddi1 contain N-terminal UbLs that are recognized by proteasome subunits Rpn1, Rpn10, and Rpn13 (Elsasser et al., 2002 ; Funakoshi et al., 2002 ; Kaplun et al., 2005 ; Husnjak et al., 2008 ; Zhang et al., 2009 ; Gomez et al., 2011 ).

[0246] The term "proteasome" or "proteasome system" is used interchangeably herein and refers to a protease complex that is naturally present in vivo or artificially designed to selectively degrade polypeptides having substrates recognized by the proteasome. The components, structure and function of the proteasome system are well known in the art (for a review, see Tanaka Proc Jpn Acad Ser B Phys Biol Sci. 2009 January; 85 (1): 12–36.) One of ordinary skill in the art will understand how to purify the proteasome system from a eukaryotic cell preparation.

[0247] The term "signal sequence" or "signal peptide" as used herein refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a cell organelle (such as the endoplasmic reticulum) and / or to the cell surface. The signal peptide directs the nascent protein into the endoplasmic reticulum. This is critical if the receptor is to be glycosylated and anchored in the cell membrane. In general, the signal peptide is naturally attached to the amino terminus.

[0248] In some embodiments, the signal peptide is cleaved after passing through the endoplasmic reticulum (ER), i.e., a cleavable signal peptide. In some embodiments, a restriction site is located at the carboxyl terminus of the signal peptide to facilitate cleavage.

[0249] For example, the signal sequence contains at least about 10-30 amino acid residues, preferably about 15-25 amino acid residues, more preferably about 18-20 amino acid residues, even more preferably about 19 amino acid residues, and has at least about 35-65%, preferably about 38-50%, more preferably about 40-45% hydrophobic amino acid residues (e.g., valine, leucine, isoleucine or phenylalanine).

[0250] In some embodiments, the polypeptides of the present disclosure include peptide linkers (sometimes referred to as linkers). Peptide linkers can separate any peptide domain / region described herein. Peptide linkers can have any of a variety of amino acid sequences. Domains and regions can be connected by peptide linkers that are generally flexible, but other chemical bonds are not excluded. Linkers can be peptides of about 6 to about 40 amino acids in length, or peptides of about 6 to about 25 amino acids in length. These linkers can be produced by coupling proteins using synthetic, encoding linker oligonucleotides.

[0251] Peptide linkers with some flexibility can be used. Peptide linkers can have virtually any amino acid sequence, but it is important to remember that suitable peptide linkers will have sequences that result in generally flexible peptides. The use of small amino acids, such as glycine and alanine, helps to form flexible peptides. Creating such sequences is routine for those skilled in the art.

[0252] Suitable linkers can be readily selected and can be of any suitable different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0253] Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, and (GGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.

[0254] II. Nucleic Acids

[0255] As used interchangeably herein, the term "polynucleotide" or "nucleic acid" refers to a nucleotide polymer of any length, and includes, for example, DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or may be incorporated into any substrate in a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides, such as methylated nucleotides and analogs thereof. Nucleic acids may be in single-stranded or double-stranded form. As used herein, unless otherwise indicated, "nucleic acid" also includes nucleic acid mimics, such as locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholinos.

[0256] Nucleic acids (also called polynucleotides) can be or can include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, a-LNA with aL-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-a-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) or chimeras or combinations thereof.

[0257] As used herein, the term "non-naturally occurring" is intended to mean that the nucleic acid molecule does not exist in nature when used with reference to a nucleic acid molecule as described herein. Non-naturally occurring nucleic acids encoding proteins (e.g., EGFR) comprise at least one genetic alteration or chemical modification that is not typically present in naturally occurring nucleic acids (including wild-type nucleic acids). Genetic alterations include, for example, modifications to expressible nucleic acid sequences encoding heterologous peptides or polypeptides, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, and / or other functional disruptions of coding sequences. Such modifications include, for example, modifications to coding regions of heterologous, homologous, or heterologous and homologous polypeptides and functional fragments thereof. Additional modifications include, for example, modifications to non-coding regulatory regions, wherein the modifications alter the expression of a gene or operon. Additional modifications also include, for example, incorporating nucleic acid sequences into vectors such as plasmids or artificial chromosomes. Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.

[0258] "Isolated nucleic acid" refers to nucleic acids that are substantially separated from other genomic DNA sequences and proteins or complexes (such as ribosomes and polymerases) that naturally accompany native sequences, such as RNA, DNA or mixed nucleic acids. "Isolated" nucleic acid molecules are nucleic acid molecules that are separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecules. In addition, "isolated" nucleic acid molecules (such as mRNA molecules) can be substantially free of other cell materials or culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding antigens as described herein are separated or purified. The term includes nucleic acid sequences removed from their naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates and chemically synthesized analogs or analogs synthesized by heterologous systems. Substantially pure molecules can include isolated forms of molecules.

[0259] The term "coding region" refers to the portion of a coding nucleic acid sequence that is translated into a peptide or polypeptide.

[0260] The coding nucleic acid can be a monocistronic or polycistronic. A "monocistronic sequence" refers to a polynucleotide comprising a coding sequence for a single peptide or polypeptide chain. A "polycistronic sequence" refers to a polynucleotide comprising a coding sequence for two or more peptides and / or polypeptide chains.

[0261] The term "mRNA" as used herein refers to an information RNA molecule comprising one or more coding regions, such as an open reading frame (ORF), which can be translated by a cell or organism having the mRNA to produce one or more peptide or protein products. The region containing one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule also comprises one or more untranslated regions (UTRs).

[0262] In certain embodiments, mRNA is a monocistronic mRNA comprising only one ORF. In certain embodiments, monocistronic mRNA encodes a peptide or protein comprising at least one epitope of a selected antigen (e.g., a pathogenic antigen or a tumor-associated antigen). In other embodiments, mRNA is a polycistronic mRNA comprising two or more ORFs. In certain embodiments, polycistronic mRNA encodes two or more peptides or proteins that may be identical or different from each other. In certain embodiments, each peptide or protein encoded by polycistronic mRNA comprises at least one epitope of a selected antigen. In certain embodiments, different peptides or proteins encoded by polycistronic mRNA each comprise at least one epitope of a different antigen. In any embodiment described herein, at least one epitope may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 epitopes of an antigen.

[0263] Also provided herein is a nucleic acid (e.g., RNA molecule) comprising an open reading frame encoding the above-mentioned polypeptide. Also provided herein is, for example, using it to produce EGFR mutant-specific immune effector cells; forming a pro-inflammatory environment in the tissue surrounding a population of diseased cells expressing mutant EGFR; inducing an immune response in a subject against target cells expressing mutant EGFR; eliminating diseased cells expressing mutant EGFR in a subject; or treating a cancer expressing mutant EGFR in a subject or delaying the progress of the cancer.

[0264] The inventors unexpectedly found that the nucleic acids (e.g., RNA molecules) according to the present disclosure can induce strong specific immune responses; enhanced innate immune responses; increased secretion of proinflammatory cytokines; significantly higher percentages of phagocytic macrophages; significantly increased antigen-specific T cell activation, leading to effective anti-tumor therapy. The inventors also unexpectedly found that the nucleic acids (e.g., RNA molecules) according to the present disclosure are used in combination with other therapies (such as α-mPD-1) to exhibit synergistically enhanced specific anti-tumor effects.

[0265] In some embodiments, the nucleic acid is an RNA molecule.

[0266] Untranslated region (UTR)

[0267] The untranslated region (UTR) is a polynucleotide nucleic acid portion located before the start codon (5'UTR) and after the stop codon (3'UTR), which is not translated. In some embodiments, the polynucleotide of the present invention {e.g., ribonucleic acid (RNA), e.g., messenger RNA (mRNA)) comprising an open reading frame (ORF) encoding one or more activated EGFR mutant peptides further comprises UTR {e.g., 5'UTR or a functional fragment thereof, 3'UTR or a functional fragment thereof, or a combination thereof).

[0268] The UTR may be homologous or heterologous to the coding region in the polynucleotide. In some embodiments, the UTR is homologous to the ORF encoding one or more activated EGFR mutant polypeptides. In some embodiments, the UTR is heterologous to the ORF encoding one or more activated EGFR mutant polypeptides. In some embodiments, the polynucleotide comprises two or more 5'UTRs or their functional fragments, each 5'UTR or its functional fragment has the same or different nucleotide sequence. In some embodiments, the polynucleotide comprises two or more 3'UTRs or their functional fragments, each 3'UTR or its functional fragment has the same or different nucleotide sequence.

[0269] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof is sequence optimized.

[0270] In some embodiments, the 5'UTR or a functional fragment thereof, the 3'UTR or a functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, such as 5-methoxyuracil.

[0271] The UTR may have features that provide regulatory effects, such as increasing or decreasing stability, localization, and / or translation efficiency. The polynucleotide comprising the UTR may be administered to a cell, tissue, or organism, and one or more regulatory features may be measured using conventional methods. In some embodiments, the functional fragment of the 5'UTR or 3'UTR comprises one or more regulatory features of the full-length 5' or 3'UTR, respectively.

[0272] Natural 5'UTRs have features that play a role in translation initiation. They contain features similar to Kozak sequences, which are generally considered to be involved in the process by which ribosomes initiate translation of many genes. Kozak sequences have a consensus of CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another 'G. 5'UTRs are also known to form secondary structures that are involved in the binding of elongation factors.

[0273] By engineering features commonly found in genes that are abundantly expressed in specific target organs, the stability of polynucleotides and the production of proteins can be enhanced. For example, the introduction of a 5'UTR of a liver-expressed mRNA (such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can enhance the expression of polynucleotides in hepatocyte cell lines or the liver. Similarly, for muscle (e.g., MyoD, myosin, myoglobin, myogenin, Herculin), endothelial cells (e.g., Tie-1, CD36), myeloid cells (e.g., C / EBP, AML1, G-CSF, GM-CSF, CD1 1b, MSR, Fr-1, i-NOS), leukocytes (e.g., CD45, CD18), adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (e.g., SP-A / B / C / D), it is possible to use 5'UTRs from other tissue-specific mRNAs to improve expression in that tissue.

[0274] In some embodiments, the UTR is selected from a family of transcripts whose proteins have a common function, structure, characteristic, or property. For example, the encoded polypeptide may belong to a family of proteins (i.e., sharing at least one function, structure, characteristic, location, origin, or expression pattern) that are expressed in a particular cell, tissue, or at a certain time during development. The UTR of any gene or mRNA may be exchanged with any other UTR of the same or different protein family to generate a new polynucleotide.

[0275] In some embodiments, the 5'UTR and 3'UTR may be heterologous. In some embodiments, the 5'UTR may be derived from a different species than the 3'UTR. In some embodiments, the 3'UTR may be derived from a different species than the 5'UTR.

[0276] Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the present invention. In some embodiments, the UTR can be altered relative to the wild-type or native UTR to produce a variant UTR, such as by changing the orientation or position of the UTR relative to the ORF; or by the inclusion of additional nucleotides, deletion of nucleotides, exchange of nucleotides, or transposition. In some embodiments, variants of the 5' or 3' UTR can be utilized, such as mutants of the wild-type UTR, or variants that add or remove one or more nucleotides at the end of the UTR.

[0277] In addition, one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, for example, Mandal and Rossi, Nat. Protoc. 2013 8 (3): 568-82, and sequences available at www.addgene.org / Derrick_Rossi / , each of which is incorporated herein by reference in its entirety. UTRs or portions thereof can be placed in the same orientation as the transcript from which they are selected, or can change orientation or position. Therefore, 5' and / or 3' UTRs can be inverted, shortened, extended, or combined with one or more other 5' UTRs or 3' UTRs.

[0278] In some embodiments, the polynucleotide comprises multiple UTRs, such as double, triple or quadruple 5'UTRs or 3'UTRs. For example, the double UTR comprises two tandem or substantially tandem copies of the same UTR. For example, double β-globin 3'UTRs can be used (see US2010 / 0129877, the contents of which are incorporated herein by reference in their entirety).

[0279] In certain embodiments, the polynucleotides of the invention comprise a 5'UTR and / or a 3'UTR selected from any of the UTRs disclosed herein.

[0280] In certain embodiments, the 5'UTR sequence of the invention comprises a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO:34.

[0281] In certain embodiments, the 3'UTR sequence of the invention comprises a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NO:35.

[0282] In some embodiments, the 5'UTR comprises the sequence of SEQ ID NO:34.

[0283] In some embodiments, the 3'UTR comprises the sequence of SEQ ID NO:35.

[0284] 5'-cap structure

[0285] Without being bound by theory, it is expected that the 5'-cap structure of the polynucleotide participates in nuclear export and increases polynucleotide stability, and binds to mRNA cap binding protein (CBP), which is responsible for polynucleotide stability in cells and causes translational competence by forming mature circular mRNA species through the association of CBP with poly-A binding protein. The 5'-cap structure further assists in the removal of 5'-proximal introns during mRNA splicing. Therefore, in some embodiments, the nucleic acid molecules of the present disclosure include a 5'-cap structure.

[0286] Nucleic acid molecules can be 5'-capped by the cell's endogenous transcription machinery to generate a 5'-ppp-5'-triphosphate bond between the terminal guanylate cap residue and the sense nucleotide transcribed at the 5' end of the polynucleotide. The 5'-guanylate cap may then be methylated to generate an N7-methylguanylate residue. The ribose sugars of the terminal and / or pre-terminal transcribed nucleotides at the 5' end of the polynucleotide may also be optionally 2'-O-methylated. 5'-decapping by hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules (such as mRNA molecules) for degradation.

[0287] In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more alterations of a native 5'-cap structure produced by an endogenous process. Without being bound by theory, modifications to the 5'-cap can increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and can improve the translation efficiency of the polynucleotide.

[0288] Exemplary changes to the natural 5'-cap structure include generating a non-hydrolyzable cap structure to prevent decapping, thereby increasing the half-life of the polynucleotide. In some embodiments, since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester bond, in some embodiments, modified nucleotides can be used during the capping reaction. For example, in some embodiments, vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) can be used with α-thioguanosine nucleotides according to the manufacturer's instructions to create a thiophosphate bond in the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methylphosphonates and selenophosphate nucleotides, can be used.

[0289] Additional exemplary changes to the native 5'-cap structure include modifications at the 2'- and / or 3'-positions of the capped guanosine triphosphate (GTP), the addition of a methylene moiety (CH 2 ) replacement of the sugar ring oxygen (generating a carbocycle), modification in the triphosphate bridge portion of the cap structure, or modification in the nucleobase (G) portion.

[0290] Additional exemplary changes to the natural 5'-cap structure include, but are not limited to, 2'-O-methylation of the ribose of the 5'-terminal and / or 5'-preterminal nucleotides of the polynucleotide on the 2'-hydroxyl of the sugar (as described above). A variety of different 5'-cap structures can be used to generate the 5'-cap of a polynucleotide (such as an mRNA molecule). Additional exemplary 5'-cap structures that can be used in conjunction with the present disclosure also include those 5'-cap structures described in International Patent Publication Nos. WO2008127688, WO2008016473, and WO 2011015347, the entire contents of each of which are incorporated herein by reference.

[0291] In various embodiments, the 5'-terminal cap may include a cap analog. Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, have a chemical structure that differs from a natural (i.e., endogenous, wild-type, or physiological) 5'-cap while retaining cap function. Cap analogs can be synthesized and / or attached to a polynucleotide chemically (i.e., non-enzymatically) or enzymatically.

[0292] For example, the antiretroviral cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, one of which contains an N7-methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 G-3'mppp-G, which can be equivalently named 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of another unchanged guanosine is attached to the 5'-terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7- and 3'-O-methylated guanosine provides the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap structure is mCAP, which is similar to ARCA, but has a 2'-O-methyl group on the guanosine (i.e., N7, 2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, i.e., m 7 Gm-ppp-G).

[0293] In some embodiments, the cap analog may be a dinucleotide cap analog. As a non-limiting example, a dinucleotide cap analog may be modified with a borophosphate group or a phosphoselenate group at different phosphate positions, such as the dinucleotide cap analogs described in U.S. Pat. No. 8,519,110, the entire contents of which are incorporated herein by reference in their entirety.

[0294] In some embodiments, the cap analog can be a N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, e.g., various cap analogs and methods for synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the entire contents of the document are incorporated herein by reference). In other embodiments, the cap analog that can be used in conjunction with the nucleic acid molecules disclosed herein is a 4-chloro / bromophenoxyethyl analog.

[0295] In various embodiments, the cap analog may include a guanosine analog. Useful guanosine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0296] Without being bound by theory, it is expected that although cap analogs allow concomitant capping of polynucleotides in in vitro transcription reactions, up to 20% of transcripts remain uncapped. This phenomenon, as well as the structural differences between cap analogs and the natural 5'-cap structure of polynucleotides produced by the cell's endogenous transcription machinery, may lead to reduced translational capacity and reduced cellular stability.

[0297] Therefore, in some embodiments, an enzyme can also be used to perform post-transcriptional capping on the nucleic acid molecules of the present disclosure to produce a more realistic 5'-cap structure. The phrase "more realistic" as used herein refers to a feature that closely reflects or mimics an endogenous or wild-type feature in structure or function. That is, compared with the synthetic features or analogs of the prior art, the "more realistic" features are better able to represent endogenous, wild-type, natural or physiological cellular functions and / or structures, or are superior to the corresponding endogenous, wild-type, natural or physiological features in one or more aspects. Non-limiting examples of more realistic 5'-cap structures that can be used in conjunction with the nucleic acid molecules of the present disclosure are structures that have enhanced binding to cap-binding proteins, increased half-life, reduced sensitivity to 5'-endonucleases, and / or reduced 5'-decapping, compared to synthetic 5'-cap structures known in the art (or compared to wild-type, natural or physiological 5'-cap structures). For example, in some embodiments, the recombinant vaccinia virus capping enzyme and the recombinant 2'-O-methyltransferase can establish a canonical 5'-5'-triphosphate bond between the 5'-terminal nucleotide of the polynucleotide and the guanosine cap nucleotide, wherein the cap guanosine contains N7-methylation and the 5'-terminal nucleotide of the polynucleotide contains a 2'-O-methyl group. This structure is referred to as the cap 1 structure. Compared to other 5' cap analog structures known in the art, for example, this cap causes higher translational capacity, cellular stability, and reduced activation of cellular proinflammatory cytokines. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).

[0298] Without being bound by theory, it is contemplated that the nucleic acid molecules of the present disclosure can be capped after transcription, and because this method is relatively efficient, nearly 100% of the nucleic acid molecules can be capped.

[0299] Polyadenylation (Poly-A) region

[0300] During natural RNA processing, long chains of adenosine nucleotides (poly-A regions) are often added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3' end of the transcript is cleaved, releasing the 3'-hydroxyl. Next, poly-A polymerase adds a string of adenosine nucleotides to the RNA. The process is called polyadenylation, and poly-A regions of between 100 and 250 residues in length are added. Without being bound by theory, it is expected that poly-A regions can confer a number of advantages to the nucleic acid molecules of the present disclosure.

[0301] Therefore, in some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise polyadenylation signals. In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise one or more polyadenylation (poly-A) regions. In some embodiments, the poly-A region is entirely composed of adenine nucleotides or functional analogs thereof. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 3' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end and at least one poly-A region at its 3' end.

[0302] According to the present disclosure, in different embodiments, the poly-A region may have different lengths. Specifically, in some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 30 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 35 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 40 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 45 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 50 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 55 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 60 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 65 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 70 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 75 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 80 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 85 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 90 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 95 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 100 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 110 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 120 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 130 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 140 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 150 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 160 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 170 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 180 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 190 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 200 nucleotides in length.In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 225 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 250 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 275 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 300 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 350 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 400 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 450 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 500 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 600 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 700 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 800 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 900 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1000 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1100 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1200 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1300 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1400 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1500 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1600 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1700 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 1800 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 1900 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 2000 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 2250 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 2500 nucleotides in length.In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 2750 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 3000 nucleotides in length.

[0303] In some embodiments, the length of the poly-A region in the nucleic acid molecule can be selected based on the overall length of the nucleic acid molecule, or a portion thereof (such as the length of the coding region of the nucleic acid molecule or the length of the open reading frame, etc.). For example, in some embodiments, the poly-A region accounts for about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total length of the nucleic acid molecule containing the poly-A region.

[0304] Without being bound by theory, it is expected that certain RNA binding proteins can bind to the poly-A region located at the 3' end of the mRNA molecule. These poly-A binding proteins (PABPs) can regulate mRNA expression, such as interacting with the translation initiation machinery in the cell and / or protecting the 3'-poly-A tail from degradation. Therefore, in some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure include at least one binding site for a poly-A binding protein (PABP). In other embodiments, the nucleic acid molecules are conjugated or complexed with the PABP before being loaded into a delivery vehicle (e.g., lipid nanoparticle).

[0305] In some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise poly-AG quadruplexes. G quadruplexes are cyclic hydrogen-bonded arrays of four guanosine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this embodiment, the G quadruplexes are incorporated into the ends of the poly-A region. The stability, protein production, and other parameters including the half-life at various time points of the resulting polynucleotides (e.g., mRNA) can be measured. Studies have found that the protein yield produced by the polyA-G quadruplex structure is equivalent to at least 75% of the protein yield observed using a 120-nucleotide poly-A region alone.

[0306] In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure may include a poly-A region and may be stabilized by adding a 3'-stabilizing region. In some embodiments, a 3'-stabilizing region that can be used to stabilize nucleic acid molecules (e.g., mRNA) includes a poly-A or poly-AG quadruplex structure as described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entirety.

[0307] In other embodiments, the 3'-stabilizing region that can be used in conjunction with the nucleic acid molecules of the present disclosure includes chain terminating nucleosides such as, but not limited to, 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymidine, 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymidine, 2'-deoxynucleosides or O-methyl nucleosides, 3'-deoxynucleosides, 2',3'-dideoxynucleosides 3'-O-methyl nucleosides, 3'-O-ethyl nucleosides, 3'-arabinoside, and other alternative nucleosides known in the art and / or described herein.

[0308] Secondary structure

[0309] Without being bound by theory, it is expected that the stem-loop structure can guide RNA folding, protect the structural stability of nucleic acid molecules (e.g., mRNA), provide recognition sites for RNA binding proteins, and serve as substrates for enzymatic reactions. For example, the incorporation of miR sequences and / or TEE sequences will change the shape of the stem-loop region, thereby increasing and / or decreasing translation (Kedde et al. A Pumilio-induced RNA structure switch in p27-3'UTR controls miR-221and miR-222accessibility. Nat Cell Biol., October 2010; 12(10): 1014-20, the contents of which are incorporated herein by reference in their entirety).

[0310] Therefore, in some embodiments, nucleic acid molecules (e.g., mRNA) described herein or parts thereof can present a stem-loop structure, such as, but not limited to, histone stem-loops. In some embodiments, the stem-loop structure is formed by a stem-loop sequence having a length of about 25 or about 26 nucleotides, such as, but not limited to, those described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entirety. Additional examples of stem-loop sequences include sequences described in International Patent Publication No. WO2012 / 019780 and International Patent Publication No. WO201502667, the contents of which are incorporated herein by reference. In some embodiments, the stem-loop sequence includes TEE as described herein. In some embodiments, the stem-loop sequence includes a miR sequence as described herein. In a specific embodiment, the stem-loop sequence may include a miR-122 seed sequence.

[0311] In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure include a stem-loop sequence located upstream of the coding region in the nucleic acid molecule (at the 5' end). In some embodiments, the stem-loop sequence is located within the 5'-UTR of the nucleic acid molecule. In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure include a stem-loop sequence located downstream of the coding region in the nucleic acid molecule (at the 3' end). In some embodiments, the stem-loop sequence is located within the 3'-UTR of the nucleic acid molecule. In some cases, the nucleic acid molecule may contain more than one stem-loop sequence. In some embodiments, the nucleic acid molecule includes at least one stem-loop sequence in the 5'-UTR and at least one stem-loop sequence in the 3'-UTR.

[0312] In some embodiments, the nucleic acid molecule comprising a stem-loop structure further comprises a stabilizing region. In some embodiments, the stabilizing region comprises at least one chain terminating nucleoside, which acts to slow down degradation and thereby increase the half-life of the nucleic acid molecule. Exemplary chain terminating nucleosides that can be used in conjunction with the nucleic acid molecules of the present disclosure include, but are not limited to, 3'-deoxyadenosine (cordycepin); 3'-deoxyuridine; 3'-deoxycytosine; 3'-deoxyguanosine; 3'-deoxythymidine; 2',3'-dideoxynucleosides, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymidine; 2'-deoxynucleosides; or O-methyl nucleosides; 3'-deoxynucleosides; 2',3'-dideoxynucleosides; 3'-O-methyl nucleosides; 3'-O-ethyl nucleosides; 3'-arabinoside, as well as other alternative nucleosides known in the art and / or described herein. In other embodiments, the stem-loop structure can be stabilized by altering the 3'-region of the polynucleotide, which alterations can prevent and / or inhibit the addition of oligo(U) (International Patent Publication No. WO2013 / 103659, which is herein incorporated by reference in its entirety).

[0313] In some embodiments, the nucleic acid molecules of the present disclosure include at least one stem-loop sequence and a poly-A region or a polyadenylation signal. Non-limiting examples of polynucleotide sequences including at least one stem-loop sequence and a poly-A region or a polyadenylation signal include sequences described in International Patent Publication No. WO2013 / 120497, International Patent Publication No. WO2013 / 120629, International Patent Publication No. WO2013 / 120500, International Patent Publication No. WO2013 / 120627, International Patent Publication No. WO2013 / 120498, International Patent Publication No. WO2013 / 120626, International Patent Publication No. WO2013 / 120499, and International Patent Publication No. WO2013 / 120628, the contents of each of which are incorporated herein by reference in their entirety.

[0314] In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a pathogen antigen or a fragment thereof, such as the polynucleotide sequences described in International Patent Publication No. WO2013 / 120499 and International Patent Publication No. WO2013 / 120628, the contents of each of which are incorporated herein by reference in their entirety.

[0315] In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a therapeutic protein, such as the polynucleotide sequences described in International Patent Publication No. WO2013 / 120497 and International Patent Publication No. WO2013 / 120629, the contents of each of which are incorporated herein by reference in their entirety.

[0316] In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode a tumor antigen or a fragment thereof, such as the polynucleotide sequences described in International Patent Publication No. WO2013 / 120500 and International Patent Publication No. WO2013 / 120627, the contents of each of which are incorporated herein by reference in their entirety.

[0317] In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal may encode an allergenic antigen or an autoimmune autoantigen, such as the polynucleotide sequences described in International Patent Publication No. WO2013 / 120498 and International Patent Publication No. WO2013 / 120626, the contents of each of which are incorporated herein by reference in their entirety.

[0318] In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs selected from pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

[0319] In some embodiments, provided herein are compositions comprising a nucleic acid as described above and at least one lipid.

[0320] In some embodiments, the lipid comprises a first lipid according to Formula 01-I or Formula 01-II; or a compound listed in Table 01-1; or a compound according to Formula 02-I; or a compound listed in Table 02-1; or a compound according to Formula 03-I; or a compound listed in Table 03-1; or a compound according to Formula 04-I; or a compound listed in Table 04-1; optionally, the lipid further comprises a second lipid according to Formula 05-I.

[0321] In some embodiments, the present invention provides a composition comprising multiple types of nucleic acid molecules, each nucleic acid molecule encoding an EGFR domain, wherein each encoded EGFR domain comprises at least one mutant fragment of human EGFR, wherein the mutant fragment comprises one or more EGFR mutations selected from T790M, C797S, L858R and E746_A750 del, and wherein the EGFR domains encoded by at least two of the multiple types of nucleic acid molecules are different.

[0322] In some embodiments, the EGFR domain encoded by at least one of the plurality of species of nucleic acid molecules comprises two or more mutant fragments of human EGFR, wherein the two or more mutant fragments are the same or different.

[0323] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules comprises an EGFR mutation selected from any one of (a) to (k):

[0324] (a) L858R;

[0325] (b) T790M;

[0326] (c) E746_A750 del;

[0327] (d) C797S;

[0328] (e) T790M and C797S;

[0329] (f) L858R and T790M;

[0330] (g) L858R and E746_A750 del;

[0331] (h) E746_A750 del;

[0332] (i) E746_A750 del and T790M;

[0333] (j) L858R, E746_A750 del and T790M; and

[0334] (k) L858R, E746_A750 del, T790M, and C797S.

[0335] In some embodiments, the at least one mutated fragment of human EGFR comprises a L858R fragment.

[0336] In some embodiments, the at least one mutated fragment of human EGFR includes a T790M fragment.

[0337] In some embodiments, at least one mutant fragment of human EGFR includes an E746_A750 del fragment.

[0338] In some embodiments, the at least one mutated fragment of human EGFR comprises a C797S fragment.

[0339] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is a L858R fragment, wherein the L858R fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the L858R mutation.

[0340] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules include a T790M fragment, wherein the T790M fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the T790M mutation.

[0341] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is an E746_A750 del fragment, wherein the E746_A750 del fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the E746_A750 del mutation.

[0342] In some embodiments, one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is a C797S fragment, wherein the C797S fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the C797S mutation.

[0343] In some embodiments, the plurality of species of nucleic acid molecules further include at least one first species of nucleic acid molecules that encodes an EGFR domain longer than about 10 amino acids, and wherein at least one first species of nucleic acid molecules also encodes a degradation domain, wherein the degradation domain is configured to mark the EGFR domain for proteasomal degradation.

[0344] In some embodiments, the degradation domain comprises a ubiquitin peptide.

[0345] In some embodiments, the ubiquitin peptide is a ubiquitin monomer, a ubiquitin polymer, a ubiquitin-like domain (ULD), or a functional derivative thereof.

[0346] In some embodiments, the spacer peptide is located between the degradation domain and the EGFR domain; optionally wherein the spacer peptide is at least about 15 amino acids in length; optionally wherein the spacer peptide is about 15-30 amino acids in length; optionally wherein the spacer peptide is about 20 amino acids, about 25 amino acids, or about 30 amino acids in length.

[0347] In certain embodiments, at least one species of nucleic acid molecule comprises a coding sequence for a ubiquitin peptide that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NOs: 37-45.

[0348] In some embodiments, at least one species of nucleic acid molecule comprises a coding sequence for a ubiquitin peptide selected from SEQ ID NOs: 37-45, as shown in Table 1 below.

[0349] Table 1: Exemplary coding sequences for ubiquitin peptides

[0350]

[0351]

[0352]

[0353] In some embodiments, the plurality of species of nucleic acid molecules further comprises at least one second species of nucleic acid molecule encoding an EGFR domain no longer than about 10 amino acids, and wherein the at least one second species of nucleic acid molecule lacks a coding sequence for a degradation domain.

[0354] In some embodiments, the plurality of species of nucleic acid molecules include mRNA molecules.

[0355] In some embodiments, at least one of the plurality of species of nucleic acid molecules further comprises a 3'-UTR and / or a 5'-UTR; optionally wherein the 5'-UTR comprises the sequence of SEQ ID NO:34; optionally wherein the 3'-UTR comprises the sequence of SEQ ID NO:35.

[0356] In some embodiments, the plurality of species of nucleic acid molecules include RNA molecules comprising a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, and 32.

[0357] In some embodiments, the plurality of species of nucleic acid molecules include RNA molecules comprising a sequence selected from the group consisting of SEQ ID NOs: 8, 11, 14, 17, 20, 23, 26, 29, and 32.

[0358] In some embodiments, the plurality of species of nucleic acid molecules include DNA molecules comprising a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33.

[0359] In some embodiments, the plurality of nucleic acid molecules includes a DNA molecule comprising a sequence selected from the group consisting of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33.

[0360] In some embodiments, at least one of the plurality of species of nucleic acid molecules comprises one or more functional nucleotide analogs selected from pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

[0361] In some embodiments, the composition further comprises at least one lipid. Optionally, the lipid comprises a first lipid according to Formula 01-I or Formula 01-II; or a compound listed in Table 01-1; or a compound according to Formula 02-I; or a compound listed in Table 02-1; or a compound according to Formula 03-I; or a compound listed in Table 03-1; or a compound according to Formula 04-I; or a compound listed in Table 04-1; optionally, the lipid further comprises a second lipid according to Formula 05-I.

[0362] In some embodiments, the composition is formulated as a lipid nanoparticle encapsulating the nucleic acid.

[0363] In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0364] In some embodiments, the composition is a vaccine.

[0365] In some embodiments, the composition is administered in combination with a second therapeutic agent; optionally, the second therapeutic agent comprises EGFR-TKI, PD1 antibody. Optionally, the EGFR-TKI is erlotinib, gefitinib, or dacomitinib.

[0366] As used herein, when referring to polypeptides, the term "domain" refers to a polypeptide motif having one or more identifiable structural or functional characteristics or properties (eg, binding ability, serving as a site for protein-protein interactions).

[0367] As used herein, when referring to polypeptides, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" as it relates to amino acid-based embodiments. As used herein, when referring to polynucleotides, the term "site" is used synonymously with "nucleotide" as it relates to nucleotide-based embodiments. A site refers to a position within a peptide, polypeptide, or polynucleotide that can be modified, manipulated, altered, derivatized, or varied within a polypeptide- or polynucleotide-based molecule.

[0368] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of the polypeptide of interest. For example, any protein fragment of a reference protein of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more than 100 amino acids in length is provided herein (referring to a polypeptide sequence that is at least one amino acid residue shorter than a reference polypeptide sequence but is otherwise identical). In another embodiment, any protein comprising 10, 20, 30, 40, 50 or 100 amino acid segments that are 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% identical to any sequence described herein can be utilized according to the present disclosure. In some embodiments, the polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations as shown in any sequence provided or cited herein. In another embodiment, any protein comprising a 20, 30, 40, 50 or 100 amino acid stretch having greater than 80%, 90%, 95% or 100% identity to any sequence described herein can be utilized in accordance with the present disclosure, wherein the protein has a 5, 10, 15, 20, 25 or 30 amino acid stretch having less than 80%, 75%, 70%, 65% or 60% identity to any sequence described herein.

[0369] The polypeptide or polynucleotide molecules disclosed herein can share a certain degree of sequence similarity or identity with a reference molecule (e.g., a reference polypeptide or a reference polynucleotide), such as with a molecule described in the art (e.g., an engineered or designed molecule or a wild-type molecule). The term "identity" as known in the art refers to the relationship between two or more polypeptide or polynucleotide sequences, as determined by comparing sequences. In the art, identity also refers to the degree of sequence relatedness between them, which is determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smaller sequences in two or more sequences with gap comparisons (if any) processed by a specific mathematical model or computer program (e.g., "algorithm"). The identity of related peptides can be easily calculated by known methods.

[0370] "Percent identity" applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid sequence or nucleic acid sequence of a second sequence after alignment and introduction of gaps (if necessary) to achieve maximum percent identity. Methods and computer programs for comparison are well known in the art. It should be understood that identity depends on the calculation of percent identity, but its value may be different due to gaps and penalties introduced in the calculation. Typically, a variant of a specific 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 the specific reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such alignment tools include those in the BLAST suite (Stephen F. Altschul et al. (1997), "Gapped BLAST 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 & 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 & 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.). Recently, a fast optimal global sequence alignment algorithm (FOGSAA) has been developed that is claimed to produce global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, particularly in the definition of "identity" below.

[0371] The term "homology" as used herein refers to the overall correlation between polymer molecules, such as nucleic acid molecules (such as DNA molecules and / or RNA molecules) and / or polypeptide molecules. Polymer molecules (such as nucleic acid molecules (such as DNA molecules and / or RNA molecules) and / or polypeptide molecules) that share a threshold level of similarity or identity determined by the comparison of matching residues are referred to as homologous. Homology is a qualitative term that describes the relationship between molecules and can be based on quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence matching between two comparison sequences. In some embodiments, if the sequence of a polymer molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical or similar, the polymer molecules are considered to be "homologous" to each other. The term "homologous" necessarily refers to the comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95% or even 99% homologous over at least a stretch of at least 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode stretches of at least 4-5 uniquely specified amino acid sequences. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode stretches of at least 4-5 uniquely specified amino acids. Two protein sequences are considered homologous if they are at least 50%, 60%, 70%, 80% or 90% identical over at least a stretch of 20 amino acids.

[0372] Homology means that the compared sequences diverged from a common origin during evolution. The term "homolog" refers to a first amino acid sequence or nucleic acid sequence (e.g., a gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by inheritance from a common ancestral sequence. The term "homolog" can apply to relationships between genes and / or proteins that have been separated by a speciation event, or to relationships between genes and / or proteins that have been separated by a gene duplication event.

[0373] The term "identity" refers to the overall relatedness between polymer molecules, such as polynucleotide molecules (such as DNA molecules and / or RNA molecules) and / or polypeptide molecules. For example, the percentage identity of the two polynucleic acid sequences can be calculated by comparing two polynucleic acid sequences to achieve the best comparison purpose (for example, a gap can be introduced in one or both of the first and second nucleic acid sequences to achieve the best comparison, and different sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence compared for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides on the corresponding nucleotide positions are then compared. When a certain position in the first sequence occupies the same nucleotide as the corresponding position in the second sequence, the two molecules are identical in this position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, wherein the number of gaps and the length of each gap are taken into account, which needs to be introduced for the best comparison of the two sequences. The comparison of sequences and the determination of the percentage identity between the two sequences can be completed using a mathematical algorithm. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; all of which are incorporated herein by reference.For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleic acid sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Methods commonly used to determine percent identity between sequences include, but are not limited to, the method disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988); which is incorporated herein by reference. Techniques for determining identity are incorporated into public computer programs. Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN and FASTA Altschul, SF et al., J. Molec. Biol, 215, 403 (1990)).

[0374] The term "degenerate sequence" refers to a situation where even if one or more nucleotide sequences in a specific nucleotide sequence are changed, the encoded amino acid sequence remains unchanged.

[0375] The term "vector" refers to a polynucleotide that can replicate within a biological system or can be moved between such systems. Vector polynucleotides typically contain elements such as an origin of replication, a polyadenylation signal, or a selectable marker that function to promote replication or maintenance of these polynucleotides in a biological system. Examples of such biological systems can include cells, viruses, animals, plants, and reconstructed biological systems that utilize biological components that can replicate vectors. The polynucleotides that make up the vector can be DNA or RNA molecules or hybrids thereof.

[0376] Table 2 below shows the amino acid, DNA and RNA sequences of exemplary constructs according to the present disclosure.

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390] Functional nucleotide analogs

[0391] In some embodiments, the payload nucleic acid molecules described herein contain only canonical nucleotides selected from A (adenosine), G (guanosine), C (cytosine), U (uridine), and T (thymidine). Without being bound by theory, it is expected that certain functional nucleotide analogs can confer useful properties to nucleic acid molecules. In the context of the present disclosure, examples of these useful properties include, but are not limited to, increasing the stability of nucleic acid molecules, reducing the immunogenicity of nucleic acid molecules in inducing innate immune responses, enhancing the production of proteins encoded by nucleic acid molecules, increasing intracellular delivery and / or retention of nucleic acid molecules, and / or reducing the cytotoxicity of nucleic acid molecules, etc.

[0392] Therefore, in some embodiments, the effective load nucleic acid molecule comprises at least one functional nucleotide analog as described herein. In some embodiments, the functional nucleotide analog contains at least one chemical modification to a nucleobase, a sugar group and / or a phosphate group. Therefore, the effective load nucleic acid molecule comprising at least one functional nucleotide analog contains at least one chemical modification to a nucleobase, a sugar group and / or an internucleoside bond. Exemplary chemical modifications to the nucleobase, sugar group or internucleoside bond of a nucleic acid molecule are provided herein.

[0393] As described herein, nucleotides ranging from 0% to 100% of all nucleotides in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70 In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein, and the nucleotides of the present invention are functional nucleotide analogs as described herein. In any one of these embodiments, the functional nucleotide analogs can be present in any position of the nucleic acid molecule, including the 5'-end, the 3'-end and / or one or more internal positions. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and / or different types of internucleoside linkages (eg, backbone structures).

[0394] As described herein, ranging from 0% to 100% of all nucleotides of one type (e.g., all purine-containing nucleotides as a type, or all pyrimidine-containing nucleotides as a type, or all A, G, C, T, or U as a type) in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 95%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 10% to about 100%, about 20% to about 25%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 10% to about 80%, In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, the nucleotides of the present invention are functional nucleotide analogs as described herein. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and / or different types of internucleoside linkages (eg, backbone structures).

[0395] III. Chemical Modification

[0396] Modified nucleotide sequence encoding activating EGFR mutant polypeptide

[0397] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one activating EGFR mutant polypeptide, wherein the RNA comprises at least one chemical modification.

[0398] The terms "chemical modification" and "chemically modified" refer to modifications of at least one of adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides in their position, pattern, percentage or population. In general, these terms do not refer to ribonucleotide modifications in the naturally occurring 5'-terminal mRNA cap moiety.

[0399] The modification of polynucleotides includes but is not limited to the modification described herein, and includes but is not limited to the modification including chemical modification. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) can include naturally occurring modifications, non-naturally occurring modifications, or polynucleotides can include naturally occurring modifications and combinations of non-naturally occurring modifications. Polynucleotides can include any useful modification, such as modification of sugars, core bases or internucleoside bonds (e.g., connecting phosphates, phosphodiester bonds or phosphodiester backbones).

[0400] For polypeptides, the term "modification" refers to modifications relative to the canonical set of 20 amino acids. Polypeptides as provided herein are also considered "modified" if they contain amino acid substitutions, insertions, or a combination of substitutions and insertions.

[0401] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise various (more than one) different modifications. In some embodiments, a specific region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified RNA polynucleotides (e.g., modified mRNA polynucleotides) introduced into a cell or organism exhibit reduced degradation in the cell or organism relative to unmodified polynucleotides, respectively. In some embodiments, the modified RNA polynucleotides (e.g., modified mRNA polynucleotides) introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively.

[0402] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are included in non-natural modified nucleotides introduced during or after the synthesis of the polynucleotides to achieve desired functions or characteristics. Modifications can be present on internucleotide bonds, purine or pyrimidine bases, or sugars. Modifications can be introduced at the end of a chain or at any position in a chain by chemical synthesis or polymerase. Any region of a polynucleotide can be chemically modified.

[0403] In some embodiments, the polynucleotides of the present invention (e.g., RNA, e.g., mRNA) comprise chemically modified nucleobases. The present invention includes modified polynucleotides containing polynucleotides described herein (e.g., polynucleotides comprising nucleotide sequences encoding one or more activated EGFR mutant polypeptides). The modified polynucleotides can be chemically and / or structurally modified. When the polynucleotides of the present invention are chemically and / or structurally modified, the polynucleotides may be referred to as "modified polynucleotides".

[0404] The present disclosure provides the modified nucleosides and nucleotides of the polynucleotides (for example, RNA polynucleotides, such as mRNA polynucleotides) encoding one or more activated EGFR mutant polypeptides. "Nucleoside" refers to a compound containing a sugar molecule (for example pentose or ribose) or its derivatives and an organic base (for example purine or pyrimidine) or its derivatives (also referred to herein as "nucleobase") combination. "Nucleotide" refers to a nucleoside comprising a phosphate group. The modified nucleotides can be synthesized by any useful method such as chemical method, enzymatic method or recombinant method to include one or more modified or non-natural nucleosides. Polynucleotides can include one or more regions connecting nucleosides. These regions can have variable main chain bonds. The bonds can be standard phosphodiester bonds, in which case the polynucleotides will include nucleotide regions.

[0405] Modified polynucleotides disclosed herein can include various modifications. In some embodiments, the modified polynucleotides contain one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotides introduced into cells can exhibit one or more desired properties, such as improved protein expression, reduced immunogenicity or reduced cell degradation, compared to unmodified polynucleotides.

[0406] In some embodiments, the polynucleotides of the present invention (e.g., polynucleotides comprising a nucleotide sequence encoding one or more activated EGFR mutant polypeptides) are structurally modified. As used herein, "structural" modification refers to inserting, deleting, duplicating, inverting or randomizing two or more connected nucleosides in a polynucleotide, without significant chemical modification to the nucleotide itself. Because chemical bonds are bound to break and reform to achieve structural modification, structural modification has chemical properties and is therefore chemically modified. However, structural modification will result in different nucleotide sequences. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified to "ATCCCG" from "ATCG". Dinucleotide "CC" is inserted here, resulting in structural modification of the polynucleotide.

[0407] In some embodiments, the polynucleotides of the invention are chemically modified. The term "chemical modification" or "chemical modification" as used herein with respect to polynucleotides refers to modification of one or more of adenosine (A), guanosine (G), uridine (U) or cytidine (C) ribose or deoxyribonucleosides in their position, pattern, percentage or population. In general, in this article, these terms are not intended to refer to ribonucleotide modifications in the naturally occurring 5'-terminal mRNA cap portion.

[0408] In some embodiments, polynucleotides of the invention may have uniform chemical modifications of all or any of the same nucleoside types, or a population of modifications generated by simple downward titration of the same starting modification in all or any of the same nucleoside types, or a measured percentage of chemical modifications of all or any of the same nucleoside types but randomly incorporated, such as where all uridines are replaced by uridine analogs (e.g., pseudouridine or 5-methoxyuridine). In another embodiment, polynucleotides may have uniform chemical modifications of two, three or four of the same nucleoside types throughout the polynucleotide (such as all uridines and all cytosines, etc., are modified in the same manner).

[0409] Modified nucleotide base pairing not only encompasses standard adenosine-thymine, adenosine-uracil or guanosine-cytosine base pairs, but also encompasses base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonds to be formed between non-standard bases and standard bases or between two complementary non-standard base structures, such as in polynucleotides with at least one chemical modification. An example of such non-standard base pairing is base pairing between modified nucleotides inosine and adenine, cytosine or uracil. Any combination of bases / sugars or linkers may be incorporated into the polynucleotides of the present disclosure.

[0410] Those skilled in the art will understand that, unless otherwise indicated, the polynucleotide sequences described in this application will reference "T" in representative DNA sequences, but when the sequence represents RNA, "T" will replace "U".

[0411] In some embodiments, mRNA is a uracil modified sequence comprising an ORF encoding one or more activated EGFR mutant polypeptides, wherein the mRNA comprises a chemically modified nucleobase, such as 5-methoxyuracil. In certain aspects of the invention, when a 5-methoxyuracil base is linked to a ribose (such as in a polynucleotide), the resulting modified nucleoside or nucleotide is referred to as 5-methoxyuracil. In some embodiments, the uracil in the polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99% or about 100% 5-methoxyuracil. In one embodiment, the uracil in the polynucleotide is at least 95% 5-methoxyuracil. In another embodiment, the uracil in the polynucleotide is 100% 5-methoxyuracil.

[0412] In embodiments where the uracil in the polynucleotide is at least 95% 5-methoxyuracil, the total uracil content can be adjusted so that the mRNA provides a suitable protein expression level while inducing little or no immune response. In some embodiments, the uracil content of the ORF is about 105% to about 145%, about 105% to about 140%, about 110% to about 140%, about 110% to about 145%, about 115% to about 135%, about 105% to about 135%, about 110% to about 135%, about 115% to about 145%, or about 115% to about 140% of the theoretical minimum uracil content (% Utm) in the corresponding wild-type ORF. In other embodiments, the uracil content of the ORF is about 117% to about 134% or 118% to 132% of % Utm. In some embodiments, the uracil content of the ORF encoding one or more activating EGFR mutant polypeptides is about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, or about 150% of % Utm. In this context, the term "uracil" may refer to 5-methoxyuracil and / or naturally occurring uracil.

[0413] In some embodiments, the uracil content in the ORF of the mRNA encoding one or more activated EGFR mutant polypeptides of the present invention is less than about 50%, about 40%, about 30%, about 20%, about 15% or about 12% of the total nucleobase content in the ORF. In some embodiments, the uracil content in the ORF is about 12% to about 25% of the total nucleobase content in the ORF. In some embodiments, the uracil content in the ORF is about 15% to about 17% of the total nucleobase content in the ORF. In one embodiment, the uracil content in the ORF of the mRNA encoding one or more activated EGFR mutant polypeptides is less than about 20% of the total nucleobase content in the open reading frame. In this context, the term "uracil" can refer to 5-methoxyuracil and / or naturally occurring uracil.

[0414] In a further embodiment, the ORF of the mRNA encoding one or more activating EGFR mutant polypeptides of the present invention comprises 5-methoxyuracil and has an adjusted uracil content, wherein the adjusted uracil content comprises less uracil pairs (UU) and / or uracil triplets (UUU) and / or uracil quadruplets (UUUU) than the corresponding wild-type nucleotide sequence encoding one or more activating EGFR mutant polypeptides. In some embodiments, the ORF of the mRNA encoding one or more activating EGFR mutant polypeptides of the present invention does not contain uracil pairs and / or uracil triplets and / or uracil quadruplets. In some embodiments, uracil pairs and / or uracil triplets and / or uracil quadruplets are reduced to below a certain threshold, for example, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times in the ORF of the mRNA encoding one or more activating EGFR mutant polypeptides. In a specific embodiment, the ORF of the mRNA encoding one or more activating EGFR mutant polypeptides of the present invention contains less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 non-phenylalanine uracil pairs and / or triplets. In another embodiment, the ORF of the mRNA encoding one or more activating EGFR mutant polypeptides does not contain non-phenylalanine uracil pairs and / or triplets.

[0415] In a further embodiment, the ORF of the mRNA encoding one or more activating EGFR mutant polypeptides of the present invention comprises 5-methoxyuracil and has an adjusted uracil content, the adjusted uracil content comprising fewer uracil-rich clusters than the corresponding wild-type nucleotide sequence encoding one or more activating EGFR mutant polypeptides. In some embodiments, the ORF of the mRNA encoding one or more activating EGFR mutant polypeptides of the present invention comprises a uracil-rich cluster whose length is shorter than the corresponding uracil-rich cluster in the corresponding wild-type nucleotide sequence encoding one or more activating EGFR mutant polypeptides.

[0416] In some embodiments, the chemically modified nucleosides in the open reading frame are selected from the group consisting of uridine, adenine, cytosine, guanine, and any combination thereof.

[0417] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides with modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halogenated-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C) and 2-thio-5-methyl-cytidine.

[0418] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides with modified uridine include 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methoxyuridine, 2-thiouridine, 5-cyanouridine, 2'-0-methyluridine and 4'-thiouridine. In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides with modified adenine include 7-deazaadenine, 1-methyladenosine, 2-methyladenine (m2A), N6-methyl-adenosine (m6A) and 2,6-diaminopurine.

[0419] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with modified guanine include inosine (I), 1-methyl-inosine (m il), wyosine (imG), methyl wyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ l), 7-methyl-guanosine (m7G), 1-methyl-guanosine (mlG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0420] In some embodiments, the nucleobase modified nucleotide in a polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) is 5-methoxyuridine.

[0421] In some embodiments, a polynucleotide (eg, an RNA polynucleotide, such as an mRNA polynucleotide) includes a combination of at least two (eg, 2, 3, 4, or more) modified nucleobases.

[0422] In some embodiments, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) includes 5-methoxyuridine (5mo5U) and 5-methylcytidine (m5C).

[0423] In some embodiments, for a particular modification, a polynucleotide (e.g., an RNA polynucleotide, such as an mRNA polynucleotide) is uniformly modified (e.g., completely modified, modified throughout the sequence). For example, a polynucleotide can be uniformly modified with 5-methoxyuridine, meaning that substantially all uridine residues in the mRNA sequence are replaced with 5-methoxyuridine. Similarly, any type of nucleoside residue present in a polynucleotide sequence can be uniformly modified by replacing it with a modified residue (such as any of the residues described above).

[0424] In some embodiments, the modified nucleobase is a modified cytosine.

[0425] In some embodiments, the modified nucleobase is a modified uracil. Examples of nucleobases and nucleosides having modified uracil include 5-methoxyuracil.

[0426] In some embodiments, the modified nucleobase is a modified adenine.

[0427] In some embodiments, the modified nucleobase is a modified guanine.

[0428] In some embodiments, the polynucleotide may include any useful linker between nucleosides. Such linkers (including backbone modifications) that can be used in the compositions of the present disclosure include, but are not limited to, the following: 3'-alkylene phosphonates, 3'-aminophosphoramides, olefin-containing backbones, aminoalkylphosphoramides, aminoalkylphosphotriesters, boranephosphonates, -CH 2 -ON(CH 3 )-CH 2 -、-CH 2 -N(CH 3 )-N(CH 3 )-CH 2 -、-CH 2 -NH-CH 2 -, chiral phosphonates, chiral phosphorothioates, formyl and thioformyl backbones, methylene (methylimino), methylene formyl and thioformyl backbones, methyleneimino and methylenehydrazine backbones, morpholino bonds, -N(CH 3 )-CH 2 -CH2 -, oligonucleosides with heteroatom internucleoside linkages, phosphinates, phosphoamidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphotriesters, PNA, siloxane backbones, sulfamate backbones, sulfide sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thioalkylphosphonates, thioalkylphosphotriesters, and thioalkylphosphothioates.

[0429] In general, RNA comprises a sugar group ribose, which is a 5-membered ring containing oxygen. Exemplary non-limiting modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or an alkylene group, such as methylene or ethylene); addition of double bonds (e.g., replacement of ribose with a cyclopentenyl or cyclohexenyl group); ring contraction of ribose (e.g., forming a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., forming a 6- or 7-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexyl, cyclohexenyl, and morpholine, which also have a phosphoamidoester backbone); polycyclic forms (e.g., tricyclic; and "unlocked" forms, such as glycol nucleic acids (GNA) (e.g., R-GNA or S-GNA, where The sugar group may also include one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, a polynucleic acid molecule may include nucleotides containing, for example, arabinose or L-ribose as sugars. Such sugar modifications are taught in International Patent Publication Nos. WO2013052523 and WO2014093924, the contents of each of which are incorporated herein by reference in their entirety.

[0430] The polynucleotides of the present invention (e.g., polynucleotides comprising a nucleotide sequence encoding one or more activated EGFR mutant polypeptides or functional fragments or variants thereof) may include combinations of modifications to sugars, nucleobases and / or internucleoside bonds. These combinations may include any one or more modifications described herein.

[0431] The polynucleotides of the present disclosure may be partially or completely modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotides (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in the polynucleotides of the present disclosure or in a given predetermined sequence region thereof (e.g., in an mRNA including or not including a polyA tail). In some embodiments, all nucleotides X in the polynucleotides of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may be any one of the nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, or A+G+C.

[0432] The polynucleotides may contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, U, or C), or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 10 ... %, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). It will be appreciated that any remaining percentages are due to the presence of unmodified A, G, U, or C. Polynucleotide can contain minimum 1% and maximum 100% modified nucleotides, or any intermediate percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides or at least 90% modified nucleotides. For example, polynucleotide can contain modified pyrimidines, such as modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% uracil in polynucleotide is replaced by modified uracil (for example, 5-substituted uracil). Modified uracil can be replaced by compounds with a single unique structure, or can be replaced by a variety of compounds with different structures (for example, 2,3,4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the polynucleotide are replaced by modified cytosines (e.g., 5-substituted cytosines). The modified cytosines can be replaced by compounds having a single unique structure, or can be replaced by multiple compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0433] IV. Lipids

[0434] As used herein and unless otherwise specified, the term "lipid" refers to a group of organic compounds, including but not limited to esters of fatty acids, and is generally characterized by being poorly soluble in water, but soluble in many non-polar organic solvents. Although lipids are generally poorly soluble in water, there are certain classes of lipids (e.g., lipids modified by polar groups, such as DMG-PEG2000) that have limited water solubility and can be soluble in water under certain conditions. Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be divided into at least three categories: (1) "simple lipids", which include fats and oils and waxes; (2) "complex lipids", which include phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derivatized lipids", such as steroids. Further, as used herein, lipids also encompass lipid-like compounds. The term "lipid-like compound", also referred to as "lipid-like", refers to a lipid-like compound (e.g., an amphiphilic compound having lipid-like physical properties).

[0435] The term "lipid nanoparticle" or "LNP" refers to a particle with at least one nanometer (nm) size (e.g., 1 to 1,000nm), which contains one or more types of lipid molecules. LNP provided herein may further contain at least one non-lipid payload molecule (e.g., one or more types of nucleic acid molecules). In some embodiments, LNP comprises a non-lipid payload molecule partially or completely encapsulated in the lipid shell. In particular, in some embodiments, wherein the payload is a negatively charged molecule (e.g., mRNA encoding a viral protein), and the lipid component of LNP comprises at least one cationic lipid. Without being bound by theory, it is expected that cationic lipids can interact with negatively charged payload molecules and promote payload binding and / or encapsulation into LNP during LNP formation. Other lipids that can form a part of LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as steroids, polymer-conjugated lipids and various zwitterionic lipids. In certain embodiments, LNPs according to the present disclosure comprise one or more lipids of the 01, 02, 03, and 04 series, such as one or more lipids of formulas (01-I), (01-II), (02-I), (03-I), and (04-I) (and subformulas thereof) described herein.

[0436] The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or can be positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., the environment of its intended use). Therefore, the term "cation" encompasses "permanent cations" and "cationizable". In certain embodiments, the positive charge in the cationic lipid is due to the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that exhibits positive charge in the environment of its intended use (e.g., at physiological pH). In certain embodiments, the cationic lipid is one or more lipids of the 01, 02, 03, and 04 series, such as one or more lipids of formula (01-I), (01-II), (02-I), (03-I) and (04-I) (and its subformula) described herein. The term "anionic lipid" refers to a lipid that is negatively charged at any pH value or hydrogen ion activity of its environment, or can be negatively charged in response to the pH value or hydrogen ion activity of its environment (e.g., the environment of its intended use). Exemplary anionic lipids include one or more negatively charged phosphate groups, eg, at physiological pH.

[0437] The term "polymer-conjugated lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a pegylated lipid (PEG-lipid), wherein the polymer portion comprises polyethylene glycol.

[0438] The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or in a neutral zwitterionic form at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment of the intended use of the lipid, such as physiological pH. As non-limiting examples, neutral lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylamino)ethyl hydrogen phosphate (DOCP), sphingomyelin (SM), ceramides, steroids such as sterols, and derivatives thereof. The neutral lipids provided herein can be synthetic or derived from (isolated or modified from) natural sources or compounds.

[0439] The term "charged lipid" encompasses any lipid molecule that exists in a positively or negatively charged form within a selected pH value or a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH conditions in the environment of the intended lipid use, such as physiological pH. As a non-limiting example, the neutral lipids that can be used in conjunction with the present disclosure include but are not limited to phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-racemic-glycerol) sodium salt (DOPG-Na) and 1,2-dioleoyl-sn-glycero-3-phospho-sodium salt (DOPA-Na). Charged lipids as provided herein can be synthetic or derived from (isolated or modified from) natural sources or compounds.

[0440] As used herein and unless otherwise specified, the term "alkyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, which is saturated. In one embodiment, an alkyl group has, for example, 1 to 24 carbon atoms (C 1 -C 24 alkyl), 4 to 20 carbon atoms (C 4 -C 20 alkyl), 6 to 16 carbon atoms (C 6 -C 16 alkyl), 6 to 9 carbon atoms (C 6 -C 9 alkyl), 1 to 15 carbon atoms (C 1 -C 15 alkyl), 1 to 12 carbon atoms (C 1 -C 12 alkyl), 1 to 8 carbon atoms (C 1 -C 8 Alkyl) or 1 to 6 carbon atoms (C 1 -C 6 Alkyl) and is attached to the rest of the molecule by a single bond. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, alkyl is optionally substituted.

[0441] As used herein and unless otherwise indicated, the term "alkenyl" refers to a straight or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing one or more carbon-carbon double bonds. As understood by those of ordinary skill in the art, the term "alkenyl" also encompasses groups having "cis" and "trans" configurations or, alternatively, "E" and "Z" configurations. In one embodiment, an alkenyl group has, for example, 2 to 24 carbon atoms (C 2 -C 24 alkenyl), 4 to 20 carbon atoms (C 4 -C 20 alkenyl), 6 to 16 carbon atoms (C 6 -C 16 alkenyl), 6 to 9 carbon atoms (C 6 -C 9 alkenyl), 2 to 15 carbon atoms (C 2 -C 15 alkenyl), 2 to 12 carbon atoms (C 2 -C 12 alkenyl), 2 to 8 carbon atoms (C 2 -C 8 alkenyl) or 2 to 6 carbon atoms (C 2 -C 6 The term "alkenyl" refers to a group consisting of a 1-alkenyl group and a 1-alkenyl group, and is connected to the rest of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, vinyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, and the like. Unless otherwise specified, alkenyl groups are optionally substituted.

[0442] As used herein and unless otherwise indicated, the term "alkynyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing one or more carbon-carbon triple bonds. In one embodiment, an alkynyl group has, for example, 2 to 24 carbon atoms (C 2 -C 24 Alkynyl), 4 to 20 carbon atoms (C 4 -C 20 Alkynyl), 6 to 16 carbon atoms (C 6 -C 16 Alkynyl), 6 to 9 carbon atoms (C 6 -C 9 Alkynyl), 2 to 15 carbon atoms (C 2 -C 15 Alkynyl), 2 to 12 carbon atoms (C 2 -C 12 Alkynyl), 2 to 8 carbon atoms (C 2 -C 8 Alkynyl) or 2 to 6 carbon atoms (C 2 -C 6Alkynyl) and is connected to the rest of the molecule by a single bond. Examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise specified, alkynyl is optionally substituted.

[0443] As used herein and unless otherwise indicated, the term "alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical, which consists only of carbon and hydrogen and is saturated. In one embodiment, an alkylene group has, for example, 1 to 24 carbon atoms (C 1 -C 24 alkylene), 1 to 15 carbon atoms (C 1 -C 15 alkylene), 1 to 12 carbon atoms (C 1 -C 12 alkylene), 1 to 8 carbon atoms (C 1 -C 8 alkylene), 1 to 6 carbon atoms (C 1 -C 6 alkylene), 2 to 4 carbon atoms (C 2 -C 4 alkylene), 1 to 2 carbon atoms (C 1 -C 2 Alkylene). Examples of alkylene include, but are not limited to, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is connected to the rest of the molecule by a single bond and to the radical by a single bond. The point of attachment of the alkylene chain to the rest of the molecule and to the radical may be through one carbon or any two carbons within the chain. Unless otherwise indicated, the alkylene chain is optionally substituted.

[0444] As used herein and unless otherwise indicated, the term "alkenylene" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a radical, consisting solely of carbon and hydrogen and containing one or more carbon-carbon double bonds. In one embodiment, an alkenylene group has, for example, 2 to 24 carbon atoms (C 2 -C 24 alkenyl), 2 to 15 carbon atoms (C 2 -C 15 alkenyl), 2 to 12 carbon atoms (C 2 -C 12 alkenyl), 2 to 8 carbon atoms (C 2 -C 8 alkenyl), 2 to 6 carbon atoms (C 2 -C 6 alkenylene) or 2 to 4 carbon atoms (C 2 -C 4Examples of alkenylene include, but are not limited to, vinylene, propenylene, n-butenyl, etc. Alkenylene is connected to the rest of the molecule by a single bond or a double bond, and to the radical by a single bond or a double bond. The point of attachment of alkenylene to the rest of the molecule and to the radical may be through one carbon or any two carbons within the chain. Unless otherwise indicated, alkenylene is optionally substituted.

[0445] As used herein and unless otherwise indicated, the term "cycloalkyl" refers to a saturated, non-aromatic, monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms. Cycloalkyls may include fused or bridged ring systems. In one embodiment, a cycloalkyl has, for example, 3 to 15 ring carbon atoms (C 3 -C 15 cycloalkyl), 3 to 10 ring carbon atoms (C 3 -C 10 Cycloalkyl) or 3 to 8 ring carbon atoms (C 3 -C 8 Cycloalkyl). The cycloalkyl is connected to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyls include, but are not limited to, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, and the like. Unless otherwise indicated, cycloalkyls are optionally substituted.

[0446] As used herein and unless otherwise specified, the term "cycloalkylene" is a divalent cycloalkyl group. Unless otherwise specified, a cycloalkylene group is optionally substituted.

[0447] As used herein and unless otherwise indicated, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting only of carbon and hydrogen atoms and including one or more carbon-carbon double bonds. Cycloalkenyl groups may include fused or bridged ring systems. In one embodiment, a cycloalkenyl group has, for example, 3 to 15 ring carbon atoms (C 3 -C 15 cycloalkenyl), 3 to 10 ring carbon atoms (C 3 -C 10 Cycloalkenyl) or 3 to 8 ring carbon atoms (C 3 -C 8 Cycloalkenyl). The cycloalkenyl is connected to the rest of the molecule by a single bond. Examples of monocyclic cycloalkenyls include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise indicated, the cycloalkenyl is optionally substituted.

[0448] As used herein and unless otherwise specified, the term "cycloalkenylene" is a divalent cycloalkenyl group. Unless otherwise specified, a cycloalkenylene group is optionally substituted.

[0449] As used herein and unless otherwise indicated, the term "heterocyclyl" refers to a non-aromatic radical monocyclic or polycyclic moiety containing one or more (e.g., one, one or two, one to three or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorus and sulfur. The heterocyclyl can be connected to the main structure at any heteroatom or carbon atom. The heterocyclyl can be a monocyclic, bicyclic, tricyclic, tetracyclic or other polycyclic system, wherein the polycyclic system can be a condensed ring, a bridged ring or a spirocyclic system. The heterocyclyl polycyclic system can contain one or more heteroatoms in one or more rings. The heterocyclyl can be saturated or partially unsaturated. Saturated heterocycloalkyl can be referred to as "heterocycloalkyl". Partially unsaturated heterocycloalkyl can be referred to as "heterocycloalkenyl" when the heterocyclyl contains at least one double bond, or can be referred to as "heterocycloalkynyl" when the heterocyclyl contains at least one triple bond. In one embodiment, the heterocyclyl has, for example, 3 to 18 ring atoms (3 to 18-membered heterocyclyl), 4 to 18 ring atoms (4 to 18-membered heterocyclyl), 5 to 18 ring atoms (3 to 18-membered heterocyclyl), 4 to 8 ring atoms (4 to 8-membered heterocyclyl), or 5 to 8 ring atoms (5 to 8-membered heterocyclyl). Numerical ranges such as "3 to 18" appearing herein refer to each integer in the given range; for example, "3 to 18 ring atoms" means that the heterocyclyl can be composed of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, etc., up to and including 18 ring atoms. Examples of heterocyclic groups include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thienyl, pyridyl, piperidinyl, quinolyl, and isoquinolyl. Unless otherwise indicated, heterocyclic groups are optionally substituted.

[0450] As used herein and unless otherwise specified, the term "heterocyclylene" is a divalent heterocyclyl group. Unless otherwise specified, a heterocyclylene group is optionally substituted.

[0451] As used herein and unless otherwise specified, the term "aryl" refers to a monocyclic aromatic group and / or a polycyclic monovalent aromatic group containing at least one aromatic hydrocarbon ring. In certain embodiments, an aryl group has 6 to 18 ring carbon atoms (C 6 -C 18 aryl), 6 to 14 ring carbon atoms (C 6 -C 14 aryl) or 6 to 10 ring carbon atoms (C 6 -C 10The term "aryl" also refers to a bicyclic, tricyclic or other polycyclic hydrocarbon ring in which at least one ring is aromatic and the other rings may be saturated, partially unsaturated or aromatic, such as dihydronaphthyl, indenyl, indanyl or tetrahydronaphthyl (tetrahydronaphthyl). Unless otherwise indicated, aryl is optionally substituted.

[0452] As used herein and unless otherwise specified, the term "arylene" is a divalent aromatic group. Unless otherwise specified, an arylene group is optionally substituted.

[0453] The term "heteroaryl" as used herein, unless otherwise indicated, refers to a monocyclic aromatic group and / or a polycyclic aromatic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one, one or two, one to three or one to four) heteroatoms independently selected from O, S and N. Heteroaryl can be connected to the main structure by any heteroatom or carbon atom. In certain embodiments, heteroaryl has 5 to 20, 5 to 15 or 5 to 10 ring atoms. The term "heteroaryl" also refers to a bicyclic, tricyclic or other polycyclic ring, wherein at least one ring is aromatic, and the other rings can be saturated, partially unsaturated or aromatic, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S and N. Examples of monocyclic heteroaryls include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl and triazinyl. Examples of bicyclic heteroaryls include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolyl, tetrahydroisoquinolyl, isoquinolyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromone, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl and tetrahydroquinolyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise specified, heteroaryl groups are optionally substituted.

[0454] As used herein and unless otherwise specified, the term "heteroarylene" is a divalent heteroaryl group. Unless otherwise specified, a heteroarylene group is optionally substituted.

[0455] When groups described herein are referred to as "substituted", they may be substituted with any suitable substituent. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments provided herein, as well as: halogen atoms, such as F, Cl, Br or I; cyano; oxo (=O); hydroxyl (-OH); alkyl; alkenyl; alkynyl; cycloalkyl; aryl; -(C=O)OR'; -O(C=O)R'; -C(=O)R'; -OR'; -S(O) x R';-S-SR';-C(=O)SR';-SC(=O)R';-NR'R';-NR'C(=O)R';-C(=O)NR'R';-NR'C(=O)NR'R';-OC(=O)NR'R';-NR'C(=O)OR';-NR'S(O) x NR'R';-NR'S(O) x R'; and -S(O) x NR'R', wherein: R' is independently H, C 1 -C 15 alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments, the substituent is C 1 -C 12 In other embodiments, the substituent is a cycloalkyl. In other embodiments, the substituent is a halo, such as fluorine. In other embodiments, the substituent is an oxo. In other embodiments, the substituent is a hydroxyl. In other embodiments, the substituent is an alkoxy (-OR'). In other embodiments, the substituent is a carboxyl. In other embodiments, the substituent is an amino (-NR'R').

[0456] As used herein and unless otherwise indicated, the term "prodrug" of a biologically active compound refers to a compound that can be converted into a biologically active compound under physiological conditions or by solvolysis. In one embodiment, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a biologically active compound. Prodrugs may be inactive when administered to a subject in need, but are converted into biologically active compounds in vivo. Prodrugs are generally rapidly converted into parent biologically active compounds in vivo, such as by hydrolysis in the blood. Prodrug compounds generally provide advantages of solubility, tissue compatibility or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, pp. 21-24 (Elsevier, Amsterdam)). Discussions on prodrugs are provided in Higuchi, T. et al., ACSSymposium Series, Vol. 14; and Bioreversible Carriers in Drug Design, Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press, 1987.

[0457] In one embodiment, the term "prodrug" also includes any covalently bound carrier that releases the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound can be prepared by modifying the functional groups present in the compound by cleavage to the parent compound by conventional manipulation or in vivo. Prodrugs include compounds in which a hydroxyl, amino or sulfhydryl group is bonded to any group that, when the prodrug of the compound is administered to a mammalian subject, is cleaved to form a free hydroxyl, free amino or free sulfhydryl group, respectively.

[0458] Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols or amide derivatives of amine functional groups in the compounds provided herein.

[0459] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts.

[0460] Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, acid), dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, etc.

[0461] Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared by adding an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. In one embodiment, the inorganic salt is an ammonium salt, a sodium salt, a potassium salt, a calcium salt, and a magnesium salt. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, denanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, phenethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. In one embodiment, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0462] The compounds provided herein may contain one or more asymmetric centers, and thus may produce enantiomers, diastereomers, and other stereoisomeric forms, which may be defined as (R)- or (S)-, or as (D)- or (L)- for amino acids, according to absolute stereochemistry. Unless otherwise specified, the compounds provided herein are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing / separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefin double bonds or other geometrically asymmetric centers, unless otherwise specified, it is contemplated that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included.

[0463] As used herein and unless otherwise indicated, the term "isomer" refers to different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the arrangement of their atoms in space. "Atropisomers" are stereoisomers that arise due to hindered rotation about a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any ratio may be referred to as a "racemic" mixture. "Diastereomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0464] "Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

[0465] "Tautomers" refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment in which the compound is located, and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution.

[0466] It should also be noted that the compounds described herein may contain unnatural proportions of atomic isotopes at one or more atoms. For example, the compounds may be modified with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I), sulfur-35( 35 S) or carbon-14 ( 14 C) may be radiolabeled, or may be isotopically enriched, such as deuterium ( 2H), carbon-13 ( 13 C) or nitrogen-15( 15 N). As used herein, an "isotope" is an isotopically enriched compound. The term "isotopically enriched" refers to an atom having an isotopic composition other than the natural isotopic composition of the atom. "Isotopically enriched" may also refer to a compound containing at least one atom having an isotopic composition different from the natural isotopic composition of the atom. The term "isotopic composition" refers to the amount of each isotope present in a given atom. Radiolabeled and isotopically enriched compounds can be used as therapeutic agents, such as cancer therapeutics; research reagents, such as binding assay reagents; and diagnostic agents, such as in vivo imaging agents. All isotopic variants of the compounds described herein, whether or not radioactive, are intended to be included within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds described herein are provided, for example, isotopologues are enriched in deuterium, carbon-13, and / or nitrogen-15. As used herein, "deuterated" refers to at least one hydrogen (H) in a compound that has been replaced with deuterium (as D or 2 H represents) substitution, that is, the compound is enriched in deuterium at at least one position.

[0467] It should be noted that if there is a discrepancy between a depicted structure and the name of the structure, the depicted structure shall prevail.

[0468] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for use in humans or livestock.

[0469] The term "composition" is intended to encompass a product containing the specified ingredients (eg, mRNA molecules provided herein), optionally in the specified amounts.

[0470] Nanoparticle composition

[0471] In one aspect, nucleic acid molecules as described herein are formulated for in vitro and in vivo delivery. In particular, in some embodiments, nucleic acid molecules are formulated to contain lipid compositions. In some embodiments, lipid compositions are formed to enclose nucleic acid molecules in lipid nanoparticles in lipid shells. In some embodiments, lipid shells protect nucleic acid molecules from degradation. In some embodiments, lipid nanoparticles also promote the transport of enclosed nucleic acid molecules to intracellular compartments and / or mechanisms to exert expected treatment or preventive functions. In certain embodiments, when present in lipid nanoparticles, nucleic acid resists degradation of nucleases in aqueous solution. Lipid nanoparticles comprising nucleic acids and methods for making the same are known in the art, such as those disclosed in, for example, U.S. Patent Publication No. 2004 / 0142025, U.S. Patent Publication No. 2007 / 0042031, PCT Publication No. WO 2017 / 004143, PCT Publication No. WO 2015 / 199952, PCT Publication No. WO 2013 / 016058, and PCT Publication No. WO2013 / 086373, the complete disclosure of each of which is incorporated herein by reference in its entirety for all purposes.

[0472] In some embodiments, the maximum size of the nanoparticle composition provided herein is 1 μm or shorter (e.g., ≤1 μm, ≤900nm, ≤800nm, ≤700nm, ≤600nm, ≤500nm, ≤400nm, ≤300nm, ≤200nm, ≤175nm, ≤150nm, ≤125nm, ≤100nm, ≤75nm, ≤50nm or shorter), such as when measured by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy or another method. In one embodiment, at least one dimension of the lipid nanoparticle provided herein is in the range of about 40 to about 200nm. In one embodiment, at least one dimension is in the range of about 40nm to about 100nm.

[0473] Nanoparticle compositions that can be used in conjunction with the present disclosure include, for example, lipid nanoparticles (LNPs), nanolipoprotein particles, liposomes, lipid vesicles, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayer can include one or more ligands, proteins, or channels.

[0474] In some embodiments, the nanoparticle composition comprises a lipid component comprising at least one lipid, such as a compound according to one of lipid series 01, 02, 03, and 04, for example, a compound according to one of formulas (01-I), (01-II), (02-I), (03-I), and (04-I) (and subformulas thereof) described herein. For example, in some embodiments, the nanoparticle composition may comprise a lipid component comprising one of the compounds provided herein. The nanoparticle composition may also comprise one or more other lipid or non-lipid components as described below.

[0475] Cationic lipids

[0476] Cationic lipids include the following 01-04 series lipids (and their subformulae).

[0477] Lipid Series 01

[0478] In one embodiment, the cationic lipid contained in the composition, nanoparticle composition, or nanoparticle described herein is a cationic lipid described in International Patent Publication No. WO2021204175, which is incorporated herein by reference in its entirety.

[0479] In one embodiment, provided herein is a compound of formula (01):

[0480]

[0481] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0482] G 1 and G 2 Each independently is a bond, C 2 -C 12 Alkylene or C 2 -C 12 alkenylene, wherein one or more -CH 2 - is optionally substituted with -O-;

[0483] L 1 Yes-OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR aC(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c )、-(C 6 -C 10 arylene)-R 1 、-(6- to 10-membered heteroarylene)-R 1 or R 1 ;

[0484] L 2 is -OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f )、-(C 6 -C 10Arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0485] R 1 and R 2 Each independently is C 6 -C 32 Alkyl or C 6 -C 32 alkenyl;

[0486] R a , R b , R d and R e Each independently is H, C 1 -C 24 Alkyl or C 2 -C 24 alkenyl;

[0487] R c and R f Each independently is C 1 -C 32 Alkyl or C 2 -C 32 alkenyl;

[0488] G 3 It is C 2 -C 24 Alkylene, C 2 -C 24 Alkenylene, C 3 -C 8 Cycloalkylene or C 3 -C 8 Cycloalkenylene;

[0489] R 3 Yes-N(R 4 )R 5 ;

[0490] R 4 It is C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl, 4 to 8 membered heterocyclic group or C 6 -C 10 Aryl; or R 4 , G 3 or G 3 A portion of together with the nitrogen to which they are attached forms a ring portion;

[0491] R 5 It is C 1 -C 12Alkyl or C 3 -C 8 Cycloalkyl; or R 4 , R 5 Together with the nitrogen to which they are attached they form a ring portion;

[0492] x is 0, 1, or 2; and

[0493] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene and cyclic moiety is independently optionally substituted.

[0494] In one embodiment, provided herein is a compound of formula (01-II):

[0495]

[0496] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0497] is a single bond or a double bond;

[0498] G 1 and G 2 Each independently is a bond, C 2 -C 12 Alkylene or C 2 -C 12 alkenylene, wherein one or more -CH 2 - is optionally substituted with -O-;

[0499] L 1 Yes-OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c )、-(C 6 -C 10 Arylene)-R 1 , -(6- to 10-membered heteroarylene)-R 1 or R 1 ;

[0500] L 2 Yes-OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f )、-(C 6 -C 10 Arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0501] R 1 and R 2 Each independently is C 6 -C 32 Alkyl or C 6-C 32 alkenyl;

[0502] R a , R b , R d and R e Each independently is H, C 1 -C 24 Alkyl or C 2 -C 24 alkenyl;

[0503] R c and R f Each independently is C 1 -C 32 Alkyl or C 2 -C 32 alkenyl;

[0504] G 4 Yes key, C 1 -C 23 Alkylene, C 2 -C 23 Alkenylene, C 3 -C 8 Cycloalkylene or C 3 -C 8 Cycloalkenylene;

[0505] R 3 Yes-N(R 4 )R 5 ;

[0506] R 4 It is C 1 -C 12 Alkyl, C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl, 4 to 8 membered heterocyclic group or C 6 -C 10 Aryl; or R 4 , G 3 or G 3 A portion of together with the nitrogen to which they are attached forms a ring portion;

[0507] R 5 It is C 1 -C 12 Alkyl or C 3 -C 8 Cycloalkyl; or R 4 , R 5 Together with the nitrogen to which they are attached they form a ring portion;

[0508] x is 0, 1, or 2; and

[0509] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene and cyclic moiety is independently optionally substituted.

[0510] In one embodiment, the compound is a compound in Table 01-1, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0511] Table 01-1.

[0512]

[0513] Lipid Series 02

[0514] In one embodiment, provided herein is a compound of formula (02-1):

[0515]

[0516] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0517] G 1 and G 2 Each independently is C 2 -C 12 Alkylene or C 2 -C 12 Alkenylene, wherein the G 1 and G 2 One or more -CH 2 - is optionally substituted by -O-, -C(=O)O- or OC(=O)-;

[0518] Each L 1 Independently -OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b Rc 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c ),-NR a P(=O)(OR b )(OR c );

[0519] Each L 2 Independently -OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f ),-NR d P(=O)(OR e )(OR f );

[0520] R 1 and R 2 Each is independently C 6 -C 24 Alkyl or C 6 -C 24 alkenyl;

[0521] R a , R b , R d and R e Each independently is H, C 1 -C 24 Alkyl or C 2 -C 24 alkenyl;

[0522] R c and R f Each independently is C 1 -C 24 Alkyl or C 2 -C 24 alkenyl;

[0523] G 3 C 2 -C 12 Alkylene or C 2 -C 12 Alkenylene, wherein part or all of the alkylene or alkenylene is replaced by C 3 -C 8 Cycloalkylene or C 3 -C 8 The cycloalkenylene group is optionally substituted.

[0524] R 3 Yes-N(R 4 )R 5 、-OR 6 or -SR 6 ;

[0525] R 4 It is C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl, C 6 -C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0526] R 5 H, C 1 -C 12 Alkyl, C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl, C 6 -C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0527] R6为 Hydrogen, C 1 -C 12 Alkyl, C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl or C 6 -C 10 Aryl;

[0528] x is 0, 1, or 2; and

[0529] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene and cycloalkenylene is independently optionally substituted.

[0530] In one embodiment, the compound is a compound in Table 02-1, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0531] Table 02-1.

[0532]

[0533]

[0534] Lipid Series 03

[0535] In one embodiment, the cationic lipid contained in the particle or composition provided herein is a cationic lipid described in International Patent Publication No. WO2022152109, which is incorporated herein by reference in its entirety.

[0536] In one embodiment, provided herein is a compound of formula (03-I):

[0537]

[0538] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0539] G 1 and G 2 Each independently is a bond, C 2 -C 12 Alkylene or C 2 -C 12 Alkenylene, wherein the G 1 and G 2 One or more -CH 2 - is optionally replaced by -O-;

[0540] Each L 1 Independently -OC(=O)R 1 、-C(=O)OR 1、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c ),-NR a P(=O)(OR b )(OR c )、-(C 6 -C 10 Arylene)-R 1 , -(6- to 10-membered heteroarylene)-R 1 ,-(4- to 8-membered heterocyclylene)-R 1 or R 1 ;

[0541] Each L 2 Independently -OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR dC(=O)NR e R f 、-OC(=O)NR e R f ,-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f ),-NR d P(=O)(OR e )(OR f )、-(C 6 -C 10 Arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 , -(4 to 8-membered heterocyclylene)-R 2 or R 2 ;

[0542] R 1 and R 2 Each independently is C 6 -C 24 Alkyl or C 6 -C 24 alkenyl;

[0543] R a , R b , R d and R e Each independently is H, C 1 -C 24 Alkyl or C 2 -C 24 alkenyl;

[0544] R c and R f Each independently is C 1 -C 24 Alkyl or C 2 -C 24 alkenyl;

[0545] G 3 C 2 -C 12 Alkylene or C 2 -C 12 Alkenylene, wherein part or all of the alkylene or alkenylene group is optionally replaced by C 3 -C 8 Cycloalkylene, C 3 -C8 Cycloalkenylene, C 3 -C 8 Cycloalkynylene, 4- to 8-membered heterocyclylene, C 6 -C 10 arylene or 5- to 10-membered heteroarylene;

[0546] R 3 For hydrogen, C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 2 -C 12 Alkynyl, C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl, C 3 -C 8 Cycloalkynyl, 4 to 8 membered heterocyclic group, C 6 -C 10 aryl or 5- to 10-membered heteroaryl; or R 3 , G 1 or G 1 together with the nitrogen to which they are attached, form a cyclic moiety; or R 3 , G 3 or G 3 A portion of together with the nitrogen to which they are attached forms a ring portion;

[0547] R 4 It is C 1 -C 12 Alkyl or C 3 -C 8 Cycloalkyl;

[0548] x is 0, 1, or 2;

[0549] n is 1 or 2;

[0550] m is 1 or 2; and

[0551] wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0552] In one embodiment, the compound is a compound in Table 03-1, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0553] Table 03-1.

[0554]

[0555]

[0556] Lipid Series 04

[0557] In one embodiment, the cationic lipid contained in the particle or composition provided herein is a cationic lipid described in International Patent Publication No. 2022247755, which is herein incorporated by reference in its entirety.

[0558] In one embodiment, provided herein is a compound of formula (04-I):

[0559]

[0560] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0561] G 1 and G 2 Each independently is a bond, C 2 -C 12 Alkylene or C 2 -C 12 alkenylene;

[0562] L 1 Yes-OC(=O)R 1 、-C(=O)OR 1 、-OC(=O)OR 1 、-C(=O)R 1 、-OR 1 、-S(O) x R 1 、-S-SR 1 、-C(=O)SR 1 、-SC(=O)R 1 、-NR a C(=O)R 1 、-C(=O)NR b R c 、-NR a C(=O)NR b R c 、-OC(=O)NR b R c 、-NR a C(=O)OR 1 、-SC(=S)R 1 、-C(=S)SR 1 、-C(=S)R 1 、-CH(OH)R 1 、-P(=O)(OR b )(OR c )、-(C6 -C 10 Arylene)-R 1 , -(6- to 10-membered heteroarylene)-R 1 or R 1 ;

[0563] L 2 Yes-OC(=O)R 2 、-C(=O)OR 2 、-OC(=O)OR 2 、-C(=O)R 2 、-OR 2 、-S(O) x R 2 、-S-SR 2 、-C(=O)SR 2 、-SC(=O)R 2 、-NR d C(=O)R 2 、-C(=O)NR e R f 、-NR d C(=O)NR e R f 、-OC(=O)NR e R f 、-NR d C(=O)OR 2 、-SC(=S)R 2 、-C(=S)SR 2 、-C(=S)R 2 、-CH(OH)R 2 、-P(=O)(OR e )(OR f )、-(C 6 -C 10 Arylene)-R 2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0564] R 1 and R 2 Each independently is C 5 -C 32 Alkyl or 5 -C 32 alkenyl;

[0565] R a , R b , R d and R e Each independently is H, C 1 -C 24 Alkyl or C2 -C 24烯基 ;

[0566] R c and R f Each independently is C 1 -C 32 Alkyl or C 2 -C 32 alkenyl;

[0567] R 0 It is C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl, C 6 -C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0568] G 3 It is C 2 -C 12 Alkylene or C 2 -C 12 alkenylene;

[0569] R 4 C 1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl, C 6 -C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0570] R 5 It is C 1 -C 12 Alkyl, C 3 -C 8 Cycloalkyl, C 3 -C 8 Cycloalkenyl, C 6 -C 10 Aryl or 4- to 8-membered heterocycloalkyl;

[0571] x is 0, 1, or 2;

[0572] s is 0 or 1; and

[0573] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene and heteroarylene is independently optionally substituted.

[0574] In one embodiment, the compound is a compound in Table 04-1, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0575] Table 04-1.

[0576]

[0577] It should be understood that any embodiment of the compound provided herein as stated above, and any specific substituent and / or variable of the compound provided herein as stated above can be independently combined with other embodiments and / or substituents and / or variables of the compound to form the above embodiments not specifically stated. In addition, in the case of listing the substituent and / or variable list of any specific group or variable, it should be understood that each individual substituent and / or variable can be deleted from a specific embodiment and / or technical solution, and the remaining substituent and / or variable list will be considered to be within the scope of the embodiment provided herein.

[0578] It is understood that in the present specification, combinations of substituents and / or variables in the various formulae depicted are permissible only if such contributions result in stable compounds.

[0579] Other ionizable lipids

[0580] As described herein, in some embodiments, in addition to compounds according to the 01, 02, 03 and 04 series, such as lipids according to formula (01-I), (01-II), (02-I), (03-I) and (04-I) (and subformulae thereof), the nanoparticle compositions provided herein further comprise one or more charged or ionizable lipids. Without being bound by theory, it is expected that certain charged or zwitterionic lipid components of the nanoparticle compositions are similar to lipid components in cell membranes, thereby improving cellular uptake of the nanoparticles. Exemplary charged or ionizable lipids that can form part of the nanoparticle compositions of the present invention include, but are not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-trioctadecane (KL25), 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid triheptadecanoyl-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DO DMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLin DMA(2R)), (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z-,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)), (12Z,15Z)-N,N-dimethyl-2-nonylheneicosadeca-12,15-dien-1-amine, N,N-dimethyl-1-{(1S,2R)-2-octylcyclopropyl}heptadecan-8-amine.Additional exemplary charged or ionizable lipids that can form part of the nanoparticle compositions of the invention include lipids (e.g., Lipid 5) described in Sabnis et al., “A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates”, Molecular Therapy, Vol. 26, No. 6, 2018, the entirety of which is incorporated herein by reference.

[0581] In some embodiments, suitable cationic lipids include N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1); N1-[2-((1S)-1-[(3-aminopropyl)amino]-4- [Bis(3-amino-propyl)amino]butylformamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); dioctadecylamido-glycyl tetramine (DOGS); 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol); dioctadecyldimethylammonium bromide (DDAB); SAINT-2,N-methyl-4-(dioleyl)methylpyridinium; 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI); dialkylated amino acids (DILA 2)(e.g., C18:1-norArg-C16); dioleyldimethylammonium chloride (DODAC); 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (MOEPC); (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl); (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G); and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-amineium chloride (DOTAPen). Cationic lipids with a charged head group at physiological pH are also suitable, such as primary amines (e.g., DODAG N', N'-dioctadecyl-N-4,8-diaza-10-aminodecanoyl glycine amide) and guanidinium head groups (e.g., bis-guanidinium-sperm triamine-cholesterol (BGSC), bis-guanidinium-tren-cholesterol (BGTC), PONA, and (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G)). Another suitable cationic lipid is (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-Cl). In certain embodiments, the cationic lipid is a specific enantiomer or racemic form, and includes various salt forms (e.g., chlorides or sulfates) of the above cationic lipids. For example, in some embodiments, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP-Cl) or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium sulfate (DOTAP-sulfate). In some embodiments, the cationic lipid is an ionizable cationic lipid, such as dioctadecyl dimethyl ammonium bromide (DDAB); 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); 4-(dimethylamino)butyric acid triheptadecanoyl-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA); 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP); 1,2-dioleyloxy-3-dimethylaminopropane (DODMA); and N-morpholinyl cholesterol (Mo-CHOL). In certain embodiments, the lipid nanoparticle comprises a combination of two or more cationic lipids (e.g., two or more of the above cationic lipids).

[0582] In addition, in some embodiments, the charged or ionizable lipids that may form part of the nanoparticle compositions of the present invention are lipids comprising cyclic amine groups. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330, and WO2015011633, the entire contents of each patent being incorporated herein by reference in their entirety. In addition, in some embodiments, the charged or ionizable lipids that may form part of the nanoparticle compositions of the present invention are lipids comprising cyclic amine groups. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330, and WO2015011633, the entire contents of each patent being incorporated herein by reference in their entirety.

[0583] Polymer-conjugated lipids

[0584] In some embodiments, the lipid component of the nanoparticle composition can include one or more polymer-conjugated lipids, such as PEGylated lipids (PEG lipids). Without being bound by theory, it is expected that the polymer-conjugated lipid component in the nanoparticle composition can improve colloidal stability and / or reduce the protein absorption of nanoparticles. Exemplary cationic lipids that can be used in conjunction with the present disclosure include but are not limited to PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof. For example, PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000 or Chol-PEG2000.

[0585] In one embodiment, the polymer-conjugated lipid is a pegylated lipid. For example, some embodiments include pegylated diacylglycerol (PEG-DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), pegylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerol (PEG-S-DAG) (such as 4-O-(2',3'-di-tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), pegylated ceramide (PEG-cer) or PEG dialkoxypropylcarbamate (such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di-tetradecanoyl)propyl)carbamate or 2,3-di-tetradecanoyl)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate).

[0586] In one embodiment, the polymer-conjugated lipid is present at a concentration in the range of 1.0 to 2.5 mol %. In one embodiment, the polymer-conjugated lipid is present at a concentration of about 1.7 mol %. In one embodiment, the polymer-conjugated lipid is present at a concentration of about 1.5 mol %.

[0587] In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is in the range of about 35: 1 to about 25: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is in the range of about 100: 1 to about 20: 1.

[0588] In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is in the range of about 35: 1 to about 25: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is in the range of about 100: 1 to about 20: 1.

[0589] In one embodiment, the PEGylated lipid has the formula:

[0590]

[0591] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

[0592] R 12 and R 13 Each is independently a linear or branched saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and

[0593] The average value of w ranges from 30 to 60. In one embodiment, R12 and R13 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, the average value of w ranges from 42 to 55, for example, the average value of w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some specific embodiments, the average value of w is about 49.

[0594] In one embodiment, the PEGylated lipid has the formula:

[0595]

[0596] The average value of w is about 49.

[0597] Lipid Series 05

[0598] In one embodiment, provided herein is a compound of formula (05-I):

[0599]

[0600] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0601] L is lipid;

[0602] X is the connector;

[0603] Each R 3 independently H or C 1 -C 6 alkyl;

[0604] Each Y 1 is independently a bond, O, S or NR a ;

[0605] Each G 4 independently a bond or C 1 -C 12 Alkylene, one or more -CH 2 - optionally independently through -O-, -S- or -NR a - Substitution;

[0606] Each G 5 independently a bond or C 1 -C 12 Alkylene, one or more -CH 2 - optionally independently through -O-, -S- or -NR a - Substitution;

[0607] Each R a Independently H, C 1 -C 12 Alkyl or C 2 -C 12 alkenyl;

[0608] Z 1 and Z 2 One of them is positively charged, and Z 1 and Z 2 The other part is the negatively charged part;

[0609] n is an integer from 2 to 100;

[0610] T is hydrogen, halogen, alkyl, alkenyl, -OR", -SR", -COOR", -OCOR", -NR"R", -N + (R”) 3 , -P + (R”) 3, -SC(=S)-SR", -SC(=S)-OR", -SC(=S)-NR"R", -SC(=S)-aryl, cyano, azido, aryl, heteroaryl or targeting group, wherein R" is independently hydrogen or alkyl at each occurrence; and wherein each alkyl, alkenyl, alkylene, aryl and heteroaryl is independently optionally substituted.

[0611] Structural lipids

[0612] In some embodiments, the lipid component of nanoparticle composition can include one or more structural lipids. Not bound by theory, it is expected that structural lipids can stabilize the amphipathic structure of nanoparticles, such as but not limited to the lipid bilayer structure of nanoparticles. Exemplary structural lipids that can be used in conjunction with the present disclosure include but are not limited to cholesterol, fecal sterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, tomatine, ursolic acid, alpha-tocopherol and mixtures thereof. In certain embodiments, structural lipid is cholesterol. In some embodiments, structural lipid includes cholesterol and corticosteroid (such as prednisolone, dexamethasone, prednisone and hydrocortisone) or their combination.

[0613] In one embodiment, the lipid nanoparticle provided herein comprises a steroid or a steroid analog. In one embodiment, the steroid or the steroid analog is cholesterol. In one embodiment, the steroid exists in a concentration within the range of 39 to 49 mol %, 40 to 46 mol %, 40 to 44 mol %, 40 to 42 mol %, 42 to 44 mol % or 44 to 46 mol %. In one embodiment, the steroid exists in a concentration of 40, 41, 42, 43, 44, 45 or 46 mol %.

[0614] In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol is in the range of about 5:1 to 1:1. In one embodiment, the steroid is present at a concentration in the range of 32 to 40 mole % steroid.

[0615] In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol is in the range of about 5:1 to 1:1. In one embodiment, the steroid is present at a concentration in the range of 32 to 40 mole % steroid.

[0616] Phospholipids

[0617] In some embodiments, the lipid component of the nanoparticle composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Without being bound by theory, it is expected that the phospholipids can assemble into one or more lipid bilayer structures. Exemplary phospholipids that can form part of the nanoparticle compositions of the present invention include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-arachidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racem-(1-glycerol) sodium salt (DOPG) and sphingomyelin. In certain embodiments, the nanoparticle composition includes DSPC. In certain embodiments, the nanoparticle composition includes DOPE. In some embodiments, the nanoparticle composition includes both DSPC and DOPE.

[0618] Additional exemplary neutral lipids include, for example, dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

[0619] In one embodiment, the neutral lipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG).

[0620] In addition, phospholipids that may form part of the nanoparticle compositions of the present invention also include those described in WO2017 / 112865, the entire contents of which are incorporated herein by reference in their entirety.

[0621] preparation

[0622] According to the present disclosure, the nanoparticle compositions described herein may include at least one lipid component and one or more additional components, such as therapeutic and / or prophylactic agents (e.g., therapeutic nucleic acids described herein). Nanoparticle compositions may be designed for one or more specific applications or targets. The ingredients of the nanoparticle compositions may be selected based on a specific application or target, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more ingredients. Similarly, the specific formulation of the nanoparticle composition may be selected for a specific application or target, based on, for example, the efficacy and toxicity of a specific combination of each ingredient.

[0623] The nanoparticle composition is an aqueous formulation or a frozen formulation thereof (e.g., an aqueous formulation stored at about -20°C or lower, such as about -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, or -80°C).

[0624] The lipid component of the nanoparticle composition may include, for example, lipids according to one of series 01, 02, 03 and 04, for example compounds according to one of formulas (01-I), (01-II), (02-I), (03-I) and (04-I) (and subformulas thereof) described herein, phospholipids (such as unsaturated lipids, e.g., DOPE or DSPC), PEG lipids and structural lipids. The ingredients of the lipid component may be provided in specific fractions.

[0625] In one embodiment, provided herein are nanoparticle compositions comprising cationic or ionizable lipid compounds, therapeutic agents, and one or more excipients as provided herein. In one embodiment, cationic or ionizable lipid compounds comprise compounds according to series 01, 02, 03, and 04 as described herein, for example compounds according to one of formulas (01-I), (01-II), (02-I), (03-I), and (04-I) (and subformulas thereof), and optionally one or more other ionizable lipid compounds. In one embodiment, one or more excipients are selected from neutral lipids, steroids, and polymer-conjugated lipids as described herein. In one embodiment, the therapeutic agent is encapsulated in or associated with the lipid nanoparticles.

[0626] In one embodiment, the present invention provides a nanoparticle composition (lipid nanoparticle) comprising:

[0627] i) a cationic lipid (ie, a first lipid);

[0628] ii) neutral lipids (e.g. phospholipids);

[0629] iii) steroids;

[0630] iv) a polymer-conjugated lipid (ie, a second lipid); and

[0631] v) Therapeutic agents.

[0632] As used herein, "mol %" refers to the mole % of a component relative to the total moles of all lipid components in the LNP (ie, the total moles of cationic lipids, neutral lipids, steroids, and polymer-conjugated lipids).

[0633] In one embodiment, the lipid nanoparticle comprises about 20 to about 65 mol%, about 30 to about 60 mol%, about 40 to about 55 mol%, 45 to 50 mol%, 50 to 55 mol%, 55 to 60 mol%, 60 to 65 mol%, 40 to 45 mol%, 35 to 40 mol%, 30 to 35 mol%, 25 to 30 mol% or 20 to 25 mol% of cationic lipid. In one embodiment, the lipid nanoparticle comprises about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 mol% of cationic lipid. In one embodiment, the lipid nanoparticle comprises about 47.5, 47.6, 47.7, 47.8, 47.9, 48.0, 48.1, 48.2, 48.3, 48.4 or 48.5 mol% of cationic lipid. In one embodiment, the lipid nanoparticles comprise about 48.0 mol% cationic lipids.

[0634] In one embodiment, the neutral lipid (e.g., phospholipid) is present at a concentration of about 5 to about 40 mol%, 6 to 30 mol%, about 7 to about 20 mol%, or 8 to 15 mol%. In one embodiment, the neutral lipid (e.g., phospholipid) is present at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol%. In one embodiment, the neutral lipid (e.g., phospholipid) is present at a concentration of about 9.5, 10, or 10.5 mol%. In one embodiment, the lipid nanoparticle comprises about 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5 mol% of phospholipid. In one embodiment, the lipid nanoparticle comprises about 10.0 mol% of phospholipid. In one embodiment, the molar ratio of the cationic lipid to the neutral lipid (e.g., phospholipid) ranges from about 13: 1 to about 1: 2, from about 10: 1 to about 5: 1, or from about 7.5: 1 to 5: 1. In one embodiment, the molar ratio of the cationic lipid to the neutral lipid (e.g., phospholipid) is 7.5: 1, 7.25: 1, 7.0: 1, 6.75: 1, 6.5: 1, 6.25: 1, 6.0: 1, 5.75: 1, 5.5: 1, 5.25: 1, or 5.0: 1. In one embodiment, the molar ratio of the cationic lipid to the neutral lipid (e.g., phospholipid) ranges from about 4.1: 1.0 to about 4.9: 1.0, about 4.5: 1.0 to about 4.8: 1.0, or about 4.7: 1.0 to 4.8: 1.0.

[0635] In one embodiment, steroid is present in a concentration within the range of about 20 to about 50 mol%, about 30 to about 45 mol%, about 35 to about 43 mol%, 37 to 42 mol%, 30 to 35 mol%, 35 to 40 mol%, 40 to 45 mol% or 45 to 50 mol%. In one embodiment, steroid is present in a concentration of 35, 36, 37, 48, 39, 40, 41, 42, 43, 44 or 45 mol%. In one embodiment, the molar ratio of cationic lipid to steroid is within the range of 1.0:0.9 to 1.0:1.2 or 1.0:1.0 to 1.0:1.2. In one embodiment, lipid nanoparticles include about 40.0, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9 or 41.0 mol% of steroid. In one embodiment, the lipid nanoparticles comprise about 40.5 mol% steroid.

[0636] In one embodiment, the polymer-conjugated lipid is present in a concentration ranging from about 0.1 to about 10 mol %, about 0.2 to about 7.5 mol %, 0.3 to 5 mol %, 0.4 to 2.5 mol %, 0.5 to 2 mol % or 1 to 2 mol %. In one embodiment, the polymer-conjugated lipid is present in a concentration of 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25 or 2.5 mol %. In one embodiment, the lipid nanoparticle comprises about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mol % of polymer-conjugated lipid. In one embodiment, the lipid nanoparticle comprises about 1.5 mol % of polymer-conjugated lipid.

[0637] In one embodiment, the ratio of therapeutic agent to lipid in the LNP (i.e., N / P, N represents the number of moles of cationic lipid and P represents the number of moles of phosphate present as part of the nucleic acid backbone) ranges from 2: 1 to 30: 1, such as 3: 1 to 22: 1. In one embodiment, the N / P range is from 6: 1 to 20: 1 or 2: 1 to 12: 1. Exemplary N / P ranges include about 3: 1, about 6: 1, about 12: 1, and about 22: 1.

[0638] In one embodiment, provided herein is a lipid nanoparticle comprising:

[0639] i) a cationic lipid having an effective pKa greater than 6.0 (ie, a first lipid);

[0640] ii) 5 to 15 mol% neutral lipids (e.g., phospholipids);

[0641] iii) 1 to 15 mol % of anionic lipids;

[0642] iv) 30 to 45 mol % of a steroid;

[0643] v) 0.5 to 3 mol% of a polymer-conjugated lipid (ie, a second lipid); and

[0644] vi) a therapeutic agent or a pharmaceutically acceptable salt or prodrug thereof,

[0645] where mole % is determined based on the total moles of lipid present in the lipid nanoparticles.

[0646] In one embodiment, the cationic lipid can be any of a variety of lipid species with a net positive charge at a selected pH (such as physiological pH). Exemplary cationic lipids are described below. In one embodiment, the pKa of the cationic lipid is greater than 6.25. In one embodiment, the pKa of the cationic lipid is greater than 6.5. In one embodiment, the pKa of the cationic lipid is greater than 6.1, greater than 6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than 6.45, greater than 6.55, greater than 6.6, greater than 6.65 or greater than 6.7.

[0647] In one embodiment, the lipid nanoparticle comprises 40 to 45 mol% of cationic lipid. In one embodiment, the lipid nanoparticle comprises 45 to 50 mol% of cationic lipid.

[0648] In one embodiment, the molar ratio of cationic lipid to neutral lipid ranges from about 2: 1 to about 8: 1. In one embodiment, the lipid nanoparticle comprises 5 to 10 mol% neutral lipid.

[0649] Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), or 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG).

[0650] In one embodiment, the lipid nanoparticle comprises 1 to 10 mol% of anionic lipids. In one embodiment, the lipid nanoparticle comprises 1 to 5 mol% of anionic lipids. In one embodiment, the lipid nanoparticle comprises 1 to 9 mol%, 1 to 8 mol%, 1 to 7 mol% or 1 to 6 mol% of anionic lipids. In one embodiment, the molar ratio of anionic lipids to neutral lipids is in the range of 1:1 to 1:10.

[0651] In one embodiment, the steroid is cholesterol. In one embodiment, the molar ratio of cationic lipid to cholesterol is in the range of about 5: 1 to 1: 1. In one embodiment, the lipid nanoparticles comprise a steroid in the range of 32 to 40 mol% steroid.

[0652] In one embodiment, the sum of the mole % of neutral lipids and the mole % of anionic lipids is in the range of 5 to 15 mole %. In one embodiment, the sum of the mole % of neutral lipids and the mole % of anionic lipids is in the range of 7 to 12 mole %.

[0653] In one embodiment, the molar ratio of anionic lipid to neutral lipid is in the range of 1 : 1 to 1 : 10. In one embodiment, the sum of the mole % of neutral lipid and the mole % of steroid is in the range of 35 to 45 mole %.

[0654] In one embodiment, the lipid nanoparticle comprises:

[0655] i) about 20 to about 65 mol % of a cationic lipid (ie, a first lipid); ii) about 5 to about 40 mol % of a phospholipid; iii) about 20 to about 50 mol % of a steroid; and iv) a polymer-conjugated lipid (ie, a second lipid).

[0656] In one embodiment, the lipid nanoparticle comprises:

[0657] i) about 40 to about 55 mol % of a cationic lipid (i.e., a first lipid); ii) about 5 to about 15 mol % of a phospholipid; iii) about 35 to about 50 mol % of a steroid; and iv) about 2 to about 10 mol % of a polymer-conjugated lipid (i.e., a second lipid).

[0658] In one embodiment, the lipid nanoparticle comprises:

[0659] i) about 45 to about 55 mol % of a cationic lipid (i.e., a first lipid); ii) about 6 to about 10 mol % of a phospholipid; iii) about 40 to about 48 mol % of a steroid; and iv) about 1 to about 2.5 mol % of a polymer-conjugated lipid (i.e., a second lipid).

[0660] In one embodiment, the lipid nanoparticle comprises 1.0 to 2.5 mol% polymer-conjugated lipid.

[0661] In one embodiment, the neutral lipid is present at a concentration in the range of 5 to 15 mol%, 7 to 13 mol%, or 9 to 11 mol%. In one embodiment, the neutral lipid is present at a concentration of about 9.5, 10 or 10.5 mol%. In one embodiment, the molar ratio of cationic lipid to neutral lipid ranges from about 4.1: 1.0 to about 4.9: 1.0, about 4.5: 1.0 to about 4.8: 1.0, or about 4.7: 1.0 to 4.8: 1.0.

[0662] In one embodiment, the steroid is cholesterol. In some embodiments, the steroid is present at a concentration within the range of 39 to 49 mol %, 40 to 46 mol %, 40 to 44 mol %, 40 to 42 mol %, 42 to 44 mol % or 44 to 46 mol %. In one embodiment, the steroid is present at a concentration of about 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5 or 46 mol %. In certain embodiments, the molar ratio of cationic lipid to steroid is within the range of 1.0:0.9 to 1.0:1.2 or 1.0:1.0 to 1.0:1.2.

[0663] In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 5:1 to 1:1.

[0664] In one embodiment, the lipid nanoparticle comprises 1.0 to 2.5 mol% of the polymer-conjugated lipid. In one embodiment, the polymer-conjugated lipid is present at a concentration of about 1.5 mol%.

[0665] In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is in the range of about 100: 1 to about 20: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is in the range of about 35: 1 to about 25: 1.

[0666] In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is in the range of about 100: 1 to about 20: 1. In one embodiment, the molar ratio of the cationic lipid to the polymer-conjugated lipid is in the range of about 35: 1 to about 25: 1.

[0667] In one embodiment, the lipid nanoparticles have an average diameter ranging from 50 nm to 100 nm, or from 60 nm to 85 nm.

[0668] In one embodiment, compositions comprise cationic lipid, DSPC, cholesterol, and PEG-lipid provided herein, and mRNA.In one embodiment, cationic lipid, DSPC, cholesterol, and PEG-lipid provided herein have a molar ratio of about 50:10:38.5:1.5.

[0669] Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and / or preventive agents (such as RNA) to specific cells, tissues, organs or systems or groups thereof in mammals. The physicochemical properties of nanoparticle compositions can be changed to increase selectivity for specific body targets. For example, the particle size can be adjusted according to the window size of different organs. The therapeutic and / or preventive agents contained in the nanoparticle compositions can also be selected according to the desired one or more delivery targets. For example, therapeutic and / or preventive agents can be selected for specific indications, illnesses, diseases or conditions and / or for delivery to specific cells, tissues, organs or systems or groups thereof (e.g., local or specific delivery). In certain embodiments, nanoparticle compositions can include mRNA encoding a polypeptide of interest, which can be translated intracellularly to produce a polypeptide of interest. Such compositions can be designed to be specifically delivered to a specific organ. In certain embodiments, the composition can be designed to be specifically delivered to the liver of a mammal.

[0670] The amount of therapeutic and / or preventive agents in the nanoparticle composition can depend on the size, composition, desired target and / or application of the nanoparticle composition, or other characteristics, and the characteristics of the therapeutic and / or preventive agents. For example, the amount of RNA that can be used in the nanoparticle composition can depend on the size, sequence and other characteristics of the RNA. The relative amount of therapeutic and / or preventive agents and other ingredients (such as lipids) in the nanoparticle composition can also vary. In some embodiments, the wt / wt ratio of lipid components and therapeutic and / or preventive agents in the nanoparticle composition can be about 5:1 to about 60:1, such as about 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 22:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1 and 60:1. For example, the wt / wt ratio of the lipid component to the therapeutic and / or prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0671] In some embodiments, the nanoparticle composition comprises one or more RNAs, and one or more RNAs, lipids and their amounts can be selected to provide a specific N: P ratio. The N: P ratio of the composition refers to the molar ratio of the number of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N: P ratio is selected. One or more RNAs, lipids and their amounts can be selected to provide an N: P ratio of about 2: 1 to about 30: 1, such as 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 22: 1, 24: 1, 26: 1, 28: 1 or 30: 1. In certain embodiments, the N: P ratio can be about 2: 1 to about 8: 1. In other embodiments, the N: P ratio is about 5: 1 to about 8: 1. For example, the N:P ratio can be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N:P ratio can be about 5.67:1.

[0672] The physical properties of a nanoparticle composition can depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid can have different properties than a nanoparticle composition comprising a different structural lipid. Similarly, the characteristics of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher mole fraction of phospholipids can have different characteristics than a nanoparticle composition comprising a lower mole fraction of phospholipids. Characteristics can also vary depending on the method and conditions of preparation of the nanoparticle composition.

[0673] Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of nanoparticle compositions. Zeta potential can be measured using dynamic light scattering or potentiometric methods (e.g., potentiometric titration). Dynamic light scattering can also be used to determine particle size. Instruments such as Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure multiple features of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0674] In various embodiments, the average size of the nanoparticle composition can be between tens of nanometers and hundreds of nanometers. For example, the average size can be about 40nm to about 150nm, such as about 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm or 150nm. In some embodiments, the average size of the nanoparticle composition may be from about 50nm to about 100nm, from about 50nm to about 90nm, from about 50nm to about 80nm, from about 50nm to about 70nm, from about 50nm to about 60nm, from about 60nm to about 100nm, from about 60nm to about 90nm, from about 60nm to about 80nm, from about 60nm to about 70nm, from about 70nm to about 100nm, from about 70nm to about 90nm, from about 70nm to about 80nm, from about 80nm to about 100nm, from about 80nm to about 90nm, or from about 90nm to about 100nm. In certain embodiments, the average size of the nanoparticle composition may be from about 70nm to about 100nm. In some embodiments, the average size may be about 80nm. In other embodiments, the average size may be about 100nm.

[0675] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A small polydispersity index (e.g., less than 0.3) generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

[0676] The zeta potential of a nanoparticle composition can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charge (positive or negative) are generally desirable because higher charged materials may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the nanoparticle composition can be between about -10 mV and about +20 mV, about -10 mV and about +15 mV, about -10 mV and about +10 mV, about -10 mV and about +5 mV, about -10 mV and about 0 mV, about -10 mV and about -5 mV, about -5 mV and about +20 mV, about -5 mV and about +15 mV, about -5 mV and about +10 mV, about -5 mV and about +5 mV, about -5 mV and about 0 mV, about 0 mV and about +20 mV, about 0 mV and about +15 mV, about 0 mV and about +10 mV, about 0 mV and about +5 mV, about +5 mV and about +20 mV, about +5 mV and about +15 mV, or about +5 mV and about +10 mV.

[0677] The encapsulation efficiency of therapeutic and / or prophylactic agents describes the amount of therapeutic and / or prophylactic agents encapsulated or otherwise associated with nanoparticle compositions after preparation relative to the initial amount provided. It is desirable that the encapsulation efficiency is high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agents in a solution containing nanoparticle compositions before and after the nanoparticle compositions are destroyed with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agents (e.g., RNA) in a solution. For nanoparticle compositions described herein, the encapsulation efficiency of therapeutic and / or prophylactic agents can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0678] The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated as a tablet having a capsule, film, or coating. The capsule, film, or tablet containing the composition described herein may have any useful size, tensile strength, hardness, or density.

[0679] V. Uses

[0680] The terms "disease" or "disorder" are used interchangeably herein and refer to any change in the state of the body or certain organs that interrupts or interferes with the performance of functions and / or causes symptoms such as discomfort, dysfunction, suffering or even death in the patient or those who come into contact with the patient. A disease or disorder may also be associated with a distemper, infirmity, ailment, chronic illness, disease, ailment, discomfort, ailment or illness.

[0681] The term "effective amount" as used herein refers to an amount of a therapeutic agent (e.g., modified RNA) or pharmaceutical composition sufficient to alleviate at least one or more symptoms of a disease or disorder or provide a desired effect. For example, it can be an amount that induces a significant reduction in the treatment of symptoms or clinical markers associated with wound healing.

[0682] The term "delivery" means providing an entity to a destination. For example, delivering a therapeutic agent to a subject may involve administering to the subject a pharmaceutical composition comprising at least one nanoparticle containing a modified RNA (e.g., via an intradermal route). Administering a pharmaceutical composition comprising at least one nanoparticle to a mammalian tissue or subject may involve contacting one or more cells with the pharmaceutical composition.

[0683] The disclosed methods of enhancing a subject's immune response to an antigen of interest can be used in a variety of clinical or therapeutic applications.

[0684] Therefore, in one aspect, the present invention provides a method for producing EGFR mutant-specific immune effector cells, which comprises providing a starting population of immune effector cells, and contacting the starting population of immune effector cells with a polypeptide as described above, a nucleic acid as described above, or a composition as described above under suitable conditions, wherein upon contact, EGFR mutant-specific immune effector cells are produced.

[0685] Thus, in one aspect, provided herein is a method for forming a pro-inflammatory environment in tissue surrounding a population of diseased cells expressing mutant EGFR, comprising contacting the tissue with an effective amount of a polypeptide as described above, a nucleic acid as described above, or a composition as described above under suitable conditions.

[0686] Therefore, in one aspect, the present invention provides a method for inducing an immune response against a target cell expressing a mutant EGFR in a subject, wherein the method comprises administering a polypeptide as described above, a nucleic acid as described above, or a composition as described above to the subject. In a specific embodiment, a mutant EGFR antigen mRNA construct is administered to the subject, and the mutant EGFR antigen mRNA construct encodes a sequence shown in any one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33, or comprises a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33.

[0687] Therefore, in one aspect, the present invention provides a method for eliminating diseased cells expressing mutant EGFR in a subject, comprising administering an effective amount of a polypeptide as described above, a nucleic acid as described above, or a composition as described above. In a specific embodiment, a mutant EGFR antigen mRNA construct is administered to a subject, wherein the mutant EGFR antigen mRNA construct encodes a sequence shown in any one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33, or comprises a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33.

[0688] Therefore, in one aspect, the present invention provides a method for treating a cancer expressing a mutant EGFR in a subject or delaying the progression of the cancer, wherein the method comprises administering a polypeptide as described above, a nucleic acid as described above, or a composition as described above to the subject. In a specific embodiment, a mutant EGFR antigen mRNA construct is administered to the subject, and the mutant EGFR antigen mRNA construct encodes a sequence shown in any one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33, or comprises a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33.

[0689] For example, the method can be used to stimulate anti-tumor immunity in a subject with a tumor. Therefore, in one aspect, the present disclosure relates to a method for stimulating an immunogenic response to a tumor in a subject in need thereof, the method comprising administering to the subject a composition of the present disclosure (or its lipid nanoparticle or its pharmaceutical composition), the composition comprising at least one mRNA construct, the encoding mRNA construct: (i) at least one tumor antigen of interest, and (ii) a polypeptide that enhances the immune response to the tumor antigen of interest, so that the immune response to the tumor antigen of interest is enhanced. Suitable tumor antigens of interest include those described herein (e.g., tumor neoantigens, including mutated EGFR antigens). In certain embodiments, a mutant EGFR antigen mRNA construct is administered to a subject, wherein the mutant EGFR antigen mRNA construct encodes a sequence shown in any one of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33, or comprises a nucleotide sequence that is at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the sequence of SEQ ID NOs: 9, 12, 15, 18, 21, 24, 27, 30, and 33.

[0690] In one aspect, the present disclosure relates to the use of a polypeptide as described above, a nucleic acid as described above, or a composition as described above in the manufacture of a medicament or a kit for treating a cancer expressing mutant EGFR in a subject or delaying the progression of the cancer.

[0691] The present disclosure also provides a method for treating or preventing cancer in a subject in need thereof, the method comprising providing or administering to the subject at least one mRNA composition described herein (i.e., an immunopotentiator mRNA and an antigen encoding mRNA in the same or separate mRNA construct). In a related embodiment, a nanoparticle (e.g., a lipid nanoparticle) comprising mRNA is provided or administered to the subject. In a further related embodiment, a pharmaceutical composition of the present disclosure is provided or administered to the subject. In a specific embodiment, the pharmaceutical composition comprises mRNA encoding an antigen as described herein and an immunostimulatory polypeptide, or it comprises a nanoparticle containing mRNA. In a specific embodiment, mRNA is present in a nanoparticle (e.g., a lipid nanoparticle). In a specific embodiment, mRNA or nanoparticles are present in a pharmaceutical composition.

[0692] In certain embodiments, the subject in need has been diagnosed with cancer, or is considered to have a risk of cancer. In some embodiments, cancer is liver cancer, colorectal cancer, melanoma cancer, pancreatic cancer, NSCLC, cervical cancer or head and neck cancer. In specific embodiments, liver cancer is hepatocellular carcinoma. In some embodiments, colorectal cancer is a primary tumor or a metastatic tumor. In some embodiments, cancer is a hematopoietic cancer. In some embodiments, cancer is acute myeloid leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, bone marrow dystrophy syndrome (including refractory anemia and refractory cytopenia) or myeloproliferative tumors or diseases (including polycythemia vera, essential thrombocythemia and primary myelofibrosis). In other embodiments, cancer is blood-based cancer or hematopoietic cancer. The selectivity of specific cancer types can be achieved by combining appropriate LNP preparations (e.g., targeting specific cell types) with appropriate regulatory sites (e.g., microRNA) engineered into mRNA constructs.

[0693] In some embodiments, the mRNA, nanoparticle or pharmaceutical composition is administered to a patient parenterally. In certain embodiments, the subject is a mammal, such as a human. In various embodiments, an effective amount of mRNA is provided to the subject.

[0694] The method for treating cancer can also include treating the subject with an additional agent (e.g., chemotherapeutic agent) that enhances the anti-tumor response of the subject and / or has cytotoxicity to the tumor. The therapeutic agent suitable for combination therapy includes small molecule chemotherapeutic agents, including protein tyrosine kinase inhibitors, such as afatinib, apatinib, axitinib, ceritinib, crizotinib, dacomitinib, erlotinib, gefitinib, imatinib, osimertinib, pelitinib, vatalanib and vandetanib, and biological anticancer agents, such as anticancer antibodies, including but not limited to those discussed further below. Combination therapy can include administering immune checkpoint inhibitors to the subject to enhance anti-tumor immunity, such as PD-1 inhibitors, PD-L1 inhibitors and CTLA-4 inhibitors. Other regulators of immune checkpoints can target OX-40, OX-40L or ICOS. In one embodiment, the agent regulating immune checkpoints is an antibody. In another embodiment, the agent regulating immune checkpoints is a protein or small molecule regulator. In another embodiment, the antibody regulator of the agent (such as mRNA) encoding immune checkpoints. Non-limiting examples of immune checkpoint inhibitors that can be used in combination therapy include pembrolizumab, alemtuzumab, nivolumab, pidilizumab, ofatumumab, rituximab, MEDI0680 and PDR001, AMP-224, PF-06801591, BGB-A317, REGN2810, SHR-1210, TSR-042, affimer, avelumab (MSB0010718C), atezolizumab (MPDL3280A), durvalumab (MEDI4736), BMS936559, ipilimumab, tremelimumab, AGEN1884, MEDI6469, and MOXR0916.

[0695] The pharmaceutical composition comprising one or more mRNA of the present disclosure can be applied to the subject by any suitable route. In some embodiments, the composition of the present disclosure is applied by one or more of a variety of routes, including parenteral (e.g., subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection, and any suitable infusion technique), oral, transdermal or intradermal, interdermal, rectal, intravaginal, local (e.g., by powder, ointment, cream, gel, lotion and / or drops), mucosa, nasal cavity, oral cavity, intestinal, vitreous, tumor, sublingual, intranasal; by intratracheal instillation, bronchial instillation and / or inhalation; as oral spray and / or powder, nasal spray and / or aerosol, and / or by portal vein catheter. In some embodiments, the composition can be applied by intravenous, intramuscular, intradermal, intraarterial, tumor, subcutaneous or inhalation. In some embodiments, the composition is intramuscularly administered. However, in view of the possible progress of drug delivery science, the present disclosure encompasses the delivery of the compositions of the present disclosure by any appropriate route. In general, the most suitable route of administration will depend on a variety of factors, including the properties of the pharmaceutical composition comprising one or more mRNAs (e.g., its stability in various body environments such as blood and the gastrointestinal tract) and the patient's condition (e.g., whether the patient can tolerate a specific route of administration).

[0696] In certain embodiments, the compositions of the present disclosure may be administered at a dose sufficient to deliver about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg. In some embodiments, the present invention provides a dosage level of about 0.005mg / kg to about 5mg / kg, about 0.01mg / kg to about 5mg / kg, about 1mg / kg to about 10mg / kg, about 1mg / kg to about 5mg / kg, about 2mg / kg to about 5mg / kg, about 0.0001mg / kg to about 1mg / kg, about 0.001mg / kg to about 1mg / kg, about 0.005mg / kg to about 1mg / kg, about 0.01mg / kg to about 1mg / kg, or about 0.1mg / kg to about 1mg / kg given dosage, wherein a dosage of 1mg / kg provides 1mg of mRNA or nanoparticle per 1kg of subject body weight. In a specific embodiment, an mRNA or nanoparticle of the present disclosure at a dosage of about 0.005mg / kg to about 5mg / kg can be administered.

[0697] The same or different amount can be applied once or more times a day to obtain the desired level of mRNA expression and / or effect (e.g., therapeutic effect). The desired dose can be delivered, for example, three times a day, twice a day, once a day, once every other day, once every three days, once a week, once every two weeks, once every three weeks, or once every four weeks. In certain embodiments, the desired dose can be delivered using multiple administrations (e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations). In some embodiments, a single dose can be applied, for example, before or after surgery, or in the case of acute diseases, illnesses or diseases. For any particular patient, a specific treatment is effective, prevention is effective or other suitable dosage levels will depend on a variety of factors, including the severity and identification of the treated disease, if any; the specific ingredients used by one or more mRNAs; the patient's age, weight, general health, sex and diet; the administration time, route of administration and excretion rate of the specific pharmaceutical composition used; the duration of treatment; the medicine used in combination or in concert with the specific pharmaceutical composition used; and similar factors well known in the medical field.

[0698] The immunomodulatory therapeutic composition RNA (e.g., mRNA) and lipid nanoparticles disclosed herein can be administered by any approach that produces therapeutically effective results. These include, but are not limited to, intradermal, intramuscular, intranasal and / or subcutaneous administration. The present disclosure provides methods including administering the RNA composition and lipid nanoparticles disclosed herein to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the disease, a specific composition, its mode of administration, its mode of activity, etc. The RNA composition and lipid nanoparticles disclosed herein are typically formulated in dosage unit form for ease of administration and uniform dosage. However, it should be understood that the total daily dosage of the RNA (e.g., mRNA) composition can be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dosage level for any particular patient will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or concerted with the specific compounds; and like factors well known in the medical art.

[0699] As provided herein, an effective amount of the RNA composition or lipid nanoparticle of the present disclosure can be as low as 10 μg, for example, administered in a single dose or two 5 μg doses. In some embodiments, the effective amount is a total dose of 10 μg-300 μg. For example, the effective amount can be 10 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg or 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg or 300 μg of total dose. In some embodiments, the effective amount is a total dose of 10 μg-300 μg. In some embodiments, the effective amount is a total dose of 30 μg-100 μg or 50 μg-200 μg.

[0700] In some embodiments, the RNA (e.g., mRNA) composition and the lipid nanoparticles can be delivered in an amount sufficient to deliver 0.0001 mg / kg to 100 mg / kg, 0.001 mg / kg to 0.05 mg / kg, 0.005 mg / kg to 0.05 mg / kg, 0.001 mg / kg to 0.005 mg / kg, 0.05 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg. The dosage level of 0.5mg / kg to 30mg / kg, 0.01mg / kg to 10mg / kg, 0.1mg / kg to 10mg / kg or 1mg / kg to 25mg / kg of the subject's body weight / day, once a day or more, once a week, once a month, etc., is administered to obtain the desired treatment, diagnosis, prevention or imaging effect (see, for example, the unit dose range described in International Publication No. WO2013078199, which is hereby incorporated by reference in its entirety). The desired dosage can be delivered in a manner such as three times a day, twice a day, once a day, once every other day, once every three days, once a week, once every two weeks, once every three weeks, once every four weeks, once every 2 months, once every three months, once every 6 months, etc. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations). When multiple administrations are used, a split dosing regimen such as described herein can be used. In exemplary embodiments, the RNA (e.g., mRNA) composition can be administered at a dosage level sufficient to deliver 0.0005 mg / kg to 0.01 mg / kg, e.g., about 0.0005 mg / kg to about 0.0075 mg / kg, e.g., about 0.0005 mg / kg, about 0.001 mg / kg, about 0.002 mg / kg, about 0.003 mg / kg, about 0.004 mg / kg, or about 0.005 mg / kg.

[0701] In some embodiments, the RNA (e.g., mRNA) composition can be administered once or twice (or more) at a dose level sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg.

[0702] In some embodiments, the RNA (e.g., mRNA) composition can be administered twice (e.g., day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later), at 0.0100 mg, 0.025 mg, 0.050 mg, 0.075mg, 0.100mg, 0.125mg, 0.150mg, 0.175mg, 0.200mg, 0.225mg, 0.250mg, 0.275mg, 0.300mg, 0.325mg, 0.350mg, 0.375mg, 0.40 0mg, 0.425mg, 0.450mg, 0.475mg, 0.500mg, 0.525mg, 0.550mg, 0.575mg, 0.600mg, 0.625mg, 0.650mg, 0.675mg, 0.700mg, 0.725mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg or a total dose of 1.0 mg or sufficient to deliver 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0 The present invention also can be used for the administration of the composition of the present invention.For example, the composition of RNA (e.g., mRNA) can be administered at a dosage level of 0.375mg, 0.400mg, 0.425mg, 0.450mg, 0.475mg, 0.500mg, 0.525mg, 0.550mg, 0.575mg, 0.600mg, 0.625mg, 0.650mg, 0.675mg, 0.700mg, 0.725mg, 0.750mg, 0.775mg, 0.800mg, 0.825mg, 0.850mg, 0.875mg, 0.900mg, 0.925mg, 0.950mg, 0.975mg or a total dosage of 1.0mg. The present disclosure encompasses higher and lower dosages and administration frequencies. For example, the RNA (e.g., mRNA) composition can be administered three or four times.

[0703] In some embodiments, the RNA (e.g., mRNA) composition or lipid nanoparticles comprising the same can be administered twice (e.g., day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later) at a total dose of 0.010 mg, 0.025 mg, 0.100 mg, or 0.400 mg or a dose level sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg, or 0.400 mg.

[0704] In some embodiments, the RNA (e.g., mRNA) composition or lipid nanoparticles comprising the same in the method for vaccinating a subject is administered to the subject in an effective amount of a single dose of a nucleic acid vaccine between 10pg / kg and 400pg / kg. In some embodiments, the RNA composition or lipid nanoparticles comprising the same in the method for vaccinating a subject is administered to the subject in an effective amount of a single dose of a nucleic acid vaccine between 10pg and 400pg. In some embodiments, the RNA (e.g., mRNA) composition or lipid nanoparticles comprising the same in the method for vaccinating a subject is administered to the subject in an effective amount of a single dose of a nucleic acid vaccine between 10pg and 400pg. In some embodiments, the RNA (e.g., mRNA) composition or lipid nanoparticles comprising the same in the method for vaccinating a subject is administered to the subject in a single dose of 25-1000pg (e.g., a single dose of an mRNA encoding an antigen). In some embodiments, the RNA composition is administered to a subject in a single dose of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 pg. For example, the RNA composition can be administered to a subject in a single dose of 25-100, 25-500, 50-100, 50-500, 50-1000, 100-500, 100-1000, 250-500, 250-1000 or 500-1000 pg. In some embodiments, the RNA (e.g., mRNA) composition or lipid nanoparticles comprising the same for use in a method of vaccinating a subject is administered to the subject in two doses, which combined equal 25-1000 pg of the RNA (e.g., mRNA) composition.

[0705] The RNA (e.g., mRNA) compositions described herein or lipid nanoparticles comprising the same can be formulated into dosage forms described herein, such as intranasal, intratracheal, or injectable dosage forms (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0706] In some embodiments, the pharmaceutical composition of the present disclosure can be used in combination with another agent (e.g., another therapeutic agent, preventive agent and / or diagnostic agent). The so-called "combination" does not imply that the agent must be used simultaneously and / or formulated together for delivery, although these delivery methods are within the scope of the present disclosure. For example, one or more compositions comprising one or more different mRNAs can be used in combination. The composition can be used simultaneously, before or after one or more other required treatments or medical procedures. In general, each agent will be used according to the dosage and / or schedule determined for the agent. In some embodiments, the present disclosure encompasses the delivery of the composition of the present disclosure, or its imaging, diagnosis or prevention composition and the combination of an agent that improves its bioavailability, reduces and / or changes its metabolism, inhibits its excretion, and / or changes its distribution in vivo.

[0707] Exemplary therapeutic agents that can be administered in combination with the compositions of the present disclosure include, but are not limited to, cytotoxic therapeutic agents, chemotherapeutic agents, and other therapeutic agents. Cytotoxic agents can include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, adriamycin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, rachelmin and analogs thereof.

[0708] In any of the foregoing or related aspects, the present invention provides lipid nanoparticles and an optional pharmaceutically acceptable carrier, or a pharmaceutical composition for treating or delaying the progression of cancer in an individual, wherein the treatment comprises administering the composition in combination with a second composition, wherein the second composition comprises a check point inhibitor polypeptide and an optional pharmaceutically acceptable carrier.

[0709] In any of the foregoing or related aspects, the present invention provides a use of a lipid nanoparticle and an optional pharmaceutically acceptable carrier in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the lipid nanoparticle and an optional pharmaceutically acceptable carrier, and wherein the treatment comprises administering the medicament in combination with a composition comprising a check point inhibitor polypeptide and an optional pharmaceutically acceptable carrier.

[0710] In any of the foregoing or related aspects, the invention provides a kit comprising a container containing lipid nanoparticles and optionally a pharmaceutically acceptable carrier or pharmaceutical composition, and a package insert containing instructions for administration of the lipid nanoparticles or pharmaceutical composition for treating or delaying progression of cancer in an individual. In some aspects, the package insert further comprises instructions for administering the lipid nanoparticles or pharmaceutical composition in combination with a composition comprising a checkpoint inhibitor polypeptide and optionally a pharmaceutically acceptable carrier to treat or delay progression of cancer in an individual.

[0711] In any of the foregoing or related aspects, the invention provides a kit comprising a drug containing lipid nanoparticles and an optional pharmaceutically acceptable carrier, or a pharmaceutical composition, and a package insert comprising instructions for administering the drug alone or in combination with a composition containing a checkpoint inhibitor polypeptide and an optional pharmaceutically acceptable carrier to treat or delay the progression of cancer in an individual. In some aspects, the kit further comprises a package insert comprising instructions for administering a first drug to treat or delay the progression of cancer in an individual before, simultaneously with, or after the administration of a second drug.

[0712] In any of the foregoing or related aspects, the present invention provides lipid nanoparticles, compositions, or uses thereof, or kits comprising lipid nanoparticles or compositions described herein, wherein the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4, or a combination thereof. In some aspects, the checkpoint inhibitor polypeptide is an antibody. In some aspects, the checkpoint inhibitor polypeptide is an antibody selected from an anti-CTLA4 antibody or an antigen-binding fragment thereof that specifically binds to CTLA4, an anti-PD1 antibody or an antigen-binding fragment thereof that specifically binds to PD1, an anti-PD-L1 antibody or an antigen-binding fragment thereof that specifically binds to PD-L1, and a combination thereof. In some aspects, the checkpoint inhibitor polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-PD1 antibody, wherein the anti-PD1 antibody is pembrolizumab.

[0713] In a related aspect, the present disclosure provides a method of reducing or decreasing tumor size or inhibiting tumor growth in a subject in need thereof, comprising administering to the subject any of the foregoing or related lipid nanoparticles of the present disclosure, or any of the foregoing or related compositions of the present disclosure.

[0714] In related aspects, the present disclosure provides a method of inducing an anti-tumor response in a subject with cancer, comprising administering to the subject any of the foregoing or related lipid nanoparticles of the present disclosure, or any of the foregoing or related compositions of the present disclosure. In some aspects, the anti-tumor response comprises a T cell response. In some aspects, the T cell response comprises CD8+T cells.

[0715] In some aspects of any of the above methods or related methods, the subject has metastatic colorectal cancer.

[0716] In some aspects of any of the above methods or related methods, the subject has non-small cell lung cancer (NSCLC).

[0717] In some aspects of any of the above methods or related methods, the subject has pancreatic cancer.

[0718] In any of the foregoing or related aspects, the present disclosure provides a pharmaceutical composition comprising lipid nanoparticles and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition is formulated for intramuscular delivery.

[0719] As used herein, "cancer cell" or "tumor cell" refers to cancerous, precancerous or transformed cells in vivo, in vitro and in tissue culture, which have spontaneous or induced phenotypic changes, not necessarily involving the uptake of new genetic material. Although transformation can be caused by infection with transforming viruses and the incorporation of new genomic nucleic acids or the uptake of exogenous nucleic acids, it can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Examples of transformation / cancer include, for example, morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant tumors, tumor-specific marker levels, invasiveness, tumor growth or inhibition in suitable animal hosts such as nude mice, in vitro, in vivo and in vitro (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd Edition, 1994)).

[0720] As used herein, the term "subject" refers to any organism to which a composition or formulation according to the present disclosure may be administered, e.g., for experimental, diagnostic, preventive and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0721] As used herein, the term "treat" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying the onset, inhibiting the progression, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of a particular infection, disease, condition, and / or illness. In some embodiments, "treating" cancer may refer to inhibiting the survival, growth, and / or spread of a tumor. Treatment may be performed on subjects who do not show signs of a disease, condition, and / or illness and / or subjects who only show early signs of a disease, condition, and / or illness, with the goal of reducing the risk of developing pathology associated with the disease, condition, and / or illness.

[0722] Example

[0723] The examples in this section are offered by way of illustration and not limitation.

[0724] General Preparative HPLC Methods: HPLC purifications were performed on a Waters 2767 equipped with a diode array detector (DAD) on an Inertsil Pre-C8 OBD column, typically using water with 0.1% TFA as solvent A and acetonitrile as solvent B.

[0725] General LCMS methods: LCMS analysis was performed on a Shimadzu (LC-MS2020) system. Chromatography was performed on a SunFire C18, typically using water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B.

[0726] Example A .

[0727] A1: Preparation of compound 02-1 (ie, compound 1 in the following scheme).

[0728]

[0729] 1 H NMR (400 MHz, CDCl 3 )δ:0.86-0.90(m,12H),1.27-1.63(m,53H),1.97-2.01(m,2H),2.28-2.64(m,14H),3.52-3.58(m,2H),4.00-4.10(m,8H). LCMS:Rt:1.080min;MS m / z(ESI):826.0[M+H] + .

[0730] The following compounds were prepared in a similar manner to compound 02-1 using the corresponding starting materials.

[0731]

[0732] A2: Preparation of compound 02-2 (ie, compound 2 in the following scheme).

[0733]

[0734] 1 H NMR (400 MHz, CDCl 3 )δ:0.86-0.90(m,12H),1.28-1.67(m,54H),1.88-2.01(m,7H),2.28-2.56(m,18H),3.16-3.20(m,1H),3.52-3.54(m,2H),4.00-4.10(m,8H). LCMS:Rt:1.060min;MS m / z(ESI):923.0[M+H] + .

[0735] A3: Preparation of compound 02-4 (ie, compound 4 in the following scheme).

[0736]

[0737] 1 H NMR (400 MHz, CDCl 3 )δ:0.86-0.90(m,9H),1.26-1.32(m,34H),1.41-1.49(m,4H),1.61-1.66(m,15H),2.00-2.03(m,1 H),2.21-2.38(m,8H),2.43-2.47(m,4H),2.56-2.60(m,2H),3.50-3.54(m,2H),4.03-4.14(m,8H). LCMS:Rt:1.030min;MS m / z(ESI):798.0[M+H] + .

[0738] A4: Preparation of compound 02-9 (ie, compound 9 in the following scheme).

[0739]

[0740] 1 H NMR (400 MHz, CDCl 3)δ:0.86-0.90(m,12H),1.28-1.30(m,33H),1.58-2.01(m,18H),2.30- 2.54(m,18H),3.10-3.19(m,1H),3.52-3.68(m,8H),4.09-4.20(m,8H). LCMS:Rt:1.677min;MS m / z(ESI):927.7[M+H] + .

[0741] The following compounds were prepared in a similar manner to compound 02-9 using the corresponding starting materials.

[0742]

[0743] A5: Preparation of compound 02-10 (ie compound 10 in the following scheme).

[0744]

[0745] Compound 10

[0746] 1 H NMR (400 MHz, CDCl 3 )δ:0.86-0.90(m,12H),1.26-1.41(m,48H),1.51-1.72(m,11H),1.94-2.03(m,1 H),2.29-2.32(m,6H),2.41-2.91(m,5H),3.51-3.76(m,2H),3.96-4.10(m,6H). LCMS:Rt:1.327min;MS m / z(ESI):782.6[M+H] + .

[0747] The following compounds were prepared in a similar manner to compound 02-10 using the corresponding starting materials.

[0748]

[0749] A6: Preparation of compound 02-12 (ie compound 12 in the following scheme).

[0750]

[0751] Compound 12

[0752] 1 H NMR (400 MHz, CDCl 3)δ:0.86-0.89(m,18H),1.25-1.35(m,53H),1.41-1.48(m,8H),1.56-1.61(m,20H),1.95-2.01(m,2 H),2.28-2.35(m,6H),2.43-2.46(m,4H),2.56-2.58(m,2H),3.51-3.54(m,2H),4.00-4.10(m,8H). LCMS:Rt:0.080min;MS m / z(ESI):1050.8[M+H] + .

[0753] A7: Preparation of compound 02-20 (ie compound 20 in the following scheme).

[0754]

[0755] Compound 20

[0756] 1 H NMR (400MHz, CDCl3) δ: 0.86-0.90(m,9H),1.25-1.36(m,48H),1.41-1.48(m,5H),1.60-1.62(m,8H),1.97-2 .00(m,1H),2.27-2.32(m,6H),2.43-2.46(m,4H),2.56-2.59(m,2H),3.52-3.54(m,2H),4.01-4.10(m,6H). LCMS: Rt:0.093min; MS m / z(ESI):782.6[M+H] + .

[0757] Example B Preparation and characterization of lipid nanoparticles

[0758] In brief, cationic lipids (e.g., compound 01-1), DSPC, cholesterol and PEG-lipids provided herein are dissolved in ethanol with an appropriate molar ratio (e.g., 50: 10: 38.5: 1.5), and mRNA is diluted in 10 to 50mM citrate buffer (pH = 4). Alternatively, cationic lipids, DSPC, cholesterol and polymer-conjugated lipids provided herein are dissolved in ethanol with an appropriate molar ratio (e.g., 50: 10: 38.5: 1.5), and mRNA is diluted in 10 to 50mM citrate buffer (pH = 4). LNPs are prepared with a total lipid to mRNA weight ratio of about 10: 1 to 30: 1 by mixing ethanol lipid solutions with mRNA aqueous solutions in a volume ratio of 1: 3 using a microfluidic device with a total flow rate range of 9-30mL / min. Ethanol is removed by dialysis and replaced with DPBS. Finally, lipid nanoparticles are filtered by a 0.2 μm sterile filter.

[0759] The lipid nanoparticle size was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) in 173° backscatter detection mode. The encapsulation efficiency of the lipid nanoparticles was determined using the Quant-it Ribogreen RNA quantification kit (Thermo Fisher Scientific, UK) according to the manufacturer's instructions.

[0760] As reported in the literature, the apparent pKa of LNP preparations is related to the delivery efficiency of nucleic acids in vivo by LNP. The apparent pKa of each preparation is determined by the fluorescence determination method based on 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS). The LNP preparations comprising cationic lipids / DSPC / cholesterol / DMG-PEG in PBS are prepared as described above. TNS is prepared as a 300uM stock solution in distilled water. The LNP preparations are diluted to 0.1mg / ml total lipid in 3mL buffer solution, and the buffer solution contains 50mM sodium citrate, 50mM sodium phosphate, 50mM sodium borate and 30mM sodium chloride, wherein the pH range is 3 to 9. An aliquot of TNS solution is added to obtain a final concentration of 0.1mg / ml, and after vortex mixing, the excitation wavelength and emission wavelength of 325nm and 435nm are used to measure the fluorescence intensity in a Molecular Devices Spectramax iD3 spectrometer at room temperature. Sigmoidal curve best-fit analysis was applied to the fluorescence data and the pKa value was measured as the pH that yielded half-maximal fluorescence intensity.

[0761] Example C : mRNA synthesis and purification.

[0762] DNA linearization. The IVT plasmid pJ241 (constructed in-house, containing a kanamycin resistance gene, a T7 promoter sequence, a poly(A) segment, and a unique type IIS restriction site downstream of the poly(A) sequence) containing the target sequence of SEQ ID NO:36 was linearized by digestion with type IIS restriction enzymes. Each 10 μg of plasmid was mixed with 10 U of Esp3I / BsmBI and incubated at 37°C for 4 hours to ensure complete linearization. The reaction was terminated by adding 1 / 10 volume of 3M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, mixed thoroughly, and cooled at -20°C for 1 hour. The linearized DNA was precipitated by centrifugation at 13800g for 15 minutes at 4°C, washed twice with 70% ethanol, and resuspended in nuclease-free H 2 In O.

[0763] In vitro transcription of mRNA The contents of a typical 20 μL reaction mixture are shown in the table below:

[0764] Table 3: Reaction mixture for mRNA transcription

[0765] <![CDATA[H without nuclease 2 O]]> Make up to 20 μL RNase inhibitor (40U / μL) 0.5μL rNTP mixture (100 mM each) 8μL (final concentration 10mM each) 10X IVT Reaction Buffer 2μL <![CDATA[1M MgCl 2 ]]> 0.8μL 0.1M DTT 2μL 100U / mL inorganic pyrophosphatase 0.8μL 100mM NaCl 1μL Linearized DNA 1 μg T7 RNA polymerase (50U / μL) 2μL

[0766] The reaction mixture was incubated at 37°C for 6 hours, and then 1 μl of DNase I (RNase-free, 1 U / μL) was added to remove the DNA template and incubated at 37°C for another 30 minutes. The synthesized RNA was purified by adding 0.5 volume of 7.5M LiCl, 50mM EDTA and incubating at -20°C for 45 minutes, followed by centrifugation at 13800g for 15 minutes at 4°C to precipitate the mRNA. The supernatant was then removed and the pellet was rinsed twice with 500 μL of ice-cold 70% ethanol, and the mRNA was resuspended in nuclease-free H 2 O, adjust the concentration to 1 mg / mL, and store at -20 °C.

[0767] mRNA capping 10 μg of each uncapped mRNA was heated at 65°C for 10 min, placed on ice for 5 min, mixed with 10 U of vaccinia capping enzyme, 50 U of mRNA Cap 2′-O-methyltransferase, 0.2 mM SAM, 0.5 mM GTP, and 1 U of RNase inhibitor, and incubated at 37°C for 60 min to generate Cap 1 modification structures. The modified mRNA was precipitated by LiCl as described above, and the RNA was resuspended in nuclease-free HO. 2 O and stored at -20 °C.

[0768] HPLC purificationRNA was purified by high performance liquid chromatography (HPLC) using a C4 column (5 μm) (10 mm×250 mm column). Buffer A contained 0.1 M triethylammonium acetate (TEAA) (pH=7.0), and buffer B contained 0.1 M TEAA (pH=7.0) and 25% acetonitrile.

[0769] As expected, mRNA molecules were successfully produced through the above in vitro transcription and maturation process and purified from the reaction system using HPLC (data not shown).

[0770] Embodiment D: Preparation of LNPs containing mRNA

[0771] LNPs containing mRNA were prepared according to the procedure provided in Example A above, using lipids prepared according to the procedure provided in Example B above and mRNA prepared according to the procedure provided in Example C above.

[0772] Example 1 .

[0773] To verify EGFR mut Whether the mRNA vaccine is immunogenic in Balb / c mice, 20 μg EGFR was injected intramuscularly on days 0, 3, and 11. mut mRNA vaccines (named EGFR (L858R), EGFR (L858R + T790M), EGFR (L858R + E746_A750), EGFR (E746_A750), EGFR (E746_A750 + T790M), EGFR (L858R + E746_A750 + T790M) -1) and negative control PBS, 3 mice in each group. On the 14th day, all spleens were collected and single cell suspensions were prepared. In addition, the cell suspension was incubated with 5g / mL EGFR L858R, E746_A750del, T790M peptide library or the corresponding WT peptide (all peptide libraries and WT peptides were synthesized by GenScript) in an ELISPOT plate (Mabtech, catalog number 3321-4HST-2) for 48 hours. After incubation, the secretion of specific IFN-γ was detected by ELISPOT. The results showed that EGFR mut mRNA vaccines can induce T790M-specific immune responses in Balb / c mice ( Figure 2A-2C ).

[0774] Example 2 .

[0775] In order to verify the ability of EGFR (L858R + E746_A750 + T790M) -1 to induce innate immune response, serum samples of HLA-A * 0201 transgenic mice (purchased from Biocytogen) were collected 3 hours after a single intramuscular injection of 20g EGFR (L858R + E746_A750 + T790M) -1, and the cytokine levels associated with innate immune activation were detected by mouse antiviral response (13-plex) legendplex (Biolegend, catalog number 740622). First, serum was diluted 2 times with assay buffer and mixed with detection beads for 2 hours. Subsequently, detection antibodies and SA-PE were added. Results were read on a flow cytometer. Compared with PBS control, innate immunity-related cytokines such as IFN-α, IP-10, TNF-α, CXCL1, MCP-1 and IL-6 were highly upregulated 3 h after intramuscular injection of EGFR(L858R+E746_A750+T790M)-1. These results show that EGFR(L858R+E746_A750+T790M)-1 can induce a strong innate immune response ( Figure 3 ).

[0776] Example 3 .

[0777] To determine the ability of EGFR(L858R+E746_A750+T790M)-1 to induce antigen-specific cellular immune responses, HLA-A*0201 (purchased from Biocytogen), HLA-A*1101 and HLA-A*0101 (purchased from TACONIC) transgenic mice were intramuscularly injected with PBS and 20 μg EGFR(L858R+E746_A750+T790M)-1 (3 mice per group) on days 0, 7 and 14, respectively. On the 3rd day after the last injection, the spleens of the mice were collected and prepared into single cell suspensions. In addition, peptide libraries of EGFR L858R, E746_A750del and T790M and corresponding WT peptides (all peptide libraries and WT peptides were synthesized by GenScript) were added to ELISPOT plates (Mabtech, catalog number 3321-4HST-2) and then cultured in an incubator for 48 hours. After incubation, the IFN-γ secretion levels induced by EGFR L858R, E746_A750del and T790M antigen-specific T cells were detected by ELISPOT. The results showed that EGFR (L858R + E746_A750 + T790M)-1 induced strong T790M antigen-specific T cell activation and other antigen-specific T cell activation in all three transgenic mice ( Figure 4A-4B). In HLA-A*0201 transgenic mice, EGFR(L858R+E746_A750+T790M)-1 induced strong T790M antigen-specific T cell activation and moderate L858R and E746_A750del antigen-specific T cell activation. In HLA-A*1101 transgenic mice, EGFR(L858R+E746_A750+T790M)-1 induced strong T790M and L858R antigen-specific T cell activation. EGFR(L858R+E746_A750+T790M)-1 induced strong T790m-specific T cell activation in HLA-A*0101 transgenic mice.

[0778] Example 4 .

[0779] In order to verify that EGFR(L858R+E746_A750+T790M)-1-induced EGFR antigen-specific T cells can kill EGFR mutant tumor cells, 12 HLA-A*0101 transgenic mice (purchased from TACONIC) were immunized intramuscularly. Different doses of EGFR(L858R+E746_A750+T790M)-1 (10μg, 20μg or 40μg)-1 were injected intramuscularly on days 0, 7 and 14. On the third day after the last injection, single cell suspensions of mouse spleens were prepared as effector cells. NCI-H1975-EGFR L858R / T790M (HLA-A*0101) (gift from Guangzhou Institute of Respiratory Health) and A549 (EGFR WT) (gift from Guangzhou Institute of Respiratory Health) tumor cell lines were labeled with CFSE as target cells and co-incubated at an E:T ratio (effector cell: target cell ratio) of 1:20. After 72 hours, dead cells were stained with a dead dye. The antigen-specific T cell killing induced by EGFR (L858R + E746_A750 + T790M) -1 was evaluated by calculating the proportion of living cells remaining in the target cells. The results showed that EGFR (L858R + E746_A750 + T790M) -1 was able to induce the anti-tumor efficacy of antigen-specific T cells in a dose-dependent manner in vitro, and had no obvious killing effect on the EGFR WT A549 cell line ( Figure 5 ).

[0780] Example 5 .

[0781] In order to verify whether EGFR(L858R+E746_A750+T790M)-1 can induce EGFR antigen-specific T cells to kill EGFR mutant tumor cells, 6 HLA-A*1101 transgenic mice (purchased from TACONIC) were immunized intramuscularly. On days 0, 7, and 14, 20 μg of EGFR(L858R+E746_A750+T790M)-1 and PBS were injected intramuscularly. On the third day after the last injection, a single cell suspension of mouse spleen was prepared as effector cells. NCI-H1975-EGFR L858R / T790M (HLA-A*1101) (Kyinno, catalog number KC-2805) and A549 (EGFR WT) (donated by Guangzhou Institute of Respiratory Health) tumor cell lines were labeled with CFSE as target cells and co-incubated at different E:T ratios (1:100, 1:25, 1:10). After 72 hours, dead cells were stained with dead dye. The antigen-specific T cell killing induced by EGFR (L858R + E746_A750 + T790M) -1 was evaluated by calculating the remaining living cell ratio in the target cells. The results showed that EGFR (L858R + E746_A750 + T790M) -1 was able to induce the anti-tumor efficacy of antigen-specific T cells in vitro ( Figure 6 ).

[0782] Example 6 .

[0783] To verify the effect of EGFR(L858R+E746_A750+T790M)-1-induced EGFR antigen-specific T cells on EGFR mutant tumors in vivo, NCI-H1975-EGFR L858R / T790M 5E6 live cells of (HLA-A*0101) were subcutaneously injected into each B-NDG immunodeficient mouse (purchased from Biocytogen) to establish a tumor model. On days 0, 7, and 14, 40 g EGFR (L858R + E746_A750 + T790M)-1 and PBS were intramuscularly immunized in HLA-A*0101 transgenic mice. Single cell suspensions were obtained from the spleen on day 3 after the last immunization. 2E7 splenocytes were intravenously transplanted into NCI-H1975-EGFR L858R / T790M In tumor-bearing mice, the group injected with PBS alone was set as a blank control. The tumor size was measured regularly, and the tumor growth inhibition rate (TGI) was calculated. The results showed that EGFR(L858R+E746_A750+T790M)-1 could induce the production of EGFR mutant antigen-specific T cells and had a certain anti-tumor effect ( Figure 7 ).

[0784] Example 7 .

[0785] To explore whether EGFR(L858R+E746_A750+T790M)-1 has synergistic effects with α-PD-1 in adoptive transfer assays, NCI-H1975-EGFR L858R / T790M (HLA-A*0101) tumor-bearing mice and spleen cells immunized with EGFR(L858R+E746_A750+T790M)-1 were treated according to Example 6 above. In addition, BIW was injected with 10mpkα-mPD-1 (Bioxcell, catalog number BP0146). The group injected with only PBS was set as the blank group, and mIgG (Bi oxcell, catalog number BE0089) was the negative control for α-mPD-1. Tumor size was measured regularly, and the tumor growth inhibition rate (TGI) was calculated. The results showed that EGFR(L858R+E746_A750+T790M)-1 can enhance the specific anti-tumor effect more than the combination of EGFR(L858R+E746_A750+T790M)-1 and mIgG ( Figure 8 ).

[0786] Example 8 .

[0787] To verify the ability of EGFR(L858R+E746_A750+T790M+C797S) to induce antigen-specific cellular immune responses, HLA-A*0201 transgenic mice (purchased from Biocytogen) were intramuscularly immunized with PBS and 20 g EGFR(L858R+E746_A750+T790M+C797S) on days 0, 7, and 14, respectively. Single cell suspensions of splenocytes were prepared on day 3 after the last injection. At the same time, EGFR C797S, T790M peptide libraries and corresponding WT peptides (synthesized by GenScript) were added to ELISPOT plates and then cultured in an incubator for 48 hours. After 48 hours of incubation, IFN-γ secretion induced by EGFR C797S antigen-specific T cells was detected by ELISPOT. The results showed that EGFR(L858R+E746_A750+T790M+C797S) induced strong C797S and T790M antigen-specific T cell activation in HLA-A*0201 transgenic mice ( Fig. 9 ).

[0788] Example 9 .

[0789] To verify whether the C797S antigen affects the specific T cell killing induced by other EGFR antigens, HLA-A*1101 transgenic mice were immunized intramuscularly. On days 0, 7, and 14, 20 g of EGFR (L858R + E746_A750 + T790M + C797S) and PBS were injected intramuscularly. On the third day after the last injection, a single cell suspension of mouse spleen was prepared as effector cells. NCI-H1975-EGFR L858R / T790M (HLA-A*1101) and A549 (EGFR WT) tumor cell lines were labeled with CFSE as target cells and co-incubated at different E:T ratios (1:100, 1:25, 1:10). After 72 hours, dead cells were stained with a dead dye. The antigen-specific T cell killing induced by EGFR (L858R + E746_A750 + T790M + C797S) was evaluated by calculating the remaining proportion of live cells in the target cells. The results showed that C797S did not reduce L858R and T790M-specific T cell killing. EGFR (L858R + E746_A750 + T790M + C797S) exhibited anti-tumor efficacy of antigen-specific T cells in vitro ( Fig.10 ).

[0790] Other Implementations

[0791] It is to be understood that, although it has been described in conjunction with the specific embodiments of the present disclosure, the foregoing description is intended to illustrate rather than limit the scope of the present disclosure, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims. All references described herein are incorporated by reference in their entirety.

[0792] References

[0793] 1.GLOBOCAN 2020 from WHO.

[0794] 2.Lee CS,Sharma S,Miao E,et al.A Comprehensive Review ofContemporary Literature for Epidermal Growth Factor Receptor Tyrosine KinaseInhibitors in Non-Small Cell Lung Cancer and TheirToxicity.[J].Lung Cancer:Targets and Therapy,2020.

[0795] 3.Karachaliou N,Fernandez-Bruno M,Bracht J,et al.EGFR first-andsecond-generation TKIs-there is still place for them in EGFR-mutant NSCLCpatients[J].Translational Cancer Research,2018(1).

[0796] 4.Xu C W,Lei L,Wang W X,et al.Molecular Characteristics and ClinicalOutcomes of EGFR Exon 19 C-Helix Deletion in Non-Small Cell Lung Cancer andResponse to EGFR TKIs[J].Translational oncology,2020,13(9):100791.

[0797] 5.Kimura S,Tanaka K,Harada T,et al.Sensitivity of epidermal growthfactor receptor with single or double uncommon mutations to afatinibconfirmed by a visual assay[J].Cancer ence.2018,109.

[0798] 6.Ma C,Wei S,Song Y.T790M and acquired resistance of EGFR TKI:aliteratre review of clinical reports[J].Journal of thoracic disease,2011,3(1):10.

[0799] 7.Tan C S,Kumarakulasinghe N B,Huang Y Q,et al.Third generation EGFRTKIs:current data and future diections[J].Molecular Cancer.2018,17(1):29.

[0800] 8.Engelman J A,Janne P A.Mechanisms of Acquired Resistance toEpidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small CellLung Cancer[J].Clinical Cancer Research,2008.

[0801] 9.Pallis A,Briasoulis E,Linardou H,et al.Mechanisms of Resistance toEpidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients withAdvanced Non-Small-Cell Lung Cancer:Clinical and Molecular Considerations[J].CMC,2011.

Claims

1. A polypeptide comprising an EGFR domain, wherein the EGFR domain comprises at least one mutant fragment of human EGFR.

2. The polypeptide of claim 1, wherein the polypeptide further comprises a degradation domain configured to mark the EGFR domain for proteasomal degradation.

3. The polypeptide of claim 2, wherein the degradation domain comprises a ubiquitin peptide.

4. The polypeptide of claim 3, wherein the ubiquitin peptide is a ubiquitin monomer, a ubiquitin polymer, a ubiquitin-like domain (ULD) or a functional derivative thereof.

5. The polypeptide of claim 4, wherein the polypeptide comprises an EGFR domain fused directly or indirectly to the C-terminus of the degradation domain, and wherein the EGFR domain is at least about 10 amino acids in length.

6. The polypeptide of claim 5, wherein the EGFR domain is located at the C-terminal portion of the polypeptide.

7. The polypeptide of claim 6, wherein the EGFR domain comprises two or more mutant fragments of human EGFR.

8. The polypeptide of claim 6, wherein the EGFR domain is fused directly to the C-terminus of the degradation domain, and wherein the EGFR domain is at least about 10 amino acids in length.

9. A polypeptide as described in claim 6, wherein the spacer peptide is located between the degradation domain and the EGFR domain; optionally wherein the length of the spacer peptide is at least about 15 amino acids; optionally wherein the length of the spacer peptide is about 15-30 amino acids; optionally wherein the length of the spacer peptide is about 20 amino acids, about 25 amino acids or about 30 amino acids.

10. The polypeptide of any one of claims 1 to 9, wherein the degradation domain consists of, consists essentially of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:

1.

11. A polypeptide as described in claim 10, wherein the degradation domain further comprises a spacer located between the ubiquitin peptide and the EGFR domain, and wherein the spacer peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

12. The polypeptide of claim 11, wherein the polypeptide further comprises one or more linker sequences.

13. The polypeptide of any one of claims 1 to 12, wherein the EGFR domain comprises at least one mutant fragment of human EGFR, wherein each mutant fragment comprises one or more EGFR mutations selected from T790M, C797S, L858R and E746_A750 del; optionally wherein the mutant fragment comprises one or more EGFR mutations selected from any one of (a) to (k): (l) L858R; (m)T790M; (n)E746_A750 del; (o)C797S; (p) T790M and C797S; (q) L858R and T790M; (r) L858R and E746_A750 del; (s)E746_A750 del; (t) E746_A750 del and T790M; (u) L858R, E746_A750 del and T790M; and (v) L858R, E746_A750 del, T790M, and C797S.

14. A polypeptide as described in claim 13, wherein the length of at least one mutant fragment of human EGFR is at least about 10 amino acids; optionally wherein the length of the human EGFR mutant fragment is about 10 amino acids, 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids or about 35 amino acids.

15. The polypeptide of claim 13 or 14, wherein the at least one mutant fragment of human EGFR comprises two or more mutant fragments of human EGFR, and wherein the two or more mutant fragments are the same or different.

16. The polypeptide of any one of claims 13 to 15, wherein the EGFR domain comprises two or more mutant fragments of human EGFR, and wherein at least two of the two or more mutant fragments are fused directly or through a linker peptide.

17. The polypeptide of any one of claims 13 to 16, wherein the at least one mutant fragment of human EGFR comprises a L858R fragment, wherein the L858R fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the L858R mutation.

18. The polypeptide of claim 17, wherein the EGFR domain comprises two or more identical copies of the L858R fragment.

19. The polypeptide of claim 17 or 18, wherein the L858R fragment has the amino acid sequence of SEQ ID NO:

3.

20. The polypeptide of any one of claims 13 to 19, wherein the at least one mutant fragment of human EGFR comprises a T790M fragment, wherein the T790M fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the T790M mutation.

21. The polypeptide of claim 20, wherein the EGFR domain comprises two or more identical copies of the T790M fragment.

22. The polypeptide of claim 20 or 21, wherein the T790M fragment has the amino acid sequence of SEQ ID NO:

4.

23. The polypeptide of any one of claims 13 to 22, wherein the at least one mutant fragment of human EGFR comprises an E746_A750 del fragment, wherein the E746_A750del fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the E746_A750 del mutation.

24. The polypeptide of claim 23, wherein the EGFR domain comprises two or more identical copies of the E746_A750 del fragment.

25. The polypeptide of claim 23 or 24, wherein the E746_A750 del fragment has the amino acid sequence of SEQ ID NO:

5.

26. The polypeptide of any one of claims 13 to 24, wherein the at least one mutant fragment of human EGFR comprises a C797S fragment, wherein the C797S fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the C797S mutation.

27. The polypeptide of claim 26, wherein the EGFR domain comprises two or more identical copies of the C797S fragment.

28. The polypeptide of claim 26 or 27, wherein the C797S fragment has the amino acid sequence of SEQ ID NO:

6.

29. The polypeptide of any one of claims 13 to 16, wherein the EGFR domain comprises: (a) six copies of the L858R fragment, wherein each L858R fragment has an amino acid sequence of SEQ ID NO: 3; (b) three copies of an L858R fragment and three T790M fragments, wherein each L858R fragment has the amino acid sequence of SEQ ID NO: 3, and wherein each T790M fragment has the amino acid sequence of SEQ ID NO: 4; (c) three L858R fragments and three E746_A750 del fragments, wherein each L858R fragment has the amino acid sequence of SEQ ID NO: 3, and wherein each E746_A750 del fragment has the amino acid sequence of SEQ ID NO: 5; (d) six E746_A750 fragments, wherein each E746_A750 fragment has an amino acid sequence of SEQ ID NO: 5; (e) three E746_A750 del fragments and three T790M fragments, wherein each T790M fragment has the amino acid sequence of SEQ ID NO: 4, and wherein each E746_A750 fragment has the amino acid sequence of SEQ ID NO: 5; (f) two L858R fragments, two E746_A750 del fragments, and two T790M fragments, wherein each L858R fragment has the amino acid sequence of SEQ ID NO:3, wherein each T790M fragment has the amino acid sequence of SEQ ID NO:4, and wherein each E746_A750 fragment has the amino acid sequence of SEQ ID NO:5; or (g) two L858R fragments, two E746_A750 del fragments, one T790M fragment and one C797S fragment, wherein each L858R fragment has the amino acid sequence of SEQ ID NO:3, wherein each T790M fragment has the amino acid sequence of SEQ ID NO:4, wherein each E746_A750 fragment has the amino acid sequence of SEQ ID NO:5, and wherein each C797S fragment has the amino acid sequence of SEQ ID NO:

6.

30. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28 and 31.

31. A composition comprising a plurality of species of polypeptides, wherein each species of polypeptide is selected from the polypeptides of any one of claims 1 to 30, wherein at least two of the plurality of species of polypeptides are different.

32. A nucleic acid comprising an open reading frame encoding the polypeptide of any one of claims 1 to 30.

33. The nucleic acid of claim 32, further comprising a 5' untranslated region (UTR); optionally wherein the 5'UTR comprises the sequence of SEQ ID NO:34 or comprises a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO:

34.

34. The nucleic acid of claim 32 or 33, further comprising a 3'UTR; optionally wherein the 3'UTR comprises the sequence of SEQ ID NO:35 or comprises a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in SEQ ID NO:

35.

35. The nucleic acid of claim 32, wherein the nucleic acid is a DNA molecule or an RNA molecule.

36. The nucleic acid of any one of claims 32 to 35, wherein the nucleic acid comprises one or more functional nucleotide analogs selected from pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.

37. A vector comprising the nucleic acid of any one of claims 32 to 35.

38. A composition comprising a nucleic acid as described in any one of claims 33 to 37 and at least one lipid; optionally, the lipid comprises a first lipid according to Formula 01-I or Formula 01-II; or a compound listed in Table 01-1; or a compound according to Formula 02-I; or a compound listed in Table 02-1; or a compound according to formula 03-I; or a compound listed in Table 03-1; Or a compound according to formula 04-I; or a compound listed in Table 04-1; Optionally, the lipid further comprises a second lipid according to formula 05-I.

39. A composition comprising multiple species of nucleic acid molecules, each of which encodes an EGFR domain, wherein each encoded EGFR domain comprises at least one mutant fragment of human EGFR, wherein the mutant fragment comprises one or more EGFR mutations selected from T790M, C797S, L858R and E746_A750 del, and wherein the EGFR domains encoded by at least two of the multiple species of nucleic acid molecules are different.

40. The composition of claim 39, wherein the EGFR domain encoded by at least one of a plurality of species of nucleic acid molecules comprises two or more mutant fragments of human EGFR, wherein the two or more mutant fragments are the same or different.

41. The composition of claim 39 or 40, wherein one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules comprises an EGFR mutation selected from any one of (a) to (K): (a) L858R; (b) T790M; (c) E746_A750 del; (d) C797S; (e) T790M and C797S; (f) L858R and T790M; (g) L858R and E746_A750 del; (h) E746_A750 del; (i) E746_A750 del and T790M; (j) L858R, E746_A750 del and T790M; and (k) L858R, E746_A750 del, T790M, and C797S.

42. A composition as described in claim 39 or 40, wherein one or more of the human EGFR mutant fragments encoded by multiple types of nucleic acid molecules is a L858R fragment, wherein the L858R fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR containing the L858R mutation.

43. The composition of claim 39 or 40, wherein one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is a T790M fragment, wherein the T790M fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the T790M mutation.

44. The composition of claim 39 or 40, wherein one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is an E746_A750 del fragment, wherein the E746_A750 del fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the E746_A750 del mutation.

45. The composition of claim 39 or 40, wherein one or more of the human EGFR mutant fragments encoded by the plurality of species of nucleic acid molecules is a C797S fragment, wherein the C797S fragment is a fragment of about 15 to 30 consecutive amino acids of human EGFR comprising the C797S mutation.

46. ​​A composition as described in any of claims 39 or 40, wherein the multiple species of nucleic acid molecules also include at least one first species of nucleic acid molecules encoding an EGFR domain longer than about 10 amino acids, and wherein at least one first species of nucleic acid molecules also encodes a degradation domain, wherein the degradation domain is configured to mark the EGFR domain for proteasomal degradation.

47. The composition of claim 46, wherein the degradation domain comprises a ubiquitin peptide.

48. The composition of claim 47, wherein the ubiquitin peptide is a ubiquitin monomer, a ubiquitin polymer, a ubiquitin-like domain (ULD) or a functional derivative thereof.

49. A composition as described in claim 48, wherein the spacer peptide is located between the degradation domain and the EGFR domain; optionally wherein the length of the spacer peptide is at least about 15 amino acids; optionally wherein the length of the spacer peptide is about 15-30 amino acids; optionally wherein the length of the spacer peptide is about 20 amino acids, about 25 amino acids or about 30 amino acids.

50. The composition of any one of claims 43 to 46, wherein the at least one species of nucleic acid molecule comprises a coding sequence for a ubiquitin peptide selected from the group consisting of SEQ ID NOs: 37-45.

51. A composition as described in any one of claims 39 to 51, wherein the plurality of species of nucleic acid molecules also includes at least one second species of nucleic acid molecules encoding an EGFR structural domain that is no longer than about 10 amino acids, and wherein the at least one second species of nucleic acid molecules lacks the coding sequence of a degradation domain.

52. A composition as described in any one of claims 39 to 52, wherein the plurality of species of nucleic acid molecules comprise mRNA molecules.

53. A composition as described in any one of claims 39 to 53, wherein at least one of the plurality of species of nucleic acid molecules further comprises a 3'-UTR and / or a 5'-UTR; optionally wherein the 5'-UTR comprises the sequence of SEQ ID NO:34; optionally wherein the 3'-UTR comprises the sequence of SEQ ID NO:

35.

54. A composition as described in claim 39, wherein the multiple species of nucleic acid molecules include RNA molecules, and the RNA molecules contain a sequence selected from SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29, 32, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29 and 32.

55. A composition as described in claim 39, wherein the multiple species of nucleic acid molecules include DNA molecules, and the DNA molecules contain a sequence selected from SEQ ID NO:9, 12, 15, 18, 21, 24, 27, 30 and 33, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown in 9, 12, 15, 18, 21, 24, 27, 30 and 33.

56. The composition of any one of claims 39 to 55, wherein at least one of the plurality of species of nucleic acids comprises one or more functional nucleotide analogs selected from pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

57. A composition as described in any one of claims 39 to 56, further comprising at least one lipid; optionally, the lipid comprises a first lipid according to Formula 01-I or Formula 01-II; or a compound listed in Table 01-1; or a compound according to Formula 02-I; or a compound listed in Table 02-1; or a compound according to formula 03-I; or a compound listed in Table 03-1; Or a compound according to formula 04-I; or a compound listed in Table 04-1; Optionally, the lipid further comprises a second lipid according to formula 05-I.

58. The composition of claims 38 to 57, wherein the composition is formulated as a lipid nanoparticle containing the nucleic acid.

59. The composition of claim 31 or 55, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

60. The composition of claim 56, wherein the composition is a vaccine.

61. The composition of claims 31, 38-39, wherein the composition is administered in combination with a second therapeutic agent; optionally, the second therapeutic agent comprises EGFR-TKI, PD1 antibody; optionally, the EGFR-TKI is erlotinib, gefitinib or dacomitinib.

62. A method for producing EGFR mutant-specific immune effector cells, comprising providing a starting population of immune effector cells, and contacting the starting population of immune effector cells with a polypeptide as described in any one of claims 1 to 30, a nucleic acid as described in any one of claims 33-37, or a composition as described in any one of claims 31 to 57 under suitable conditions, wherein EGFR mutant-specific immune effector cells are produced through the contact.

63. The method of claim 62, wherein the contacting is performed in the presence of a proteasome system, and wherein the proteasome system degrades the EGFR domain into one or more peptides of about 9 to 15 amino acids in length.

64. The method of claim 61 or 62, wherein the contacting is performed in the presence of antigen presenting cells (APCs), wherein the APCs present the EGFR domain or a degraded portion thereof on their surface.

65. The method of claim 64, wherein the APC is selected from the group consisting of dendritic cells, macrophages, Langerhans cells, and B cells.

66. The method of claim 63 or 65, wherein the APC expresses HLA class I and / or HLA class II molecules; optionally wherein the HLA class I molecule is HLA-A*0101 subtype or HLA-A*0201 subtype.

67. The method of any one of claims 61 to 66, wherein the immune effector cells are selected from T cells, peripheral blood lymphocytes, and natural killer cells; optionally wherein the T cells include CD4+T cells, CD8+T cells, antigen-specific T cells, tumor-infiltrating T cells, γδT cells, cytotoxic T cells, natural killer T cells, and mucosa-associated invariant T cells (MAIT).

68. A method for forming a pro-inflammatory environment in tissue surrounding a diseased cell population expressing mutant EGFR, comprising contacting the tissue with an effective amount of a polypeptide as described in any one of claims 1 to 31, a nucleic acid as described in any one of claims 32 to 36, or a composition as described in any one of claims 33 or 60 under suitable conditions.

69. The method of claim 68, wherein the infiltration of activated B cells, CD4+T cells, CD8+T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils and / or neutrophils in the tissue is increased.

70. The method of claim 69, wherein the concentration of a pro-inflammatory cytokine in the tissue is increased.

71. The method of claim 70, wherein the proinflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, IP-10, CXCL1, MCP-1, or any combination thereof.

72. The method of any one of claims 68 to 71, wherein presentation of an antigen originating or derived from the diseased cells by antigen presenting cells in the tissue is increased.

73. The method of any one of claims 68 to 71, wherein phagocytosis of the diseased cells in the tissue is increased.

74. The method of any one of claims 68 to 72, wherein apoptosis of the diseased cells induced by cell-mediated cytotoxicity in the tissue is increased.

75. The method of any one of claims 68 to 74, wherein apoptosis of the diseased cells induced by antibody-dependent cellular cytotoxicity in the tissue is increased.

76. The method of any one of claims 68 to 75, wherein the population of the diseased cells in the tissue is reduced.

77. The method of claim 76, wherein the population of the diseased cells in the tissue is reduced by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

78. The method of any one of claims 68 to 77, wherein the method is performed in vitro or in vivo.

79. A method of inducing an immune response against a target cell expressing a mutant EGFR in a subject, wherein the method comprises administering to the subject a polypeptide according to any one of claims 1 to 31, a nucleic acid according to any one of claims 32 to 36, or a composition according to any one of claims 32 or 60, wherein upon said administration, (a) the EGFR domain encoded by the nucleic acid molecule is expressed; (b) the EGFR domain is degraded into fragments of about 9 to 15 amino acids; (c) the EGFR domain or degradation fragment thereof associates with an HLA class I or HLA class II complex; (d) an increase in the innate immune response against the target cells; (e) increased secretion of pro-inflammatory cytokines; (f) an increase in the percentage of phagocytic macrophages in the macrophage population; (g) the percentage of dendritic cells presenting the EGFR domain or its degradation fragments in the dendritic cell population is increased; (h) an increase in the percentage of antigen-specific T cells specific for a domain of EGFR or an epitope thereof; or (i) The percentage of B cells producing antibodies specific to the EGFR domain or its epitope in the B cell population is increased.

80. The method of claim 81, wherein the innate immune response is measured by (a) activation of the NF-kB signaling pathway; or (b) increased secretion of cytokines selected from IFN-α, IP-10, TNF-α, CXCL1, MCP-1, IL-2, or any combination thereof.

81. The method of claim 80, wherein the proinflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof.

82. The method of any one of claims 79 to 81, wherein the target cell is a diseased cell expressing a mutant EGFR having a mutation selected from L858R, T790M, E746_A750 del and C797S; optionally wherein the diseased cell expresses a mutant EGFR having a double mutation of L858R and T790M.

83. A method of eliminating diseased cells expressing mutant EGFR in a subject, comprising administering an effective amount of the polypeptide of any one of claims 1 to 27, the nucleic acid of any one of claims 33 to 37, or the composition of any one of claims 31 or 58.

84. The method of claim 83, wherein the subject is a human subject having the HLA subtype HLA-A*0101 or HLA-A*0201.

85. The method of claim 83 or 84, wherein the diseased cell is a cancer cell.

86. A method of treating a cancer expressing a mutant EGFR or slowing the progression of a cancer in a subject, wherein the method comprises administering to the subject a polypeptide of any one of claims 1 to 30, a nucleic acid of any one of claims 32 to 36, or a composition of any one of claims 31 or 58.

87. The method of claim 86, wherein upon said administering, (a) the EGFR domain encoded by the nucleic acid molecule is expressed; (b) the EGFR domain is degraded into fragments of about 9 to 15 amino acids; (c) the EGFR domain or degradation fragment thereof associates with an HLA class I or HLA class II complex; (d) an increased innate immune response against the cancer; (e) increased secretion of pro-inflammatory cytokines; (f) an increase in the percentage of phagocytic macrophages in the macrophage population; (g) the percentage of dendritic cells presenting the EGFR domain or its degradation fragments in the dendritic cell population is increased; (h) an increase in the percentage of antigen-specific T cells specific for a domain of EGFR or an epitope thereof; or (i) The percentage of B cells producing antibodies specific to the EGFR domain or its epitope in the B cell population is increased.

88. The method of claim 89, wherein the innate immune response is measured by (a) activation of the NF-kB signaling pathway; or (b) increased secretion of cytokines selected from IFN-α, IP-10, TNF-α, CXCL1, MCP-1, IL-2, or any combination thereof.

89. The method of claim 87, wherein the proinflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof.

90. The method of any one of claims 86 to 89, wherein the mutant EGFR contains a mutation selected from L858R, T790M, E746_A750 del and C797S; optionally wherein the mutant EGFR contains a double mutation of L858R and T790M.

91. The method of any one of claims 86 to 89, wherein the method further comprises administering a second therapeutic agent simultaneously or sequentially.

92. The method of claim 95, wherein the second therapeutic agent comprises EGFR-TKI, PD1 antibody; optionally, the EGFR-TKI is erlotinib, gefitinib or dacomitinib.

93. The method of any one of claims 86 to 92, wherein the cancer expressing mutant EGFR is NSCLC.

Images