Treatment involving non-immunogenic RNA and PD-1 axis binding antagonists for antigen vaccination

Abstract

The present disclosure relates to methods and agents for antigen vaccination and induction of effective antigen-specific immune effector cell responses, such as T cell responses. These methods and agents are particularly useful for treating diseases characterized by disease cells expressing an antigen to which immune effector cells are directed. In some embodiments, the present disclosure relates to a method comprising administering to a subject (i) a non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope for inducing an immune response against the antigen in a subject, i.e., a non-immunogenic RNA encoding a vaccine antigen, and (ii) a PD-1 axis binding antagonist, such as an anti-PD-1 antibody and / or an anti-PD-L1 antibody.

Description

[Technical Field]

[0001] The present disclosure relates to methods and agents for antigen vaccination and the induction of effective antigen-specific immune effector cell responses, such as T cell responses. These methods and agents are particularly useful for treating diseases characterized by disease cells expressing an antigen to which immune effector cells are directed. In some embodiments, the present disclosure relates to a method comprising administering to a subject (i) a non-immunogenic RNA encoding a peptide or polypeptide containing an epitope for inducing an immune response against the antigen in a subject, i.e., a non-immunogenic RNA encoding a vaccine antigen, and (ii) a PD-1 axis binding antagonist, e.g., an anti-PD-1 antibody and / or an anti-PD-L1 antibody. Administration of the non-immunogenic RNA encoding the vaccine antigen to a subject can provide the vaccine antigen for stimulation, priming, and / or expansion of immune effector cells (after expression of the RNA by appropriate target cells), thereby inducing an immune response against the vaccine antigen (and disease-associated antigens) in the subject. In some embodiments, the immune effector cells bear an antigen receptor, such as a T cell receptor (TCR) or chimeric antigen receptor (CAR), with binding specificity for the antigen or its processing product. In some embodiments, immune effector cells are genetically modified to express an antigen receptor. Such genetic modification can be performed ex vivo or in vitro, and then the immune effector cells can be administered to a subject in need of treatment and / or can be performed in vivo in the subject in need of treatment. The immune effector cells can be derived from the subject in need of treatment or can be endogenous to the subject in need of treatment. The antigen receptor of the immune effector cells can target an antigen associated with a disease. As demonstrated herein, immune effector cells, such as T cells, induced by administration of non-immunogenic RNA express higher amounts of PD-1 compared to immune effector cells, such as T cells, induced by administration of standard RNA. As a result, immune effector cells, such as T cells, induced by administration of non-immunogenic RNA are susceptible to PD-1 / PD-L1 blockade, resulting in the expansion of immune effector cells and an enhanced immune response.Thus, administering to a subject a PD-1 axis binding antagonist, such as an anti-PD-1 antibody and / or an anti-PD-L1 antibody, can strongly enhance the immune response to vaccine antigens (and disease-associated antigens) in the subject. [Background technology]

[0002] The immune system plays an important role not only in pathogen-related diseases but also in cancer, autoimmunity, and allergies. T cells are key mediators of antitumor immune responses. CD4+ T cells can prime dendritic cells (DCs) for antitumor CD8+ T cell responses and directly target tumor cells by IFNγ-mediated MHC upregulation and proliferation inhibition. CD4+ T cells mediate the influx of various immune subsets into tumors, including CD8+ T cells, which can directly lyse tumor cells.

[0003] T cell responses are naturally induced not only against pathogens but also against tumors. Such tumor-specific T cell responses can be induced or even enhanced by therapeutic anticancer vaccination, given that antigens are delivered to DCs in an environment that allows T cell priming and proliferation, allowing them to mature into potent antigen-presenting cells.

[0004] In the context of mRNA-based vaccine platforms, mRNA can be delivered to antigen-presenting cells located in secondary lymphoid organs by liposomal formulations (RNA-lipoplexes, RNA-LPX) without the need for additional adjuvants for immune stimulation (Kreiter, S. et al. Nature 520, 692-696 (2015); Kranz, L. M. et al. Nature 534, 396-401 (2016)). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kreiter,S.et al.Nature 520,692-696(2015) [Non-patent document 2] Kranz,LMet al.Nature 534,396-401(2016) [Non-patent document 3] Holtkamp, ​​Silke et al., 2006, Blood 108(13):4009-17 [Non-patent document 4] Kuhn, AN et al., 2010, Gene Therapy 17(8):961-71 [Non-Patent Document 5] Orlandini von Niessen,Alexandra G.et al.,2019,Molecular Therapy 27(4):824-36 [Non-patent document 6] Kreiter, S. et al., 2008, The Journal of Immunology 180(1):309-18 [Non-Patent Document 7] Kranz, Lena Maseen et al., 2016, Nature 534(7607):396-401 [Non-patent document 8] Andries,Oliwia et al.,2015,Journal of Controlled Release:Official Journal of the Controlled Release Society 217:337-44 [Non-Patent Document 9] Baiersdorfer, Markus et al., 2019, Molecular Therapy-Nucleic Acids 15 (April) [Non-Patent Document 10] Pardi, Norbert et al., 2015, Journal of Controlled Release: Official Journal of the Controlled Release Society 217:345-51 Summary of the Invention [Problem to be solved by the invention]

[0006] Previous studies have optimized antigen-encoding RNAs for intracellular stability, translation efficiency (Holtkamp, ​​Silke et al., 2006, Blood 108(13):4009-17; Kuhn, A.N. et al., 2010, Gene Therapy 17(8):961-71; Orlandini von Niessen, Alexandra G. et al., 2019, Molecular Therapy 27(4):824-36), and enhanced MHC class I and II presentation (Kreiter, S. et al., 2008, The Journal of Immunology 180(1):309-18). Intraperitoneally administered liposomally formulated RNA (RNA-LPX) was designed to specifically target the translation and MHC presentation of encoded antigens to resident DCs within lymphoid organs (Kranz, Lena Mareen et al., 2016, Nature 534(7607):396-401). Internalized by DCs, RNA-LPX mimics infectious non-self and functions as a natural TLR7 / 8 ligand, triggering a potent type I IFN-dominated innate response accompanied by pro-inflammatory cytokines.

[0007] Our RNA-LPX vaccine platform consists of non-nucleoside modified RNA (standard RNA, stdRNA) that is not subjected to double-stranded RNA purification, simultaneously providing target identity, i.e., antigen, and adjuvant.

[0008] To reduce the immunogenicity of vaccine RNA, nucleosides can be modified and residual double-stranded RNA can be removed. The inventors have synthesized N1-methyl-pseudourine-modified and cellulose-purified vaccine RNA (modified RNA, modRNA) (Andries, Oliwia et al., 2015, Journal of Controlled Release: Official Journal of the Controlled Release Society 217:337-44; Baiersdorfer, Markus et al., 2019, Molecular Therapy-Nucleic Acids 15 (April); Pardi, Norbert et al., 2015, Journal of Controlled Release: Official Journal of the Controlled Release Society 217:345-51). Compared to stdRNA, modRNA limits immune activation and systemic IFNα release.

[0009] Here, we demonstrate that immune effector cells, such as T cells, induced by modRNA express higher levels of PD-1 than cells induced by standard RNA. We demonstrate that the combination of modRNA vaccination and PD-1 axis-binding antagonist treatment results in efficient vaccination, including efficient antigen-specific immune responses and antitumor activity. [Means for solving the problem]

[0010] The present invention generally encompasses immunotherapeutic treatment of a subject, including (i) administering to the subject non-immunogenic RNA encoding a peptide or polypeptide containing an epitope for inducing an immune response to an antigen in the subject, i.e., non-immunogenic RNA encoding a vaccine antigen, and (ii) administering to the subject a PD-1 axis-binding antagonist, such as an anti-PD-1 antibody and / or an anti-PD-L1 antibody, for example, by administering RNA encoding the PD-1 axis-binding antagonist or PD-1 axis-binding antagonist. The immunotherapies described herein include vaccine therapy and may further include cell-based cancer immunotherapy, such as TIL or T cell-based therapy, e.g., TCR or CAR transgenic T cell-based therapy using autologous cells. Generally, immune effector cells stimulated using the treatments described herein can target antigen-expressing cells, such as diseased cells, particularly cancer cells expressing tumor antigens. Target cells may express the antigen on their cell surface or display a processed product of the antigen. In some embodiments, the antigen is a tumor-associated antigen and the disease is cancer. Such treatment provides selective eradication of cells that express the antigen, thereby minimizing adverse effects on normal cells that do not express the antigen. Immune effector cells (which may be genetically modified to express an antigen receptor) target the antigen or its processing product, and thus target cell populations or target tissues that express the antigen. Such immune effector cells can be administered to a subject in need of treatment or can be endogenous to the subject in need of treatment. In some embodiments, the immune effector cells carry an antigen receptor, such as a T cell receptor (TCR) or chimeric antigen receptor (CAR), that has binding specificity for the target antigen or its processing product. In some embodiments, the immune effector cells are genetically modified to express the antigen receptor. Such genetic modification to express the antigen receptor can be performed ex vivo or in vitro, after which the immune effector cells can be administered to a subject in need of treatment, or can be performed in vivo in the subject in need of treatment, or can be performed by a combination of ex vivo or in vitro and in vivo modification.Non-immunogenic RNA encoding a vaccine antigen is administered to provide the antigen (after expression of the polynucleotide by appropriate target cells) for stimulation, priming, and / or expansion of immune effector cells that target the target antigen or its processing products. In some embodiments, the immune response induced in accordance with the present disclosure is an immune response against a target cell population or target tissue expressing the antigen to which the immune effector cells are directed. In some embodiments, the immune response induced in accordance with the present disclosure is a T cell-mediated immune response. In some embodiments, the immune response is an anti-tumor immune response, and the target cell population or target tissue is a tumor cell or tumor tissue.

[0011] The PD-1 axis-binding antagonist, such as an anti-PD-1 antibody and / or an anti-PD-L1 antibody, can be provided by administering a PD-1 axis-binding antagonist. Alternatively, the PD-1 axis-binding antagonist, such as an anti-PD-1 antibody and / or an anti-PD-L1 antibody, can be administered in the form of RNA encoding the PD-1 axis-binding antagonist. In some embodiments, the RNA is targeted to the liver for systemic availability. Hepatocytes can be efficiently transfected and produce large amounts of protein.

[0012] The methods and medicaments described herein may further provide for the administration or inclusion of an immunostimulatory agent or an RNA encoding an immunostimulatory agent. In some embodiments, the methods and medicaments described herein do not provide for the administration or inclusion of an immunostimulatory agent or an RNA encoding an immunostimulatory agent. In some embodiments, the methods and medicaments described herein provide for the administration or inclusion of an immunostimulatory agent or an RNA encoding an immunostimulatory agent.

[0013] The immunostimulant may be conjugated to a pharmacokinetic-modifying group (hereinafter referred to as an "extended pharmacokinetics (PK)" immunostimulant). In some embodiments, the RNA encoding the immunostimulant is targeted to the liver for systemic availability. Hepatocytes can be efficiently transfected and produce large amounts of protein.

[0014] The RNA encoding the vaccine antigen is preferably targeted to secondary lymphoid organs.

[0015] In one aspect, there is provided a method for inducing an immune response in a subject, comprising: (i) administering to a subject a non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope for inducing an immune response in the subject against an antigen; (ii) providing to the subject a PD-1 axis binding antagonist.

[0016] In some embodiments, the subject has a disease, disorder, or condition associated with expression or up-regulation of the antigen.

[0017] In one aspect, there is provided a method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: (i) administering to a subject a non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope for inducing an immune response in the subject against an antigen; (ii) providing to the subject a PD-1 axis binding antagonist.

[0018] In some embodiments, the immune response is a T cell-mediated immune response.

[0019] In some embodiments, the immune response comprises the generation of antigen-specific T cells.

[0020] In some embodiments, the antigen is a tumor-associated antigen.

[0021] In some embodiments, the disease, disorder, or condition is cancer.

[0022] In some embodiments, the method comprises: (i) a non-immunogenic RNA encoding a peptide or polypeptide containing the epitope; and (ii) administering to the subject a PD-1 axis binding antagonist, or RNA encoding a PD-1 axis binding antagonist.

[0023] In some embodiments, the method comprises: (i) a non-immunogenic RNA encoding a peptide or polypeptide containing the epitope; and (ii) administering a PD-1 axis binding antagonist to the subject.

[0024] In some embodiments, the non-immunogenic RNA, when administered, results in reduced dendritic cell activation, T cell activation and / or IFN-α secretion compared to standard RNA.

[0025] In some embodiments, the non-immunogenic RNA is made non-immunogenic by incorporating modified nucleosides and / or limiting the amount of double-stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA is made non-immunogenic by incorporating modified nucleosides and / or removing dsRNA.

[0026] In some embodiments, the modified nucleoside inhibits RNA-mediated activation of an innate immune receptor.

[0027] In some embodiments, the modified nucleoside comprises a substitution of one or more uridines with a nucleoside comprising a modified nucleobase.

[0028] In some embodiments, the modified nucleobase is a modified uracil.

[0029] In some embodiments, the nucleoside comprising a modified nucleobase is 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cm o5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine Douridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um) , 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine].

[0030] In some embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0031] In some embodiments, the nucleoside comprising a modified nucleobase is 1-methyl-pseudouridine.

[0032] In some embodiments, the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is mRNA.

[0033] In some embodiments, the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is in vitro transcribed RNA.

[0034] In some embodiments, non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope is administered in a formulation to target the lymphatic system, which in some embodiments includes secondary lymphoid organs, particularly the spleen.

[0035] In some embodiments, non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope is administered in a formulation to target dendritic cells.

[0036] In some embodiments, the dendritic cells are immature dendritic cells.

[0037] In some embodiments, non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope is administered in a formulation comprising lipoplex (LPX) particles.

[0038] In some embodiments, the PD-1 axis binding antagonist comprises a PD-1 binding antagonist.

[0039] In some embodiments, the PD-1 binding antagonist comprises an anti-PD-1 antibody.

[0040] In some embodiments, the anti-PD-1 antibody comprises nivolumab or pembrolizumab.

[0041] In some embodiments, the PD-1 axis binding antagonist comprises a PD-L1 binding antagonist.

[0042] In some embodiments, the PD-L1 binding antagonist comprises an anti-PD-L1 antibody.

[0043] In some embodiments, the anti-PD-L1 antibody comprises atezolizumab, avelumab, or durvalumab.

[0044] In some embodiments, the methods comprise administering an immunostimulant or RNA encoding an immunostimulant.

[0045] In some embodiments, the methods do not include administering an immunostimulant or RNA encoding an immunostimulant.

[0046] In some embodiments, the immunostimulant is a pro-inflammatory or anti-inflammatory immunostimulant.

[0047] In some embodiments, the immunostimulant comprises a cytokine or a variant thereof.

[0048] In some embodiments, the cytokine comprises a type I interferon or a variant thereof.

[0049] In some embodiments, the type I interferon comprises interferon-α or a variant thereof.

[0050] In some embodiments, the cytokine comprises an interleukin or a variant thereof.

[0051] In some embodiments, the cytokines support T cell priming.

[0052] In some embodiments, the cytokine comprises IL12, IL15, or a variant thereof.

[0053] In some embodiments, the cytokine supports the proliferation and / or maintenance of T cells.

[0054] In some embodiments, the cytokine comprises IL2, IL7, or a variant thereof.

[0055] In some embodiments, the immunostimulant is an extended pharmacokinetic (PK) immunostimulant. In some embodiments, the extended PK immunostimulant comprises a fusion protein. In some embodiments, the fusion protein comprises an immunostimulant portion and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof. In some embodiments, the serum albumin comprises mouse serum albumin or human serum albumin. In some embodiments, the immunoglobulin fragment comprises an immunoglobulin Fc domain.

[0056] In some embodiments, the RNA encoding the immunostimulant is present in a formulation for targeting to the lymphatic system.

[0057] In some embodiments, the RNA encoding the immunostimulant is present in a formulation for targeting to the liver.

[0058] In some embodiments, the lymphatic system is a secondary lymphoid organ, particularly the spleen.

[0059] In some embodiments, the RNA encoding the immunostimulatory agent is non-immunogenic. In some embodiments, the RNA encoding the immunostimulatory agent is mRNA. In some embodiments, the RNA encoding the immunostimulatory agent is in vitro transcribed RNA.

[0060] In some embodiments, the non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope and the PD-1 axis-binding antagonist or RNA encoding a PD-1 axis-binding antagonist are administered in a common formulation or in separate formulations.

[0061] In some embodiments, the method is for treating or preventing cancer in a subject.

[0062] In some embodiments, a non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope is transiently expressed in cells of a subject.

[0063] In some embodiments, the subject is a human.

[0064] In one aspect, (i) a non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope for inducing an immune response to an antigen in a subject; and (ii) Provided herein are pharmaceutical formulations comprising a PD-1 axis binding antagonist or RNA encoding a PD-1 axis binding antagonist.

[0065] In some embodiments, the pharmaceutical preparation is for treating a disease, disorder, or condition associated with expression or up-regulation of an antigen.

[0066] In some embodiments, the immune response is a T cell-mediated immune response.

[0067] In some embodiments, the immune response comprises the generation of antigen-specific T cells.

[0068] In some embodiments, the antigen is a tumor-associated antigen.

[0069] In some embodiments, the disease, disorder, or condition is cancer.

[0070] In some embodiments, the pharmaceutical formulation comprises: (i) a non-immunogenic RNA encoding a peptide or polypeptide containing the epitope; and (ii) includes PD-1 axis binding antagonists.

[0071] In some embodiments, the non-immunogenic RNA, when administered, results in reduced dendritic cell activation, T cell activation and / or IFN-α secretion compared to standard RNA.

[0072] In some embodiments, the non-immunogenic RNA is made non-immunogenic by incorporating modified nucleosides and / or limiting the amount of double-stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA is made non-immunogenic by incorporating modified nucleosides and / or removing dsRNA.

[0073] In some embodiments, the modified nucleoside inhibits RNA-mediated activation of an innate immune receptor.

[0074] In some embodiments, the modified nucleoside comprises a substitution of one or more uridines with a nucleoside comprising a modified nucleobase.

[0075] In some embodiments, the modified nucleobase is a modified uracil.

[0076] In some embodiments, the nucleoside comprising a modified nucleobase is 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cm o5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine Douridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um) , 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine].

[0077] In some embodiments, the nucleoside comprising a modified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0078] In some embodiments, the nucleoside comprising a modified nucleobase is 1-methyl-pseudouridine.

[0079] In some embodiments, the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is mRNA.

[0080] In some embodiments, the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is in vitro transcribed RNA.

[0081] In some embodiments, the non-immunogenic RNA encoding the epitope-containing peptide or polypeptide is present in the formulation for targeting to the lymphatic system, which in some embodiments includes secondary lymphoid organs, particularly the spleen.

[0082] In some embodiments, non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope is present in the formulation for targeting dendritic cells.

[0083] In some embodiments, the dendritic cells are immature dendritic cells.

[0084] In some embodiments, the non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope is present in a formulation comprising lipoplex (LPX) particles.

[0085] In some embodiments, the PD-1 axis binding antagonist comprises a PD-1 binding antagonist.

[0086] In some embodiments, the PD-1 binding antagonist comprises an anti-PD-1 antibody.

[0087] In some embodiments, the anti-PD-1 antibody comprises nivolumab or pembrolizumab.

[0088] In some embodiments, the PD-1 axis binding antagonist comprises a PD-L1 binding antagonist.

[0089] In some embodiments, the PD-L1 binding antagonist comprises an anti-PD-L1 antibody.

[0090] In some embodiments, the anti-PD-L1 antibody comprises atezolizumab, avelumab, or durvalumab.

[0091] In some embodiments, the pharmaceutical formulation comprises an immunostimulant or RNA encoding an immunostimulant.

[0092] In some embodiments, the pharmaceutical formulation does not comprise an immunostimulant or RNA encoding an immunostimulant.

[0093] In some embodiments, the immunostimulant is a pro-inflammatory or anti-inflammatory immunostimulant.

[0094] In some embodiments, the immunostimulant comprises a cytokine or a variant thereof.

[0095] In some embodiments, the cytokine comprises a type I interferon or a variant thereof.

[0096] In some embodiments, the type I interferon comprises interferon-α or a variant thereof.

[0097] In some embodiments, the cytokine comprises an interleukin or a variant thereof.

[0098] In some embodiments, the cytokines support T cell priming.

[0099] In some embodiments, the cytokine comprises IL12, IL15, or a variant thereof.

[0100] In some embodiments, the cytokine supports the proliferation and / or maintenance of T cells.

[0101] In some embodiments, the cytokine comprises IL2, IL7, or a variant thereof.

[0102] In some embodiments, the immunostimulant is an extended pharmacokinetic (PK) immunostimulant. In some embodiments, the extended PK immunostimulant comprises a fusion protein. In some embodiments, the fusion protein comprises an immunostimulant portion and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof. In some embodiments, the serum albumin comprises mouse serum albumin or human serum albumin. In some embodiments, the immunoglobulin fragment comprises an immunoglobulin Fc domain.

[0103] In some embodiments, the RNA encoding the immunostimulant is present in a formulation for targeting to the lymphatic system.

[0104] In some embodiments, the RNA encoding the immunostimulant is present in a formulation for targeting to the liver.

[0105] In some embodiments, the lymphatic system is a secondary lymphoid organ, particularly the spleen.

[0106] In some embodiments, the RNA encoding the immunostimulatory agent is non-immunogenic. In some embodiments, the RNA encoding the immunostimulatory agent is mRNA. In some embodiments, the RNA encoding the immunostimulatory agent is in vitro transcribed RNA.

[0107] In some embodiments, the non-immunogenic RNA encoding the epitope-containing peptide or polypeptide and the PD-1 axis-binding antagonist or RNA encoding the PD-1 axis-binding antagonist are present in a common formulation or in separate formulations.

[0108] In some embodiments, the pharmaceutical formulation is a kit.

[0109] In some embodiments, the pharmaceutical formulation comprises a non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope and a PD-1 axis binding antagonist or RNA encoding a PD-1 axis binding antagonist in a pharmaceutical composition.

[0110] In some embodiments, the pharmaceutical formulation comprises a non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope and a PD-1 axis-binding antagonist or an RNA encoding a PD-1 axis-binding antagonist in separate containers.

[0111] In some embodiments, the pharmaceutical formulation further comprises instructions for using the pharmaceutical formulation.

[0112] In some embodiments, the pharmaceutical formulation is a pharmaceutical composition.

[0113] In one aspect, provided herein is a pharmaceutical formulation as described herein for pharmaceutical use.

[0114] In some embodiments, the medical use includes the therapeutic or prophylactic treatment of a disease or disorder.

[0115] In some embodiments, the disease or disorder is cancer.

[0116] In one aspect, provided herein is a pharmaceutical formulation described herein for use in the methods described herein.

[0117] In some embodiments, the RNA described herein is a single-stranded RNA that can be translated into a respective protein upon entry into a cell, e.g., a recipient cell. In addition to an amino acid sequence, e.g., a wild-type or codon-optimized sequence encoding a pharmaceutically active peptide or polypeptide (epitope-containing peptide or polypeptide), such as an antigen sequence, the RNA may contain one or more structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized for maximum RNA effectiveness in terms of stability and translation efficiency. In some embodiments, the RNA contains all of these elements. In some embodiments, the RNA is a β-S-ARCA(D1)(m2 7,2’-O GppSpG) or m2 7,3’-O Gppp(m1 2’-O ) ApG may be used as a specific capping structure at the 5' end of the RNA drug substance. The 5'-UTR sequence may be the 5'-UTR sequence of human α-globin mRNA, which may have an optimized "Kozak sequence" to increase translation efficiency. The 3'-UTR sequence may be a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial-encoded 12S ribosomal RNA (called I) located between the coding sequence and the poly(A) tail to ensure higher maximum protein levels and long-term mRNA persistence. These were identified through an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see International Publication No. 2017 / 060314, incorporated herein by reference). Alternatively, the 3'-UTR may be two repeat 3'-UTRs of human β-globin mRNA. Additionally, a poly(A) tail measuring 110 nucleotides in length may be used, consisting of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues, designed to enhance RNA stability and translation efficiency.

[0118] An epitope-containing peptide or polypeptide may contain an amino acid sequence other than the amino acid sequence of the epitope or antigen. In some embodiments, such other amino acid sequence includes an amino acid sequence that enhances antigen processing and / or presentation. Alternatively or additionally, such other amino acid sequence includes an amino acid sequence that disrupts immune tolerance.

[0119] Nucleic acid such as RNA as described herein can be complexed with polymer, protein and / or lipid, preferably lipid, to produce nucleic acid particles for administration.When different nucleic acid combinations are used, nucleic acid can be complexed together or separately. [Brief explanation of the drawings]

[0120] [Figure 1] This figure shows that vaccination with modRNA results in enhanced PD-1 expression on vaccine-induced antigen-specific CD8+ T cells compared with vaccination with uRNA. C57BL / 6 mice (n = 3 per group and time point) were vaccinated twice intravenously on days 0 and 7 with 20 μg of RNA-LPX consisting of modRNA or uRNA encoding the H-2Kb-restricted epitope OVA257-264 (SIINFEKL). Control mice were administered NaCl. (a) Percentage of PD-1-expressing OVA-specific CD8+ T cells in the spleen 3, 5, and 7 days after each vaccination (mean ± SEM) and (b) PD-1 expression on OVA-specific CD8+ T cells (mean MFI values ​​± SEM). (c) PD-1 expression on OVA-specific CD8+ T cells in the blood 5 days after the second vaccination (mean MFI values ​​± SEM). Data were not plotted when the percentage of OVA-specific CD8+ T cells was less than 1% (a, b). Vertical dotted lines indicate treatment days (a, b). Unpaired t-test (c). **: P≦0.01. modRNA, nucleoside-modified RNA. uRNA, uridine-containing RNA. OVA, chicken ovalbumin. MFI, median fluorescence intensity. RNA, ribonucleic acid. PD-1, programmed death receptor 1. [Figure 2]Figure 1 shows the enhanced efficacy of modRNA vaccination in combination with checkpoint blockade, particularly when vaccinating against a self-antigen. (a) C57BL / 6 mice (n=5 / group) were vaccinated IV five times (days 0, 7, 14, 21, and 28) with 1 or 10 μg of RNA-LPX consisting of modRNA encoding the H-2Kb-restricted epitope OVA257-264 (SIINFEKL) and simultaneously treated IP with 200 μg of anti-PD-L1 antibody. Control mice received modRNA and isotype or NaCl. Percentage of OVA-specific CD8+ T cells among total CD8+ T cells in the blood 5 days after each vaccination (mean ± SEM). (b) C57BL / 6 mice (n = 5 / group) were vaccinated five times intravenously (on days 0, 7, 14, 21, and 28) with 20 μg of RNA-LPX, consisting of modRNA encoding TRP2180-188 (SVYDFFVWL) fused to TRP88-102 (RKFFHRTCKCTGNFA), an MHC class II-presented epitope of human TRP2, and simultaneously treated with 250 μg of anti-PD-1 antibody IP. Control mice received modRNA and isotype or NaCl. Percentage of TRP2-specific CD8+ T cells among total CD8+ T cells in the blood 5 days after each vaccination (mean ± SEM). Vertical dotted lines indicate the treatment day (a, b). Statistical significance was determined by mixed-effects analysis and Tukey's multiple comparison test (b). *: P ≤ 0.05, **: P ≤ 0.01. modRNA is nucleoside-modified RNA. RNA is ribonucleic acid. OVA is chicken ovalbumin. TRP2 is tyrosinase-related protein 2. PD-1 is programmed death receptor 1. PD-L1 is programmed death receptor ligand 1. [Figure 3]Figure 1 shows the enhanced therapeutic antitumor activity of modRNA vaccination in combination with checkpoint blockade compared to modRNA vaccination alone. C57BL / 6 mice (n=10 / group) were inoculated SC with B16-F10 tumor cells and vaccinated weekly IV with 10 μg of RNA-LPX, consisting of modRNA encoding TRP2180-188 (SVYDFFVWL) fused to TRP88-102 (RKFFHRTCKCTGNFA), an MHC class II-presented epitope of human TRP2, from day 9 to day 65 after tumor inoculation. Mice were simultaneously treated IP with anti-PD-L1 antibody or isotype control (primary treatment: 10 mg / kg, subsequent treatments: 5 mg / kg). Control mice received control RNA along with anti-PD-L1 antibody. (a) Individual tumor growth. (b) Survival. ModRNA is nucleoside-modified RNA. RNA is ribonucleic acid. TRP2 is tyrosinase-related protein 2. PD-L1 is programmed death receptor ligand 1. [Figure 4]This figure shows that the addition of IL-2 to the combination of modRNA vaccination and checkpoint blockade enhances the induction of antigen-specific CD8+ T cells compared to the dual combination. C57BL / 6 mice (n=7 / group) were vaccinated three times IV (on days 0, 7, and 14) with 20 μg of RNA-LPX, consisting of modRNA encoding TRP2180-188 (SVYDFFVWL) fused to TRP88-102 (RKFFHRTCKCTGNFA), an MHC class II-presented epitope of human TRP2. Simultaneously with the second and third vaccinations, they were treated with 10 mg / kg of anti-PD-1 antibody or isotype control IP and 3 μg of IL-2 or albumin control IV. Control mice were administered NaCl. (a) Percentage of TRP2-specific CD8+ T cells among total CD8+ T cells in the blood (left) and spleen (right) 5 days after the third vaccination. (b) Ratio of TRP2-specific CD8+ T cells to regulatory T cells in the spleen 5 days after the third vaccination. (c) Percentage of IFNγ-secreting TRP2-specific CD8+ T cells among total CD8+ T cells after ex vivo restimulation with TRP2 peptide or no peptide. Data were not plotted if the percentage of TRP2-specific CD8+ T cells was less than 0.5% (c). In the group treated with the triplicate combination, one outlier was removed (spleen; a, b). Each dot represents one mouse, and the horizontal line represents the mean (a, b). Statistical significance was determined by one-way analysis of variance and Tukey's multiple comparison test (a, b) or mixed-effects analysis and Tukey's multiple comparison test (c). ns: P > 0.05, ***: P ≤ 0.001, ****: P ≤ 0.0001. modRNA is nucleoside-modified RNA. RNA is ribonucleic acid. TRP2 is tyrosinase-related protein 2. Tregs are regulatory T cells. PD-1 is programmed death receptor 1. [Figure 5]Figure 1 shows that the addition of IL-2 to the combination of modRNA vaccination and checkpoint blockade enhances therapeutic anti-tumor activity compared to the dual combination. C57BL / 6 mice (n=10 / group) were inoculated SC with B16-F10 tumor cells and vaccinated weekly IV with 10 μg of RNA-LPX, consisting of modRNA encoding TRP2180-188 (SVYDFFVWL) fused to TRP88-102 (RKFFHRTCKCTGNFA), an MHC class II-presented epitope of human TRP2, from day 8 to day 91 post-tumor inoculation. Mice were simultaneously treated IP with anti-PD-L1 antibody (primary treatment: 10 mg / kg, subsequent treatments: 5 mg / kg) and IV with 1 μg of IL-2 or albumin control 2 days after each vaccination / anti-PD-L1 treatment. (a) Individual tumor growth. (b) Survival rate. (c) Representative images of mice experiencing vitiligo in response to treatment. modRNA is nucleoside-modified RNA. RNA is ribonucleic acid. TRP2 is tyrosinase-related protein 2. IL-2 is interleukin-2. PD-L1 is programmed death receptor ligand 1.

[0121] Array Description The following table provides a list of the specific sequences referenced herein.

[0122] [Table 1] DETAILED DESCRIPTION OF THE INVENTION

[0123] Although the present disclosure will be further described in more detail below, it should be understood that the disclosure is not limited to the specific methods, protocols and reagents described herein, and these may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0124] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The variously described examples and preferred embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiment with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0125] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques described in the art.

[0126] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprises" and variations such as "comprising" are understood to imply the inclusion of a stated feature, element, member, integer, or step or group of features, elements, members, integers, or steps, but not the exclusion of any other feature, element, member, integer, or step or group of features, elements, members, integers, or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps and to those that do not materially affect the basic and novel characteristics of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, at each occurrence in this application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of." Similarly, at each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0127] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0128] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0129] The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better describe the disclosure and does not pose a limitation on the scope of the claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0130] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where the event, circumstance, or condition occurs and cases where it does not occur.

[0131] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" should be interpreted as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0132] In the context of the present disclosure, the term "about" indicates an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the indicated numerical value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, e.g., ±0.01%. In some embodiments, "about" indicates a deviation of ±10% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±5% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±4% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±3% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±2% from the indicated numerical value. In some embodiments, "about" indicates a deviation of ±1% from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the indicated numerical value. As will be understood by one of ordinary skill in the art, such specific deviations from the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than artificial or engineered technical effects.

[0133] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein.

[0134] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0135] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Terms not defined have their art-wide accepted meanings.

[0136] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0137] As used herein, terms such as "enhance" or "enhance" refer to the ability to produce an overall increase or enhancement in a level, for example, by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0138] As used herein, "physiological pH" refers to a pH of about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0139] As used in this disclosure, "% w / v" refers to weight-to-volume percent, a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of a solution in milliliters (mL).

[0140] As used in this disclosure, "wt. %" refers to weight percent, a unit of concentration that measures the amount of a substance in grams (g) expressed as a percentage of the total weight of the entire composition in grams (g).

[0141] As used in this disclosure, "mol %" is defined as the ratio of moles of one component to the total moles of all components multiplied by 100.

[0142] As used in this disclosure, "mol %" is defined as the ratio of moles of one lipid component to the total moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like substances.

[0143] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Thus, ionic strength, I, is calculated by the formula:

[0144]

number

[0145] where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum Σ applies to all the different types of ions (i) in the solution.

[0146] According to the present disclosure, the term "ionic strength" in some embodiments refers to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, due to the presence of chelating agents, their concentration or effective concentration (presence of free ions) is, in some embodiments, sufficiently low to prevent degradation of nucleic acids. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of phosphodiester bonds between nucleotides, such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or essentially absent.

[0147] "Osmolality" refers to the concentration of a particular solute expressed as osmoles of solute per kilogram of solvent.

[0148] The term "lyophilize" or "lyophilization" refers to the lyophilization of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or 1 Pa or less) to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilize" and "freeze-dry" are used interchangeably herein.

[0149] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with the fluid to be atomized (atomized) in a vessel (spray dryer), where the solvent from the droplets formed evaporates, resulting in a dry powder.

[0150] The term "reconstitute" relates to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0151] The term "recombinant" in the context of this disclosure means "produced through genetic engineering." In some embodiments, "recombinant" in the context of this disclosure is not naturally occurring.

[0152] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects and objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0153] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C.

[0154] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given in terms of EDTA disodium salt.

[0155] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing step.

[0156] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0157] According to the present disclosure, the term "peptide" refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Although both "peptide" and "polypeptide" are protein molecules, the terms "protein" and "polypeptide" are generally used synonymously herein.

[0158] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" can refer to a contiguous or discontinuous fraction of that structure.

[0159] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure such as an amino acid sequence or protein refers to a continuous element of that structure. When used in reference to a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0160] With respect to an amino acid sequence (peptide or polypeptide), the term "fragment" refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence comprises, for example, at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence include, for example, sequences of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

[0161] As used herein, "variant" with respect to an amino acid sequence (peptide or polypeptide) means an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence can be a native or wild-type (WT) amino acid sequence, or can be a modified form of the wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences compared to the parent, e.g., 1 to about 10 or 1 to about 5 amino acid differences.

[0162] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or polypeptide has an amino acid sequence that has not been intentionally modified.

[0163] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformational variants, isoform variants, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0164] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-termini of a protein are also referred to as N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions within the amino acid sequence that are not conserved between homologous peptides or peptides and / or substitutions of amino acids with other amino acids with similar properties are preferred. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of one member of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: -Glycine, alanine; -valine, isoleucine, leucine; -Aspartic acid, glutamic acid; -Asparagine, glutamine; -Serine, threonine; -Lysine, arginine; and -Phenylalanine, tyrosine.

[0165] In some embodiments, the degree of similarity, such as identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given for, for example, at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, such as sequence identity, can be performed using tools known in the art, for example, using best sequence alignment, for example, Align, using standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0166] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0167] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. The percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually performed by comparing the sequences over a segment or "comparison window" after optimal alignment to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually, or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, or the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, or the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms available at the United States National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 28; (iii) a maximum match within the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for low-complexity regions being used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word size set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0168] The percent identity is obtained by determining the number of identical positions where the compared sequences match, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0169] In some embodiments, the degree of similarity or identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0170] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, for example at least 95%, at least 98 or at least 99% of the amino acid residues.

[0171] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or polypeptides with substitutions, additions, insertions, or deletions is described, for example, in Molecular Cloning: A Laboratory Manual, 4 th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptides, polypeptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, by solid phase synthesis and similar methods.

[0172] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" specifically refers to a variant molecule or sequence that contains an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence, yet still performs one or more functions of the parent molecule or sequence, e.g., is capable of inducing an immune response. In some embodiments, alterations to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example, the function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0173] An amino acid sequence (peptide or polypeptide) "derived from" a specified amino acid sequence (peptide or polypeptide) refers to the origin of the initial amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be modified to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0174] In some embodiments, "isolated" means removed (e.g., purified) from a natural state or from an artificial composition, such as a composition from a manufacturing process. For example, a nucleic acid, peptide, or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid, peptide, or polypeptide can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0175] The term "genetic modification" or simply "modification" includes the transfection of a cell with a nucleic acid.

[0176] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by such cells, and the cells may be present in a subject, e.g., a patient, or the cells may be present in vitro, e.g., outside the patient. Thus, according to this disclosure, cells for transfection of nucleic acids described herein can be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue, and / or body of a patient. According to this disclosure, transfection can be transient or stable. In some applications of transfection, it is sufficient for the transfected genetic material to be expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Nucleic acids introduced during the transfection process are typically not integrated into the nuclear genome, and the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that transfected nucleic acid actually remains in the genome of cell and its daughter cells, stable transfection must occur.Such stable transfection can be achieved, for example, by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding an antigen is transiently transfected into cells.Generally, the cell that is genetically modified to express antigen receptor is stably transfected with the nucleic acid encoding the receptor.RNA can be transfected into cells to transiently express its encoded protein.

[0177] The present disclosure includes analogs of peptides or polypeptides. According to the present disclosure, a peptide or polypeptide analog is a modified form of the peptide or polypeptide from which it is derived, retaining at least one functional property of the peptide or polypeptide. For example, a pharmacologically active analog of a peptide or polypeptide retains at least one pharmacological activity of the peptide or polypeptide from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecule associated with the peptide or polypeptide, such as carbohydrates, lipids, and / or peptides or polypeptides. In some embodiments, a "peptide or polypeptide analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the peptides and polypeptides.

[0178] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0179] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0180] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0181] According to various embodiments of the present disclosure, nucleic acids such as RNAs encoding a peptide or polypeptide are taken up or introduced, i.e., transfected or transduced, into cells, which may be present in vitro or in a subject, resulting in expression of the peptide or polypeptide. The cells may, for example, express the encoded peptide or polypeptide intracellularly (e.g., in the cytoplasm and / or nucleus), secrete the encoded peptide or polypeptide, and / or express it on their surface.

[0182] In accordance with the present disclosure, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms are used interchangeably herein and mean, with respect to a particular peptide or polypeptide, that the nucleic acid, when present in an appropriate environment, e.g., a cell, can be expressed to produce the peptide or polypeptide.

[0183] As used herein, the term "expression" includes the transcription and / or translation of a particular nucleotide sequence.

[0184] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA), which can then be translated into peptides or polypeptides.

[0185] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0186] The pharmaceutical preparations described herein, particularly the kits, may include instruction materials or instructions. As used herein, "instruction materials" or "instructions" include publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instruction materials of the kits of the present invention may, for example, be attached to a container containing the composition of the present invention or may be shipped together with a container containing the composition. Alternatively, the instruction materials may be shipped separately from the container, with the intention that the instruction materials and the composition will be used in conjunction with each other by the recipient.

[0187] Prodrugs of certain compounds described herein are compounds that undergo chemical conversion under physiological conditions to provide the specified compounds upon administration to an individual. Furthermore, prodrugs can be converted to the specified compounds by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the specified compounds when placed in a transdermal patch reservoir with appropriate enzymes or chemical reagents. Exemplary prodrugs are in vivo hydrolyzable esters (using alcohol or carboxy groups contained in certain compounds) or amides (using amino or carboxy groups contained in certain compounds). Specifically, any amino group contained in certain compounds that bears at least one hydrogen atom can be converted to a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0188] In this specification, the structural formula of a compound may represent a particular isomer of the compound. However, it should be understood that the present invention includes all isomers and mixtures of isomers, such as structurally occurring geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and is not limited to the description of the formula.

[0189] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or in the geometric (spatial) arrangement of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains a pair of equal amounts of enantiomers and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even when pure, spontaneously and reversibly interconvert due to the migration of individual atoms or groups of atoms; i.e., tautomers are in dynamic chemical equilibrium with each other. One example of a tautomer is keto-enol tautomerism. "Conformers" are stereoisomers that are formally interconvertible by rotation about a single bond alone, and include in particular those resulting in different three-dimensional forms of a (hetero)cyclic ring, such as the chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0190] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in a so-called Z dimension with a length dimension. 平均 , and the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is defined as this Z 平均 Used synonymously with the value of

[0191] In some embodiments, the "polydispersity index," as mentioned in the definition of "mean diameter," can be calculated based on dynamic light scattering measurements by so-called cumulant analysis. Under certain prerequisites, this can be considered as a measure of the size distribution of an ensemble of nanoparticles.

[0192] The "radius of gyration" of the particle around the axis of rotation (R g) is the radial distance from the axis of rotation of the point at which the moment of inertia of a particle about a given axis is the same as its actual mass distribution, if the entire mass of the particle were assumed to be concentrated. Mathematically, R g is the root mean square distance of a particle's components from either its center of mass or a given axis. For example, if the particle is at a fixed distance s from the center of mass, i Mass m located at i For a polymer consisting of n mass elements (i=1, 2, 3, ..., n), R g is the s over all mass elements i 2 is the mass-averaged square root of and can be calculated as follows:

[0193]

number

[0194] The radius of gyration can be determined experimentally or calculated, for example, by using light scattering. In particular, for small scattering vectors

[0195]

number

[0196] , the structure function S is defined as:

[0197]

number

[0198] where N is the number of components (Guinier's law).

[0199] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only size but also solvent effects. For example, a smaller, more hydrated charged particle may have a larger hydrodynamic radius than a larger, more weakly hydrated charged particle. This is because the smaller particle drags more water molecules with it as it moves through the solution. Since the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius is determined by the Stokes-Einstein equation:

[0200]

number

[0201] where k B is the Boltzmann constant, T is the temperature, η is the viscosity of the solvent, and D is the diffusion coefficient. The diffusion coefficient can be experimentally determined, for example, by using dynamic light scattering (DLS). Thus, one procedure for determining the hydrodynamic radius of a particle or particle population (e.g., the hydrodynamic radius of a particle contained in a sample or control composition disclosed herein, or the hydrodynamic radius of a particle peak obtained by subjecting such a sample or control composition to field-flow fractionation) is to measure the DLS signal of the particle or particle population (e.g., the DLS signal of a particle contained in a sample or control composition disclosed herein, or the DLS signal of a particle peak obtained by subjecting such a sample or control composition to field-flow fractionation).

[0202] As used herein, the expression "light scattering" refers to a physical process in which light is forced to deviate from a straight line trajectory by one or more paths due to local inhomogeneities in the medium through which it passes.

[0203] The term "UV" means ultraviolet and refers to the band of the electromagnetic spectrum having wavelengths between 10 nm and 400 nm, i.e., shorter than those of visible light but longer than X-rays.

[0204] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered at multiple angles by a sample. "Multi-angle" in this context means that the scattered light can be detected at different discrete angles, as measured, for example, by a single detector moving over a range that includes a selected specific angle, or by an array of detectors fixed at specific angular positions. In certain embodiments, the light source used in MALS is a laser source (MALLS: Multi-Angle Laser Light Scattering). Based on the MALS signal of a composition containing particles, the radius of gyration (R) can be calculated by using an appropriate format (e.g., Zimm plot, Berry plot, or Debye plot). g ), and thus it is possible to determine the size of the particles. Preferably, the Zimm plot is calculated using the following formula:

[0205]

number

[0206] where c is the mass concentration of particles in the solvent (g / mL) and A2 is the second virial coefficient (mol mL / g 2 ), P(θ) is the form factor for the angular dependence of the scattered light intensity, and R θ is the excess Rayleigh ratio (cm -1 ) and K* is 4π 2 η o (dn / dc) 2 λ0 -4 N A -1 is an optical constant equal to , where η o is the refractive index of the solvent at the incident radiation (vacuum) wavelength, λ is the incident radiation (vacuum) wavelength (nm), and N A is Avogadro's number (mol -1), and dn / dc is the differential refractive index increment (mL / g) (see, for example, Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); BH Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291)). Preferably, the Berry plot has the following terms:

[0207]

number

[0208] where c, R θ and K* is as defined above. Preferably, the Debye plot comprises the following terms:

[0209]

number

[0210] where c, R θ and K* is as defined above.

[0211] As used herein, the term "dynamic light scattering" or "DLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of particles. A monochromatic light source, usually a laser, is incident on a sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in solution strike the light and diffract it in all directions. The diffracted light from the particles can interfere constructively (bright areas) or destructively (dark areas). This process is repeated over short time intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity at each spot over time.

[0212] As used herein, the term "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's radius of gyration and / or molar mass. A high-intensity monochromatic light, usually a laser, is emitted into a solution containing the particles. One or more detectors are used to measure the scattered intensity at one or more angles. The angular dependence is necessary to obtain accurate measurements of both the molar mass and size of all macromolecules within the radius. Therefore, simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard implementation of static light scattering.

[0213] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is a mixture of DNA and RNA. The nucleic acid can exist as a single-stranded or double-stranded molecule, and as a linear or covalently closed circular molecule. A nucleic acid can be isolated. The term "isolated nucleic acid," according to the present disclosure, means that the nucleic acid has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR) for DNA or by in vitro transcription (e.g., using RNA polymerase) for RNA; (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

[0214] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be thought of as a nucleotide without the phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0215] The five standard nucleosides that commonly make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly abbreviated by their single-letter codes: U, A, T, C, and G, respectively. However, thymidine is more commonly designated "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides can be found in both RNA and DNA. In RNA, they are designated as A, C, and G, and in DNA, they are designated as dA, dC, and dG.

[0216] The modified purine (A or G) or pyrimidine (C, T, or U) base moiety may, in some embodiments, be one or more alkyl groups, e.g., one or more C 1-4 Modified by alkyl groups, e.g., one or more methyl groups. Specific examples of modified purine or pyrimidine base moieties include N 7 -Alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)-alkyl-uracil, e.g., N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil, and N(1)-C1-4 Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(1)-methyl-uracil.

[0217] As used herein, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, DNA contains all or most of the deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal DNA nucleotides or to either or both ends of the DNA. It is also contemplated herein that the nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0218] The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, in particular cDNA, which may be obtained by reverse transcription of RNA.

[0219] The term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to one or both ends of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such modified / modified nucleotides (i.e., modified / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0220] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activator RNA (e.g., small activator RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0221] As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., production of RNA) is performed acellularly. That is, IVT does not use live / cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

[0222] mRNA According to the present disclosure, the term "mRNA" means "messenger RNA" and includes "transcripts" that can be produced by using a DNA template. Generally, mRNA encodes a peptide or polypeptide.

[0223] Although mRNA is single-stranded, it may contain self-complementary sequences that allow part of the mRNA to fold back on itself and pair with itself to form a double helix.

[0224] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0225] In a preferred embodiment of the present disclosure, mRNA relates to an RNA transcript that encodes a peptide or polypeptide.

[0226] In some embodiments, preferably the mRNA encoding the peptide or polypeptide has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0227] As is well-established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. Methods for in vitro transcription are known to those skilled in the art; for example, see Molecular Cloning:4 thEdition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Additionally, various in vitro transcription kits are commercially available from, for example, Thermo Fisher Scientific (TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®, etc.), New England BioLabs Inc. (HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit, etc.), Promega (RiboMAX™, HeLaScribe®, Riboprobe® system, etc.), Jena Bioscience (SP6 or T7 transcription kit, etc.), and Epicentre (AmpliScribe™, etc.). To provide a modified mRNA, correspondingly modified nucleotides, e.g., modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be made in and / or added to the mRNA after transcription.

[0228] In some embodiments, the mRNA is in vitro transcribed mRNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0229] In some embodiments of the present disclosure, the mRNA is a "replicon mRNA" or simply a "replicon," particularly a "self-replicating mRNA" or "self-amplifying mRNA." In certain embodiments, the replicon or self-replicating mRNA is derived from or contains elements derived from a ssRNA virus, particularly a positive-strand ssRNA virus such as an alphavirus. Alphaviruses are a typical example of a positive-strand RNA virus. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication and protein modification) and structural proteins (which form the virus particle). Typically, two open reading frames (ORFs) are present in the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded together by a first ORF that begins near the 5' end of the genome, while the structural proteins of alphaviruses are encoded together by a second ORF that is found downstream of the first ORF and extends toward the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87, pp. 111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts directly like messenger RNA to translate the open reading frame encoding the nonstructural polyprotein (nsP1-nsP4).Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be replicated by the replicase in trans (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules that can be replicated by the replicase in trans must contain specific alphavirus sequence elements to enable recognition and RNA synthesis by the alphavirus replicase.

[0230] In some embodiments of the present disclosure, mRNA comprises one or more modifications, for example, to improve its stability, and / or improve translation efficiency, and / or reduce immunogenicity, and / or reduce cytotoxicity.For example, to increase mRNA expression, mRNA can be modified in the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without changing the sequence of the expressed peptide or polypeptide.Such modifications are described, for example, in International Publication No. 2007 / 036366 and PCT / EP2019 / 056502, and include: 5' cap structure; extension or truncation of naturally occurring poly(A) tail; modification of 5' and / or 3' untranslated region (UTR), for example, introduction of UTR that is not related to the coding region of the RNA; substitution of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (for example, to change, preferably increase, the GC content of RNA).

[0231] In some embodiments, the mRNA comprises a 5' cap structure. In some embodiments, the mRNA does not have an uncapped 5'-triphosphate. In some embodiments, the mRNA may comprise a conventional 5' cap and / or a 5' cap analog. The term "conventional 5' cap" refers to the cap structure found at the 5' end of an mRNA molecule, generally consisting of guanosine 5'-triphosphate (Gppp) linked via its triphosphate moiety to the 5' end of the next nucleotide of the mRNA (i.e., the guanosine is linked to the remainder of the mRNA via a 5'-5' triphosphate bond). The guanosine is N 7 can be methylated at the cap structure m 7 The term "5' cap analog" is based on the traditional 5' cap, but uses a 5' cap analog with ... 7 These include 5' caps modified at either the 2' or 3' position of the guanosine structure (such 5' cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5' cap analogs are those with one or more substitutions at the bridging and non-bridging oxygens in the phosphate bridge, such as phosphorothioate-modified 5' cap analogs at the β-phosphate (e.g., m2), as described in PCT / EP 2019 / 056502. 7,2’O G(5')ppSp(5')G (referred to as β-S-ARCA or β-S-ARCA). For example, providing an mRNA having a 5' cap structure described herein can be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5' cap compound, with the 5' cap structure being co-transcriptionally incorporated into the generated mRNA strand, or the mRNA can be generated, for example, by in vitro transcription, and the 5' cap structure can be post-transcriptionally attached to the mRNA using a capping enzyme, for example, vaccinia virus capping enzyme.

[0232] In some embodiments, the mRNA is m2 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer), m2 7,3’OG(5')ppp(5')G, and m2 7,3’-O Gppp(m1 2’-O In some embodiments, the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope described herein comprises a 5'-cap structure selected from the group consisting of m2 7,2’O G(5')ppSp(5')G (especially its D1 diastereomer).

[0233] In some embodiments, the mRNA comprises cap 0, cap 1, or cap 2, preferably cap 1 or cap 2. According to the present disclosure, the term "cap 0" refers to the structure "m 7 GpppN" where N is any nucleoside bearing an OH moiety at the 2' position. According to the present disclosure, the term "Cap 1" refers to the structure "m 7 GpppNm, where Nm is any nucleoside bearing an OCH3 moiety at the 2' position. According to the present disclosure, the term "cap 2" refers to the structure "m 7 GpppNmNm" where each Nm is independently any nucleoside bearing an OCH3 moiety at the 2' position.

[0234] The 5' cap analog beta-S-ARCA (β-S-ARCA) has the following structure:

[0235] [ka]

[0236] It has.

[0237] The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column and therefore exhibits a shorter retention time compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)). The HPLC is preferably analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably in a 5 μm, 4.6 x 250 mm format, is used for the separation, allowing a flow rate of 1.3 ml / min to be applied. In some embodiments, a gradient of methanol in ammonium acetate is used, e.g., a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH 5.9, within 15 minutes. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0238] 5' cap analog m2, a building block of cap 1 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3’O G(5')ppp(5')m 2’-O ApG) has the following structure:

[0239] [ka]

[0240] It has.

[0241] An exemplary cap 0 mRNA containing β-S-ARCA and mRNA has the following structure:

[0242] [ka]

[0243] It has.

[0244] m2 7,3’OAn exemplary cap 0 mRNA containing G(5')ppp(5')G and mRNA has the following structure:

[0245] [ka]

[0246] It has.

[0247] m2 7,3’-O Gppp(m1 2’-O ) An exemplary cap 1 mRNA containing ApG and mRNA has the following structure:

[0248] [ka]

[0249] It has.

[0250] As used herein, the term "poly A tail" or "poly A sequence" refers to a continuous or intermittent sequence of adenylate residues typically located at the 3' end of an mRNA molecule. Poly A tails or poly A sequences are known to those skilled in the art and may follow the 3'-UTR of the mRNAs described herein. A continuous poly A tail is characterized by consecutive adenylate residues. Continuous poly A tails are typical in nature. The mRNAs disclosed herein may have a poly A tail attached to the free 3' end of the mRNA by a template-independent RNA polymerase after transcription, or a poly A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0251] Poly(A) tails of approximately 120 A nucleotides have been demonstrated to have beneficial effects on the levels of mRNA in transfected eukaryotic cells and on the levels of proteins translated from open reading frames located upstream (5') of the poly(A) tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0252] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly-A tail, are A nucleotides, while allowing the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides of the poly A tail, i.e., 100% of the number of nucleotides in the poly A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0253] In some embodiments, the poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repeated dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is called a poly(A) cassette.

[0254] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324 A1 may be used in the present disclosure. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences in which the four nucleotides (dA, dC, dG, dT) are evenly distributed and have a length of, for example, 5-50 nucleotides, have been shown to support sustained propagation of plasmid DNA in Escherichia coli (E. coli) at the DNA level and, at the RNA level, are still associated with beneficial properties for supporting RNA stability and translation efficiency. As a result, in some embodiments, the poly-A tails included in the mRNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0255] In some embodiments, no nucleotides other than A nucleotides are adjacent to the polyA tail at its 3' end, i.e., the polyA tail is not masked or followed by a nucleotide other than A at its 3' end.

[0256] In some embodiments, the poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises the poly-A tail set forth in SEQ ID NO:8. In some embodiments, the polyA tail comprises at least 100 nucleotides. In some embodiments, the polyA tail comprises about 150 nucleotides. In some embodiments, the polyA tail comprises about 120 nucleotides.

[0257] In some embodiments, the mRNA used in the present disclosure includes a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" generally does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence. The incorporation of a 3'UTR into the 3' untranslated region of an RNA (preferably mRNA) molecule can result in improved translation efficiency. By incorporating two or more such 3'UTRs (preferably arranged in a head-to-tail orientation; see, for example, Holtkamp et al., Blood 108, 4009-4017 (2006)), synergistic effects can be achieved. 3'UTRs can be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as α2-globin, α1-globin, or β-globin, e.g., β-globin, e.g., human β-globin gene or mRNA. For example, an RNA (e.g., an mRNA) can be modified by replacing or inserting an existing 3'-UTR with one or more, e.g., two, copies of a 3'-UTR from a globin gene, e.g., α2-globin, α1-globin, β-globin, e.g., β-globin, e.g., human β-globin.

[0258] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 6. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 7.

[0259] In some embodiments, the RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6.

[0260] In some embodiments, the RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0261] mRNA can have modified ribonucleotides to improve its stability, and / or reduce its immunogenicity, and / or reduce its cytotoxicity.For example, in some embodiments, the uridine in the mRNA described herein is replaced by modified nucleoside (partially or completely, preferably completely).In some embodiments, the modified nucleoside is modified uridine.

[0262] In some embodiments, the modified uridine substituting for uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

[0263] In some embodiments, the modified nucleoside that replaces (partially or completely, preferably completely) uridine in the mRNA is 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s2U), 4-thiouridine (s4U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), ), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyluridine (cm5U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2-thiouridine (nm5s2U), 5- Methylaminomethyluridine (mnm5U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-methylaminomethyl-2-selenouridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm5U), 1-taurinomethyluridine 5-Taurinomethyl-2-thiouridine (m5s2U), 1-Taurinomethyl-4-thiopseudouridine), 5-Methyl-2-thiouridine (m5s2U), 1-Methyl-4-thiopseudouridine (m1s4Ψ), 4-Thio-1-methylpseudouridine, 3-Methylpseudouridine (m3Ψ), 2-Thio-1-methylpseudouridine, 1-Methyl-1-deazapseudouridine, 2-Thio-1-methyl-1-deazapseudouridine, Dihydrouridine (D), Dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m5D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5s2U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methylpseudouridine The uridine may be any one or more of uridine (Ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-aruridine, 2'-F-uridine, 2'-OH-aruridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine]uridine, or any other modified uridine known in the art.

[0264] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified." The term "mΨ-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ- or mΨ- or m5U-modified RNAs typically exhibit reduced immunogenicity compared to their unmodified forms and are therefore preferred in applications where induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudouridine, which completely replaces uridine.

[0265] The codons of the mRNA used in the present disclosure can be further optimized, for example, to increase the GC content of the RNA and / or to replace codons that are rare in a cell (or subject) in which the peptide or polypeptide of interest is to be expressed with codons that are synonymous with the cell (or subject) more frequently. In some embodiments, the amino acid sequence encoded by the mRNA used in the present disclosure is encoded by a codon-optimized coding sequence and / or a coding sequence whose G / C content is increased compared to a wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of a wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content preferably does not change the sequence of the encoded amino acid sequence.

[0266] The term "codon-optimized" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule.In the context of the present disclosure, the coding region can be codon-optimized for optimal expression in the subject treated with the mRNA described herein.Codon optimization is based on the finding that translation efficiency is also determined by the different frequencies of tRNA occurrence in cells.Therefore, the sequence of mRNA can be modified so that codons that frequently occur with available tRNAs are inserted instead of "rare codons".

[0267] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of an mRNA described herein is increased compared to the G / C content of the corresponding coding sequence of a wild-type RNA, and the amino acid sequence encoded by the mRNA is preferably unaltered compared to the amino acid sequence encoded by the wild-type RNA. This modification of the mRNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. Given the fact that several codons encode the exact same amino acid (the so-called degeneracy of the genetic code), the most favorable codons for stability can be determined (the so-called alternative codon usage). Depending on the amino acid encoded by the mRNA, there are various possibilities for modifying the mRNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons encoding the same amino acid but that do not contain A and / or U nucleotides or contain a lower content of A and / or U nucleotides.

[0268] In various embodiments, the G / C content of the coding region of the mRNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of the wild-type RNA.

[0269] Combinations of the above-mentioned modifications, i.e., incorporation of a 5' cap structure, incorporation of a polyA sequence, unmasking of a polyA sequence, modification of the 5'-UTR and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine in the case of cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) in the case of uridine), and codon optimization, have a synergistic effect on increasing the stability and translation efficiency of RNA (preferably mRNA). Thus, in some embodiments, the mRNA used in the present disclosure includes a combination of at least two, at least three, at least four, or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5' cap structure, (ii) incorporation of a polyA sequence, unmasking of a polyA sequence, (iii) modification of the 5'-UTR and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), (iv) replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine in the case of cytidine, and / or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) in the case of uridine), and (v) codon optimization.

[0270] Some aspects of the present disclosure include targeted delivery of the mRNA disclosed herein to specific cells or tissues. In some embodiments, the present disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen. When the administered mRNA encodes an antigen or epitope for inducing an immune response, targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen, is particularly preferred. In some embodiments, the target cells are spleen cells. In some embodiments, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In some embodiments, the target cells are dendritic cells in the spleen. The "lymphatic system" is part of the circulatory system and is an important part of the immune system, including a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and initiate adaptive immune responses.

[0271] Lipid-based mRNA delivery systems have an inherent selectivity for the liver. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposome and lipid or cholesterol complex). In some embodiments, the target organ is the liver, and the target tissue is liver tissue. Delivery to such target tissue is preferred, particularly when it is desired to have mRNA or encoded peptide or polypeptide present in this organ or tissue, and / or when it is desired to express a large amount of encoded peptide or polypeptide, and / or when it is desired or required to have the encoded peptide or polypeptide present in a systemic manner, particularly in a significant amount.

[0272] In some embodiments, after administration of an mRNA particle described herein, at least a portion of the mRNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the mRNA is delivered to the cytosol of the target cell. In some embodiments, the mRNA encodes a peptide or polypeptide, and the mRNA is translated by the target cell to produce the peptide or polypeptide. In some embodiments, the target cell is a liver cell. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the target cell is a tumor cell or a cell of the tumor microenvironment. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a lymph node cell. In some embodiments, the target cell is a lung cell. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a skin cell. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a NK cell. In some embodiments, the target cell is a monocyte. Therefore, the RNA particles described herein can be used to deliver mRNA to such target cells.

[0273] Nucleic acids encoding pharmaceutically active peptides or polypeptides "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0274] In some embodiments, nucleic acids such as mRNAs used in the present disclosure comprise nucleic acid sequences encoding peptides or polypeptides, preferably pharmaceutically active peptides or polypeptides. In some embodiments, nucleic acids such as mRNAs used in the present disclosure comprise nucleic acid sequences encoding peptides or polypeptides, preferably pharmaceutically active peptides or polypeptides, and are capable of expressing the peptides or polypeptides, particularly when introduced into a cell or subject. Thus, in some embodiments, nucleic acids used in the present disclosure comprise a coding region (open reading frame (ORF)) encoding a peptide or polypeptide, e.g., a pharmaceutically active peptide or polypeptide. In this regard, an "open reading frame" or "ORF" is a contiguous stretch of codons beginning with an initiation codon and ending with a stop codon. Such nucleic acids encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active nucleic acids." In particular, such mRNAs encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active mRNAs." In some embodiments, a nucleic acid, such as an mRNA, used in this disclosure comprises a nucleic acid sequence that encodes more than one peptide or polypeptide, for example, two, three, four or more peptides or polypeptides.

[0275] According to the present disclosure, the term "pharmaceutically active peptide or polypeptide" refers to a peptide or polypeptide that can be used in the treatment of an individual in whom expression of the peptide or polypeptide is beneficial, for example, to ameliorate symptoms of a disease. Preferably, a pharmaceutically active peptide or polypeptide has curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease. In some embodiments, a pharmaceutically active peptide or polypeptide, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's condition or pathology. A pharmaceutically active peptide or polypeptide can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease. The term "pharmaceutically active peptide or polypeptide" includes the entire peptide or polypeptide and can also refer to a pharmaceutically active fragment thereof. The term can also include pharmaceutically active variants and / or analogs of the peptide or polypeptide.

[0276] According to the present disclosure, the term "pharmaceutically active peptide or polypeptide" includes vaccine antigens, PD-1 axis binding antagonists, immunostimulants, and antigen receptors.

[0277] In some embodiments, nucleic acid such as RNA that encodes pharmaceutically active peptide or polypeptide is expressed in the cell of the subject that is treated to provide pharmaceutically active peptide or polypeptide.In some embodiments, nucleic acid is transiently expressed in the cell of the subject.Therefore, in some embodiments, nucleic acid is not integrated into the genome of the cell.In some embodiments, nucleic acid is RNA, preferably in vitro transcribed RNA.

[0278] In some embodiments, expression of the vaccine is at the cell surface. In some embodiments, the vaccine antigen is expressed and presented in the context of MHC. In some embodiments, RNA encoding the vaccine antigen is expressed in cells, such as antigen-presenting cells, of a subject that have been treated to provide the vaccine antigen for binding by immune effector cells, which binding results in stimulation, priming, and / or expansion of the immune effector cells.

[0279] In some embodiments, expression of the PD-1 axis-binding antagonist is in the extracellular space, ie, the PD-1 axis-binding antagonist is secreted.

[0280] In some embodiments, expression of the immunostimulant is in the extracellular space, i.e., the immunostimulant is secreted.

[0281] In some embodiments, the expression of the antigen receptor is on the cell surface.

[0282] Non-immunogenic RNA encoding vaccine antigens The present invention includes the use of non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope for inducing an immune response to an antigen in a subject. "Peptides or polypeptides comprising an epitope for inducing an immune response to an antigen in a subject" are also referred to herein as "vaccine antigens," "peptide antigens and protein antigens," or simply "antigens."

[0283] In some embodiments, the non-immunogenic RNA encoding the vaccine antigen is a single-stranded 5'-capped mRNA that is translated into the respective protein upon entry into the cells of the subject to which the RNA is administered, e.g., antigen-presenting cells (APCs). Preferably, the RNA contains structural elements (5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence) optimized for maximum effectiveness of the RNA in terms of stability and translation efficiency.

[0284] In some embodiments, β-S-ARCA(D1) is utilized as a specific capping structure at the 5' end of the RNA. In some embodiments, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6. In some embodiments, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7. In some embodiments, the poly(A) sequence is 110 nucleotides long and consists of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence and another 70 adenosine residues. This poly(A) sequence is designed to enhance RNA stability and translation efficiency in dendritic cells. In some embodiments, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0285] In some embodiments, the RNA is administered as a lipoplex particle, preferably comprising DOTMA and DOPE, as further described herein. In some embodiments, the lipoplex article targets the lymphatic system, particularly secondary lymphoid organs, particularly the spleen, more particularly dendritic cells in the spleen. In some embodiments, such particles are administered systemically, particularly intravenously.

[0286] In some embodiments, RNA encoding the vaccine antigen is expressed in cells of a subject to provide the vaccine antigen. In some embodiments, expression of the antigen is on the cell surface. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, RNA encoding the vaccine antigen is transiently expressed in cells of a subject. In some embodiments, RNA encoding the vaccine antigen is administered systemically. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in the spleen. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in antigen-presenting cells, preferably professional antigen-presenting cells. In some embodiments, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in no or essentially no expression of RNA encoding the vaccine antigen in the lung and / or liver. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in the spleen that is at least five times higher than the expression level in the lung.

[0287] Vaccine antigens comprise epitopes for inducing immune responses against antigens in subjects.Therefore, vaccine antigens comprise antigen sequences for inducing immune responses against antigens in subjects.Such antigen sequences can correspond to target antigens or disease-related antigens, such as proteins or tumor antigens of infectious agents (e.g., viral antigens or bacterial antigens), or their immunogenic variants, or immunogenic fragments or immunogenic variants of target antigens or disease-related antigens.Therefore, antigen sequences can comprise at least one epitope of target antigens or disease-related antigens or their immunogenic variants.

[0288] Antigen sequences, e.g., epitopes, suitable for use in accordance with the present disclosure may typically be derived from the target antigen, i.e., the antigen to which an immune response is elicited. For example, the antigen sequence included within a vaccine antigen may be the target antigen or a fragment or variant of the target antigen.

[0289] The antigen sequence or its processing product, e.g., a fragment thereof, can bind to an antigen receptor, such as a TCR or CAR, carried by an immune effector cell. In some embodiments, the antigen sequence is selected from the group consisting of an antigen or fragment thereof expressed by a target cell targeted by the immune effector cell, or a variant of the antigen sequence or fragment.

[0290] Vaccine antigens provided to a subject according to the present disclosure by administering RNA encoding the vaccine antigen preferably result in the induction of an immune response in the subject to which the vaccine antigen is provided, e.g., by stimulating, priming, and / or expanding immune effector cells. The immune response, e.g., stimulated, primed, and / or expanded immune effector cells, is preferably directed against a target antigen, particularly a target antigen expressed by diseased cells, tissues, and / or organs, i.e., a disease-associated antigen. Thus, the vaccine antigen may comprise a disease-associated antigen, or a fragment or variant thereof. In some embodiments, such a fragment or variant is immunologically equivalent to the disease-associated antigen.

[0291] In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that induces an immune response, e.g., stimulation, priming, and / or expansion of immune effector cells, where the immune response, e.g., stimulated, primed, and / or expanded immune effector cells, target an antigen, i.e., a disease-associated antigen, particularly when presented by diseased cells, tissues, and / or organs. Thus, a vaccine antigen may correspond to or comprise a disease-associated antigen, a fragment of a disease-associated antigen, or an antigen homologous to a disease-associated antigen or its fragment. When a vaccine antigen comprises a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may comprise an epitope of the disease-associated antigen or a sequence homologous to an epitope of the disease-associated antigen targeted by an antigen receptor of an immune effector cell. Thus, according to the present disclosure, a vaccine antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that can induce, e.g., stimulate, prime, and / or expand, an immune response against immune effector cells bearing an antigen receptor that binds to the antigen or cells expressing the antigen. Preferably, the vaccine antigen (analogous to a disease-associated antigen) provides relevant epitopes for binding by antigen receptors present on immune effector cells. In some embodiments, the vaccine antigen or a fragment thereof (analogous to a disease-associated antigen) is expressed on the surface of a cell, such as an antigen-presenting cell (optionally in the context of an MHC), to provide relevant epitopes for binding by immune effector cells. The vaccine antigen may be a recombinant antigen.

[0292] In some embodiments of all aspects of the invention, RNA encoding a vaccine antigen is expressed in cells of a subject to provide the antigen or its processing products for binding by antigen receptors expressed by immune effector cells, which binding results in stimulation, priming and / or expansion of the immune effector cells.

[0293] According to the present disclosure, an "antigen" encompasses any substance that elicits an immune response and / or any substance to which an immune response or immune mechanism, such as a cellular and / or humoral response, is directed. This also includes situations in which an immune response or immune mechanism is directed against one or more antigenic peptides, particularly when the antigen is processed into antigenic peptides and presented in the context of an MHC molecule. In particular, "antigen" relates to any substance, such as a peptide or polypeptide, that specifically reacts with an antibody or T lymphocyte (T cell). The term "antigen" can include a molecule that contains at least one epitope, such as a T cell epitope. In some embodiments, an antigen is a molecule that, optionally after processing, induces an immune response that may be specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0294] In some embodiments, the antigen is presented or present on the surface of a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or macrophage. In some embodiments, the antigen or its processing product, such as a T cell epitope, is bound by an antigen receptor. Thus, the antigen or its processing product can specifically react with immune effector cells, such as T lymphocytes (T cells).

[0295] The term "autoantigen" (or "self-antigen") refers to an antigen that originates within a subject's body (i.e., an autoantigen may also be called a "self-antigen") and that produces an abnormally vigorous immune response against this normal part of the body. Such a vigorous immune response against an autoantigen may be the cause of an "autoimmune disease."

[0296] According to the present disclosure, any suitable antigen that is a candidate for an immune response can be used, and the immune response can include a humoral or cellular immune response, or both. In the context of some embodiments of the present disclosure, the antigen is presented by cells, such as antigen-presenting cells, in association with MHC molecules, resulting in an immune response to the antigen. The antigen can be a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens can include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen can be a tumor antigen. According to the present disclosure, the antigen can correspond to a naturally occurring product, such as a viral protein, or a portion thereof.

[0297] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, e.g., tumor antigens.

[0298] In some embodiments, the antigen is a tumor antigen, i.e., a part of a tumor cell, particularly one that is present primarily intracellularly or as a surface antigen of the tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, such as an antigen derived from a virus, bacterium, unicellular organism, or parasite, e.g., a viral antigen such as a viral ribonucleoprotein or coat protein. In some embodiments, the antigen should be presented by an MHC molecule that leads to modulation, particularly activation of cells of the immune system such as CD4+ and CD8+ lymphocytes, particularly through modulation of the activity of T cell receptors.

[0299] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, or cell nucleus. In particular, this term refers to an antigen produced intracellularly or as a surface antigen on a tumor cell. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, α2-H-ferroprotein, and γ-fetoprotein, as well as various viral tumor antigens. According to some embodiments of the present disclosure, tumor antigens include any antigen that is characteristic of a tumor or cancer and tumor or cancer cells in terms of type and / or expression level.

[0300] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0301] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0302] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by T cells, particularly when presented in the context of an MHC molecule. An epitope of a protein can include a continuous or discontinuous portion of the protein and can be, e.g., about 5 to about 100, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope in the context of the present disclosure is a T cell epitope.

[0303] Terms such as "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and may refer, for example, to an incompletely displayed form of an antigen that can elicit an immune response against the antigen or a cell that expresses or contains the antigen and presents the antigen. In some embodiments, these terms relate to an immunogenic portion of an antigen. In some embodiments, this is the portion of the antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly when presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100 amino acids in length, e.g., about 5 to about 50, about 8 to about 30, or about 8 to about 25 amino acids in length; for example, an epitope can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T-cell epitopes.

[0304] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response. MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides can be effective. For class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, although longer and shorter peptides can also be effective.

[0305] Peptide and polypeptide antigens can be 2 to 100 amino acids in length, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0306] A peptide or polypeptide antigen can be any peptide or polypeptide that is capable of inducing or increasing the ability of the immune system to generate antibody and T cell responses against the peptide or polypeptide.

[0307] In some embodiments, vaccine antigens, i.e., antigens whose inoculation into a subject induces an immune response, are recognized by immune effector cells. In some embodiments, when recognized by immune effector cells, vaccine antigens can, in the presence of appropriate costimulatory signals, induce stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In the context of embodiments of the present disclosure, vaccine antigens can be presented or present on the surface of cells, such as, for example, antigen-presenting cells.

[0308] In some embodiments, the antigen is expressed on diseased cells (such as tumor cells or infected cells).

[0309] In some embodiments, the antigen is presented by a diseased cell (such as a tumor cell or an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of the antigen presented in the context of MHC. In some embodiments, binding of the TCR, when expressed by and / or present on a T cell, to an antigen presented by a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In some embodiments, binding of the TCR, when expressed by and / or present on a T cell, to an antigen presented on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, with the T cell releasing, for example, cytotoxic factors, such as perforin and granzymes.

[0310] In some embodiments, the antigen is expressed on the surface of a diseased cell (such as a tumor cell or an infected cell). In some embodiments, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In some embodiments, the CAR binds to a native epitope of the antigen present on the surface of a living cell. In some embodiments, binding of the CAR, when expressed by and / or present on a T cell, to an antigen present on a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In some embodiments, binding of the CAR, when expressed by and / or present on a T cell, to an antigen present on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, and the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0311] According to some embodiments, the amino acid sequence that enhances antigen processing and / or presentation is fused directly or via a linker to the antigenic peptide or polypeptide (antigen sequence). Thus, in some embodiments, the RNA described herein comprises at least one coding region that encodes the antigenic peptide or polypeptide and the amino acid sequence that enhances antigen processing and / or presentation.

[0312] In some embodiments, antigens for vaccination, which may be administered in the form of RNA encoding same, include naturally occurring antigens or fragments thereof, such as epitopes thereof.

[0313] Such amino acid sequences that enhance antigen processing and / or presentation are preferably, but not limited to, located at the C-terminus of the antigen peptide or polypeptide (and optionally at the C-terminus of the amino acid sequence that disrupts immune tolerance). The amino acid sequences that enhance antigen processing and / or presentation defined herein preferably improve antigen processing and presentation. In some embodiments, the amino acid sequences that enhance antigen processing and / or presentation defined herein include, but are not limited to, sequences derived from the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3), particularly sequences comprising the amino acid sequence of SEQ ID NO: 2 or a functional variant thereof.

[0314] In some embodiments, the amino acid sequence that enhances antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO:2, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:2, or a functional fragment of the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the amino acid sequence that enhances antigen processing and / or presentation comprises the amino acid sequence of SEQ ID NO:2.

[0315] Thus, in some embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or presentation, preferably fused to the C-terminus of the antigenic peptide or polypeptide, more preferably an antigenic peptide or polypeptide described herein.

[0316] Additionally, a secretory sequence, such as a sequence comprising the amino acid sequence of SEQ ID NO: 1, can be fused to the N-terminus of the antigenic peptide or polypeptide.

[0317] Amino acid sequences derived from tetanus toxoid of Clostridium tetani can be used to overcome self-tolerance mechanisms to efficiently initiate immune responses to self-antigens by providing T cell help during priming.

[0318] Tetanus toxoid heavy chains bind indiscriminately to MHC class II alleles and induce CD4 + It is known that these antigens contain epitopes that can induce memory T cells. Furthermore, the combination of tetanus toxoid (TT) helper epitopes with tumor-associated antigens inhibits CD4 T cell activation during priming. + It is known that tumor-associated antigens improve immune stimulation compared to the application of tumor-associated antigens alone by providing mediated T cell help. + To reduce the risk of stimulating T cells, the whole tetanus toxoid fragment C stimulates CD8 + The entire fragment C is not used because it is known to contain a T cell epitope. To ensure binding to as many MHC class II alleles as possible, two peptide sequences containing promiscuous binding helper epitopes were instead selected. Based on data from ex vivo studies, the known epitope p2 (QYIKANSKFIGITEL; TT 830-844 ) and p16(MTNSVDDALINSTKIYSYFPSVISKVNQGAQG;TT 578-609 ) was selected because the p2 epitope had already been used for peptide vaccination in clinical trials to enhance anti-melanoma activity.

[0319] Preclinical data demonstrated that an RNA vaccine encoding both tumor antigens and promiscuous tetanus toxoid sequences stimulated CD8 responses to tumor antigens. +This has been shown to result in enhanced T cell responses and improved tolerance breaking. Immune monitoring data from patients vaccinated with vaccines containing sequences fused in-frame with tumor antigen-specific sequences reveals that selected tetanus sequences are able to induce tetanus-specific T cell responses in nearly all patients.

[0320] According to some embodiments, the immune tolerance-disrupting amino acid sequence is fused to the antigenic peptide or polypeptide directly or via a linker, for example a linker having an amino acid sequence according to SEQ ID NO:4.

[0321] Such an amino acid sequence that breaks immune tolerance is preferably, but not limited to, located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of the amino acid sequence that enhances antigen processing and / or presentation, where the amino acid sequence that breaks immune tolerance and the amino acid sequence that enhances antigen processing and / or presentation may be fused directly or via a linker, for example a linker having an amino acid sequence according to SEQ ID NO: 5). An amino acid sequence that breaks immune tolerance as defined herein preferably improves T cell responses. In some embodiments, an amino acid sequence that breaks immune tolerance as defined herein includes, but is not limited to, a sequence derived from the helper sequences p2 and p16 from tetanus toxoid (P2P16), particularly a sequence comprising the amino acid sequence of SEQ ID NO: 3 or a functional variant thereof.

[0322] In some embodiments, the amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO:3, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3, or a functional fragment of the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO:3.

[0323] In the following, certain terms used when describing vaccine RNA embodiments and elements thereof have the following meanings: hAg-Kozak: 5'-UTR sequence of human α-globin mRNA with an optimized "Kozak sequence" to increase translation efficiency.

[0324] sec / MITD: A fusion protein tag derived from sequences encoding the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3) that has been shown to improve antigen processing and presentation. sec corresponds to a 78-bp fragment encoding a secretory signal peptide that directs translocation of the nascent polypeptide chain into the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domain of the MHC class I molecule, also known as the MHC class I transport domain.

[0325] Antigen: The sequence encoding each vaccine antigen / epitope.

[0326] Glycine-serine linker (GS): A sequence encoding a short linker peptide consisting primarily of the amino acids glycine (G) and serine (S), commonly used in fusion proteins.

[0327] P2P16: a sequence encoding a tetanus toxoid-derived helper epitope for breaking immune tolerance.

[0328] FI elements: The 3'-UTR is a combination of two sequence elements derived from the "amino-terminal enhancer of split" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified by an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression.

[0329] A30L70: a poly(A) tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence and another 70 adenosine residues designed to increase RNA stability and translation efficiency in dendritic cells.

[0330] In some embodiments, the vaccine RNA described herein has the structure: β-S-ARCA(D1)-hAg-Kozak-sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70 It has. In some embodiments, the vaccine antigens described herein have the structure: sec-GS(1)-antigen-GS(2)-P2P16-GS(3)-MITD It has.

[0331] In some embodiments, hAg-Kozak comprises the nucleotide sequence of SEQ ID NO:6. In some embodiments, sec comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, P2P16 comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, MITD comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, GS(1) comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, GS(2) comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, GS(3) comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, FI comprises the nucleotide sequence of SEQ ID NO:7. In some embodiments, A30L70 comprises the nucleotide sequence of SEQ ID NO:8.

[0332] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is located in association with the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of the cell and accessible from outside the cell, for example, by an antibody located on the outside of the cell. In this context, a portion can be, for example, at least 4, at least 8, at least 12, or at least 20 amino acids. The association can be direct or indirect. For example, the association can be through one or more transmembrane domains, one or more lipid anchors, or through interactions with any other proteins, lipids, saccharides, or other structures that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell can be a transmembrane protein having an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0333] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and thus includes the outside of a cell that is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the surface of the cell and is accessible to binding, for example, by an antigen-specific antibody added to the cell. In some embodiments, the antigen expressed on the surface of a cell is an integral membrane protein with an extracellular portion that can be recognized by a CAR.

[0334] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside the cell, for example, by binding to a molecule, such as an antibody, that is located on the outside of the cell. In some embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

[0335] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen it targets, particularly when presented on the surface of an antigen-presenting cell or diseased cell, such as a cancer cell, in association with an MHC molecule, and preferably exerts T cell effector function. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0336] The term "target" refers to an agent, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In some embodiments, target cells are cells that express an antigen and present the antigen in association with class I MHC.

[0337] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a polypeptide into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by cells such as antigen-presenting cells. Antigen-presenting cells can be distinguished as professional or non-professional antigen-presenting cells.

[0338] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules, which are necessary for interaction with naive T cells. When T cells interact with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0339] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0340] The term "dendritic cell" (DC) refers to a subtype of phagocyte belonging to the class of antigen-presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitor cells first transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Upon contact with presentable antigens, they are activated to become mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as coreceptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that directs dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. Here, they act as antigen-presenting cells, activating helper T cells, killer T cells, and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce immune responses associated with T cells or B cells. In some embodiments, the dendritic cells are splenic dendritic cells.

[0341] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf and kill foreign pathogens within the macrophage through hydrolytic and oxidative attack, resulting in the degradation of the pathogen. Peptides derived from degraded proteins are presented on the macrophage cell surface, where they can be recognized by T cells and directly interact with antibodies on the surface of B cells, leading to the activation of T cells and B cells and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In some embodiments, the macrophages are splenic macrophages.

[0342] "Antigen-responsive CTL" refers to a CD8 CTL that is responsive to an antigen or a peptide derived from the antigen, which is presented together with class I MHC on the surface of an antigen-presenting cell. + It means T cells.

[0343] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing tumor antigens. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0344] As used herein, "activation" or "stimulation" refers to the state of immune effector cells, such as T cells, that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, induced cytokine production, and detectable effector function. The term "activated immune effector cells" refers, among other things, to immune effector cells undergoing cell division.

[0345] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, resulting in differentiation into an effector cell, such as an effector T cell.

[0346] The term "expansion" refers to the process by which a particular entity multiplies. In some embodiments, the term is used in reference to an immunological response in which immune effector cells are stimulated by an antigen, causing proliferation and amplification of specific immune effector cells that recognize that antigen. In some embodiments, expansion results in differentiation of immune effector cells.

[0347] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense to refer to the integrated body's response to an antigen and may refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against," in reference to an agent such as an antigen, cell, or tissue, refer to an immune response, such as a cellular response, to the agent. An immune response can be measured by the development of antibodies against one or more antigens, as well as CD4+, which can be detected in various in vitro proliferation or cytokine production tests. + and CD8 + T lymphocytes, e.g., CD8 + The expansion of antigen-specific T lymphocytes, such as T lymphocytes, may include one or more responses selected from the group consisting of:

[0348] The terms "inducing an immune response" and "eliciting an immune response," and similar terms, in the context of the present disclosure, refer to the induction of an immune response, e.g., the induction of a cellular immune response, a humoral immune response, or both. The immune response can be protective / preventative / prophylactic and / or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, such as a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (such as influenza virus (A, B, or C), CMV, or RSV)). "Inducing" in this context can mean that there was no immune response against a particular antigen or pathogen before induction, but it can also mean that there was some immune response against a particular antigen or pathogen before induction and that the immune response was enhanced after induction. Thus, "inducing an immune response" in this context also includes "enhancing an immune response." In some embodiments, after inducing an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or cancer, or the disease state is ameliorated by inducing an immune response.

[0349] "Cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are meant to include a cellular response directed against cells characterized by expression of an antigen and / or presentation of the antigen by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (CD4 + T cells (also called T cells) play a central role by regulating the immune response and are responsible for the production of killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells, including diseased cells.

[0350] The term "humoral immune response" refers to the process in living organisms by which antibodies are produced in response to agents and organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T cells and / or B cells through membrane-bound receptors that bind to a single specific antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide, producing memory B cells and antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigen epitope recognized by its antigen receptor. Memory B lymphocytes remain dormant until subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the amplified antibody response that occurs upon re-exposure to the specific antigen.

[0351] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to an epitope on an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are called HCDR1, HCDR2, and HCDR3, and the CDRs of VL are called LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from amino terminus to carboxy terminus: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or immunologically active portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms, including polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0352] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, e.g., antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound and soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally called antibodies. Immunoglobulins generally contain several chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are mainly V L (variable light chain) domain, C L (constant light chain) domain, V H (variable heavy chain) domain, and C H (Constant heavy chain) domain C H 1. C H 2. C H 3 and C H Immunoglobulins are composed of immunoglobulin domains such as α, δ, ε, γ, and μ, which constitute the different classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy termini. Mammals have two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within different immunoglobulin isotypes, while the variable region is highly diverse and is responsible for antigen recognition.

[0353] The terms "vaccination" and "immunization" refer to the process of treating an individual for therapeutic or prophylactic reasons and, as described herein, relate to the procedure of administering to an individual one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA (particularly mRNA) encoding same, to stimulate an immune response against said one or more immunogens or antigens or against cells characterized by presenting said one or more immunogens or antigens.

[0354] "Cells characterized by antigen presentation" or "cells presenting antigen" or "MHC molecules presenting antigen on the surface of antigen-presenting cells" or similar expressions refer to cells, such as diseased cells, particularly tumor or infected cells, or antigen-presenting cells, that present antigens or antigenic peptides, either directly or after processing, in association with MHC molecules, such as MHC class I and / or MHC class II molecules. In some embodiments, the MHC molecules are MHC class I molecules.

[0355] In some embodiments, the pharmaceutically active peptide or polypeptide comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or polypeptide to a subject elicits an immune response in the subject against one or more antigens or one or more epitopes, which may be therapeutic or partially or fully protective.

[0356] In some embodiments, the RNA encodes at least one epitope, e.g., at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes.

[0357] In some embodiments, the target antigen is a tumor antigen, and the antigen sequence (e.g., epitope) is derived from the tumor antigen. The tumor antigen may be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen may also be a "neoantigen" that is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may result from one or more cancer-specific mutations in the genome of a cancer cell that result in amino acid changes. When the tumor antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of the neoantigen that contains one or more amino acid changes.

[0358] Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family such as claudin-6, claudin-18.2 and claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / MelanA, MC1R, myosin / m, MUC 1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1.

[0359] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) are attractive targets for the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) present in the spleen are particularly interesting antigen-presenting cells for RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to enhance therapeutic efficacy in tumor vaccine compositions. Rapid sequencing of tumor mutagenesis can provide multiple epitopes for personalized vaccines, which can be encoded by the mRNA described herein, for example, as a single polypeptide in which the epitopes are optionally separated by a linker. In some embodiments of the present disclosure, the mRNA encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Exemplary embodiments include mRNAs encoding at least five epitopes (called "pentatopes") and mRNAs encoding at least ten epitopes (called "decatopes").

[0360] In some embodiments, the antigen or epitope is derived from a pathogen-associated antigen, particularly a viral antigen. In some embodiments, the antigen or epitope is derived from the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof. Thus, in some embodiments, the mRNA used in the present disclosure encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0361] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a subject's immune system, the amino acid sequence induces an immune response with specificity reactive with the reference amino acid sequence. Thus, in some embodiments, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effect as the antigen targeted by the T cells with respect to stimulation, priming, and / or expansion of T cells.

[0362] The RNA encoding vaccine antigens used in the present disclosure is non-immunogenic.The RNA encoding immunostimulants can be administered according to the present disclosure to provide adjuvant effect.The RNA encoding immunostimulants can be standard RNA or non-immunogenic RNA.

[0363] As used herein, the term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA") refers to RNA that, for example, when administered to a mammal, does not induce a response by the immune system or that induces a weaker response than that induced by the same RNA that differs only in that it has not been subjected to modifications and processing that render it non-immunogenic, i.e., a weaker response than that induced by standard RNA (stdRNA). In certain embodiments, non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is made non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors, and / or by limiting the formation of double-stranded RNA (dsRNA), for example, during in vitro transcription, and / or by limiting the amount of double-stranded RNA (dsRNA), for example, by removing double-stranded RNA (dsRNA) after in vitro transcription. In certain embodiments, the non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors and / or by removing double-stranded RNA (dsRNA), for example, after in vitro transcription.

[0364] To render non-immunogenic RNA (particularly mRNA) non-immunogenic by incorporating a modified nucleoside, any modified nucleoside may be used as long as it reduces or suppresses the immunogenicity of the RNA. Modified nucleosides that suppress RNA-mediated activation of innate immune receptors are particularly preferred. In some embodiments, the modified nucleoside comprises the substitution of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is a 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3 Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5In certain embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ), or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

[0365] In some embodiments, the substitution of one or more uridines with nucleosides comprising modified nucleobases comprises substitution of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.

[0366] During the synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase, significant amounts of aberrant products, including double-stranded RNA (dsRNA), are produced due to the enzyme's unusual activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to protein synthesis inhibition. The formation of dsRNA can be limited during mRNA synthesis by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) used during synthesis. Optionally, UTP can be added once or several times during mRNA synthesis. Additionally, dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reverse-phase HPLC using, for example, a nonporous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method using Escherichia coli RNase III can be used, which specifically hydrolyzes dsRNA but not ssRNA, thereby removing dsRNA contaminants from IVT RNA preparations. Furthermore, dsRNA can be separated from ssRNA by using cellulose materials. In some embodiments, RNA preparation is contacted with cellulose material, and ssRNA is separated from the cellulose material under conditions that allow dsRNA to bind to the cellulose material and do not allow ssRNA to bind to the cellulose material.Suitable methods for providing ssRNA are disclosed in, for example, WO2017 / 182524.

[0367] "Removing" or "removal," as used herein, refers to the characteristic of a population of a first substance, such as non-immunogenic RNA, that is separated from the vicinity of a population of a second substance, such as dsRNA, where the population of the first substance is not necessarily devoid of the second substance, and the population of the second substance is not necessarily devoid of the first substance. However, the population of the first substance that is characterized by the removal of the population of the second substance has a measurably lower content of the second substance compared to an unseparated mixture of the first substance and the second substance.

[0368] In some embodiments, the amount of double-stranded RNA (dsRNA) is limited, and dsRNA (particularly mRNA) is removed from non-immunogenic RNA, for example, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in non-immunogenic RNA composition is dsRNA.In some embodiments, non-immunogenic RNA (particularly mRNA) does not contain or essentially does not contain dsRNA.In some embodiments, non-immunogenic RNA (particularly mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA. In some embodiments, the non-immunogenic RNA (particularly mRNA) composition comprises single-stranded nucleoside-modified RNA (particularly mRNA) and is substantially free of double-stranded RNA (dsRNA). In some embodiments, the non-immunogenic RNA (particularly mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, at least 99.993%, at least 99.994%, at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single-stranded nucleoside-modified RNA compared to all other nucleic acid molecules (DNA, dsRNA, etc.).

[0369] The amount of dsRNA can be determined by various methods.For example, sample can be contacted with dsRNA specific antibody, and the amount of antibody that binds to RNA can be considered as the measure of the amount of dsRNA in sample.The sample that contains known amount of dsRNA can be used as reference.

[0370] For example, RNA can be spotted on a membrane, for example, a nylon blotting membrane. For example, the membrane can be blocked in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v / v) TWEEN-20) containing 5% (w / v) skim milk powder. For detecting dsRNA, the membrane can be incubated with a dsRNA-specific antibody, for example, a dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). For example, after washing with TBS-T, the membrane can be incubated with a secondary antibody, for example, HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat. No. 715-035-150), to detect the signal provided by the secondary antibody.

[0371] In some embodiments, non-immunogenic RNA (especially mRNA) is translated in cells more efficiently than standard RNA having the same sequence. In some embodiments, translation is enhanced 2-fold compared to its unmodified counterpart. In some embodiments, translation is enhanced 3-fold. In some embodiments, translation is enhanced 4-fold. In some embodiments, translation is enhanced 5-fold. In some embodiments, translation is enhanced 6-fold. In some embodiments, translation is enhanced 7-fold. In some embodiments, translation is enhanced 8-fold. In some embodiments, translation is enhanced 9-fold. In some embodiments, translation is enhanced 10-fold. In some embodiments, translation is enhanced 15-fold. In some embodiments, translation is enhanced 20-fold. In some embodiments, translation is enhanced 50-fold. In some embodiments, translation is enhanced 100-fold. In some embodiments, translation is enhanced 200-fold. In some embodiments, translation is enhanced 500-fold. In some embodiments, translation is enhanced 1000-fold. In some embodiments, translation is enhanced 2000-fold. In some embodiments, the factor is between 10-1000-fold. In some embodiments, the fold is 10-100 fold. In some embodiments, the fold is 10-200 fold. In some embodiments, the fold is 10-300 fold. In some embodiments, the fold is 10-500 fold. In some embodiments, the fold is 20-1000 fold. In some embodiments, the fold is 30-1000 fold. In some embodiments, the fold is 50-1000 fold. In some embodiments, the fold is 100-1000 fold. In some embodiments, the fold is 200-1000 fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

[0372] In some embodiments, non-immunogenic RNA (especially mRNA) exhibits significantly lower natural immunogenicity than standard RNA having the same sequence. In some embodiments, non-immunogenic RNA (especially mRNA) exhibits a 2-fold lower natural immune response than its unmodified counterpart. In some embodiments, the natural immunogenicity is reduced 3-fold. In some embodiments, the natural immunogenicity is reduced 4-fold. In some embodiments, the natural immunogenicity is reduced 5-fold. In some embodiments, the natural immunogenicity is reduced 6-fold. In some embodiments, the natural immunogenicity is reduced 7-fold. In some embodiments, the natural immunogenicity is reduced 8-fold. In some embodiments, the natural immunogenicity is reduced 9-fold. In some embodiments, the natural immunogenicity is reduced 10-fold. In some embodiments, the natural immunogenicity is reduced 15-fold. In some embodiments, the natural immunogenicity is reduced 20-fold. In some embodiments, the natural immunogenicity is reduced 50-fold. In some embodiments, the natural immunogenicity is reduced 100-fold. In some embodiments, the natural immunogenicity is reduced 200-fold. In some embodiments, the natural immunogenicity is reduced by 500-fold, in some embodiments, the natural immunogenicity is reduced by 1000-fold, in some embodiments, the natural immunogenicity is reduced by 2000-fold.

[0373] The term "exhibiting significantly reduced natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In some embodiments, this term refers to a reduction such that an effective amount of non-immunogenic RNA (especially mRNA) can be administered without eliciting a detectable natural immune response. In some embodiments, this term refers to a reduction such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.

[0374] "Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in humans or other animals. The innate immune system is a relatively nonspecific, immediate component of the immune system. It is one of the two major components of the vertebrate immune system, along with the adaptive immune system.

[0375] PD-1 axis binding antagonists "Immune checkpoint" refers to regulators of the immune system, particularly costimulatory and inhibitory signals that regulate the magnitude and quality of T cell activity. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1 and / or PD-L2.

[0376] The "programmed death 1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on pre-activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), hPD-1 variants, isoforms, and species homologs, as well as analogs that share at least one common epitope with hPD-1. "Programmed death ligand 1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), hPD-L1 variants, isoforms, and species homologs, as well as analogs that share at least one common epitope with hPD-L1. As used herein, the term "PD-L2" includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, as well as analogs that share at least one common epitope with hPD-L2. PD-1 ligands (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages, and other immune cells. Binding of PD-1 to PD-L1 or PD-L2 downregulates T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 can switch off PD-1-expressing T cells, resulting in suppression of anti-cancer immune responses. The interaction between PD-1 and its ligands results in a reduction in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancerous cells. Immune suppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1; the effect is additive if the interaction between PD-1 and PD-L2 is also blocked.

[0377] Many immune checkpoints are regulated by the interaction between specific receptor-ligand pairs as described above. Thus, immune checkpoint proteins mediate immune checkpoint signaling. For example, checkpoint proteins directly or indirectly regulate T cell activation, T cell proliferation, and / or T cell function. Cancer cells often utilize these checkpoint pathways to protect themselves from attack by the immune system. Therefore, the function of checkpoint proteins regulated according to the present disclosure is typically to regulate T cell activation, T cell proliferation, and / or T cell function. Thus, immune checkpoint proteins regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses.

[0378] As used herein, the term "immune checkpoint modulator" or "checkpoint modulator" refers to a molecule or compound that modulates the function of one or more checkpoint proteins. Immune checkpoint modulators can typically modulate self-tolerance and / or the magnitude and / or duration of an immune response. Preferably, immune checkpoint modulators modulate the function of one or more human checkpoint proteins and are therefore "human checkpoint modulators." Specifically, human checkpoint modulators are immune checkpoint inhibitors.

[0379] As used herein, "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that, in whole or in part, reduces, inhibits, prevents, or negatively regulates one or more checkpoint proteins, or that, in whole or in part, reduces, inhibits, prevents, or negatively regulates the expression of one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to one or more molecules that regulate the checkpoint proteins.

[0380] In certain embodiments, the immune checkpoint inhibitor prevents inhibitory signals associated with an immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that interferes with inhibitory signaling associated with an immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is a small molecule that interferes with inhibitory signaling. In certain embodiments, the immune checkpoint inhibitor is a peptide-based inhibitor that interferes with inhibitory signaling.

[0381] In certain embodiments, the immune checkpoint inhibitor is an antibody, a fragment thereof, or an antibody mimetic that prevents interactions between checkpoint blocker proteins.

[0382] In some embodiments, inhibiting or blocking inhibitory immune checkpoint signaling as described herein results in the prevention or reversal of immune suppression and the establishment or enhancement of T cell immunity. In some embodiments, inhibiting immune checkpoint signaling as described herein reduces or inhibits immune system dysfunction. In some embodiments, inhibiting immune checkpoint signaling as described herein causes dysfunctional immune cells to become less dysfunctional. In some embodiments, inhibiting immune checkpoint signaling as described herein causes dysfunctional T cells to become less dysfunctional.

[0383] In certain embodiments, the inhibitory immunomodulator (immune checkpoint blocker) is a component of the PD-1 / PD-L1 or PD-1 / PD-L2 signaling pathway.

[0384] In certain embodiments, the inhibitory immunomodulator (immune checkpoint blocker) is a PD-1 axis binding antagonist.

[0385] The term "PD-1 axis-binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis-binding partner with one or more of its binding partners, so as to eliminate T cell dysfunction resulting from signaling on the PD-1 signaling axis, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, PD-1 axis-binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0386] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, e.g., PD-L1, PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In particular aspects, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In some embodiments, the PD-1 binding antagonist reduces negative costimulatory signals mediated by or through cell surface proteins expressed on T lymphocytes that are mediated by signaling through PD-1, making dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. Specific examples of PD-1 binding antagonists are provided below.

[0387] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1 and B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In particular aspects, PD-L1 binding antagonists inhibit the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, e.g., PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that is mediated by signaling through PD-L1, making dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. Specific examples of PD-L1 binding antagonists are provided below.

[0388] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In particular aspects, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, the PD-L2 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes that is mediated by signaling through PD-L2, making dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L2 binding antagonist is an immunoadhesin.

[0389] In some embodiments, PD-1 axis-binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. Other names for "PD-1" include CD279 and SLEB2. Other names for "PD-L1" include B7-H1, B7-4, CD274, and B7-H. Other names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0390] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand binding partner. In particular aspects, the PD-1 ligand binding partner is PD-L1 and / or PD-L2.

[0391] In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In particular aspects, the PD-L1 binding partner is PD-1 and / or B7-1.

[0392] In some embodiments, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In particular aspects, the PD-L2 binding partner is PD-1.

[0393] The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0394] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody).

[0395] Exemplary PD-1 binding antagonists include, but are not limited to, anti-PD-1 antibodies such as BGB-A317 (BeiGene; see U.S. Pat. No. 8,735,553, WO 2015 / 35606, and U.S. Patent Application Publication No. 2015 / 0079109), cemiplimab (Regeneron; see WO 2015 / 112800), and lambrolizumab (disclosed, for example, in WO 2008 / 156712 as hPD109A and its humanized derivatives h409A1, h409A16, and h409A17), AB137132 (Abcam), EH12.2H7, and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb; see WO 2006 / 121168), pembrolizumab (KEYTRUDA; MK-3475; Merck; see WO 2008 / 156712), pidilizumab (CT-011; CureTech; Hardy et al., 1994, Cancer Res., 54(22):5793-6 and WO 2009 / 101611), PDR001 (Novartis; WO 2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; WO 2012 / 145493), TSR-042 (WO 2014 / 179664), REGN-2810 (H4H7798N; U.S. Patent Application Publication No. 2015 / 0203579), JS001 (TAIZHOU JUNSHI PHARMA; Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70:136), AMP-224 (GSK-2661380; Li et al., 2016, Int J Mol Sci, 17(7):1151 and WO 2010 / 027827 and WO 2011 / 066342), PF-06801591 (Pfizer), BGB-A317 (BeiGene; WO 2015 / 35606 and U.S. Patent Application Publication No. 2015 / 0079109), BI 754091, SHR-1210 (WO 2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 described in WO 2006 / 121168; INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang et al., 2017, J. Hematol. Oncol.70:136), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 19846), IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and and WO 2017 / 133540), e.g., U.S. Patent No. 7,488,802, U.S. Patent No. 8,008,449, U.S. Patent No. 8,168,757, WO 03 / 042402, WO 2010 / 089411 (which further discloses anti-PD-L1 antibodies), WO 2010 / 036959, WO 2011 / 159877 (which further discloses antibodies against TIM-3), Nos. WO 2011 / 082400, WO 2011 / 161699, WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO 2012 / 145493 (which further disclose antibodies against PD-L1), WO 2015 / 035606, WO 2014 / 055648 (which further disclose anti-KIR antibodies) ), U.S. Patent Application Publication No. 2018 / 0185482 (which further discloses anti-PD-L1 and anti-TIGIT antibodies), U.S. Patent No. 8,008,449, U.S. Patent No. 8,779,105, U.S. Patent No. 6,808,710, U.S. Patent No. 8,168,757, U.S. Patent Application Publication No. 2016 / 0272708, and U.S. Patent No. 8,354,509.

[0396] In certain embodiments, the anti-PD-1 antibody comprises nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), cemiplimab (LIBTAYO, REGN2810), pidilizumab (CT-011), spartalizumab (PDR001), MEDI0680 (AMP-514), dostallimab (TSR-042), cetrelimab (JNJ 63723283), toripalimab (JS001), AMP-224 (GSK-2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, BI 754091, or SHR-1210.

[0397] In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Nivolumab (Bristol-Myers Squibb / Ono), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. In some embodiments, the anti-PD-1 antibody comprises heavy and light chain sequences: (a) The heavy chain has the amino acid sequence: QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK (SEQ ID NO: 11), (b) the light chain has the amino acid sequence: EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 12).

[0398] In some embodiments, the anti-PD-1 antibody comprises six CDR sequences derived from SEQ ID NO:11 and SEQ ID NO:12 (e.g., three heavy chain CDRs derived from SEQ ID NO:11 and three light chain CDRs derived from SEQ ID NO:12). In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable domain derived from SEQ ID NO:11 and a light chain variable domain derived from SEQ ID NO:12. In some embodiments, the anti-PD-1 antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:13, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:14. QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS (SEQ ID NO: 13) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO: 14)

[0399] In some embodiments, the anti-PD-1 antibody comprises (a) a heavy chain variable region (VH) comprising CDR-1 comprising the amino acid sequence of GITFSNSG (SEQ ID NO: 15), CDR-2 comprising the amino acid sequence of IWYDGSKR (SEQ ID NO: 16), and CDR-3 comprising the amino acid sequence ATNDDY (SEQ ID NO: 17); and (b) a light chain variable region (VL) comprising CDR-1 comprising the amino acid sequence of QSVSSY (SEQ ID NO: 18), CDR-2 comprising the amino acid sequence of DAS (SEQ ID NO: 19), and CDR-3 comprising the amino acid sequence of QQSSNWPRT (SEQ ID NO: 20).

[0400] In a specific embodiment, the anti-PD-1 antibody is nivolumab, which may be administered intravenously at a dose of 240 mg. Nivolumab is administered intravenously in accordance with institutional guidelines, published guidelines, and the respective product prescribing information, and may be administered according to this protocol.

[0401] In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO 2009 / 114335. In some embodiments, the anti-PD-1 antibody comprises heavy and light chain sequences: (a) The heavy chain has the amino acid sequence: QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 21), (b) the light chain has the amino acid sequence: EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO: 22).

[0402] In some embodiments, the anti-PD-1 antibody comprises six CDR sequences from SEQ ID NO:21 and SEQ ID NO:22 (e.g., three heavy chain CDRs from SEQ ID NO:21 and three light chain CDRs from SEQ ID NO:22). In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable domain derived from SEQ ID NO:21 and a light chain variable domain derived from SEQ ID NO:22. In some embodiments, the anti-PD-1 antibody comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:23, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:24. QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 23) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK (SEQ ID NO: 24)

[0403] In some embodiments, the anti-PD-1 antibody comprises (a) a heavy chain variable region (VH) comprising CDR-1 comprising the amino acid sequence of GYTFTNYY (SEQ ID NO: 25), CDR-2 comprising the amino acid sequence of INPSNGGT (SEQ ID NO: 26), and CDR-3 comprising the amino acids ARRDYRFDMGFDY (SEQ ID NO: 27); and (b) a light chain variable region (VL) comprising CDR-1 comprising the amino acid sequence of KGVSTSGYSY (SEQ ID NO: 28), CDR-2 comprising the amino acid sequence of LAS (SEQ ID NO: 29), and CDR-3 comprising the amino acid sequence of QHSRDLPLT (SEQ ID NO: 30).

[0404] In a specific embodiment, the anti-PD-1 antibody is pembrolizumab, which may be administered intravenously at a dose of 200 mg. Pembrolizumab may be administered intravenously in accordance with institutional guidelines, published guidelines, and the respective product prescribing information, and may be administered according to this protocol.

[0405] In certain embodiments, the anti-PD-1 antibody comprises cemiplimab.

[0406] In certain embodiments, the anti-PD-1 antibody comprises an antibody comprising a heavy chain and a light chain sequence: (a) The heavy chain has the amino acid sequence: EVQLLESGGV LVQPGGSLRL SCAASG FTFS NFG MTWVRQA PGKGLEWVSG ISGGGRDT YF ADSVKGRFTI SRDNSKNTLY LQMNSLKGED TAVYYC VKWG NIYFDY WGQG TLVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK (SEQ ID NO: 31), (b) the light chain has the amino acid sequence: DIQMTQSPSS LSASVGDSIT ITCRAS LSIN TF LNWYQQKP GKAPNLLIY A.S. SLHGGVPS RFSGSGSGTD FTLTIRTLQP EDFATYYC QQ SSNTPFT FGP GTVVDFRRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT Contains LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 32).

[0407] In certain embodiments, the immune checkpoint inhibitor comprises six CDR sequences from SEQ ID NO: 31 and SEQ ID NO: 32 (e.g., three heavy chain CDRs from SEQ ID NO: 31 and three light chain CDRs from SEQ ID NO: 32). In certain embodiments, the immune checkpoint inhibitor comprises an antibody comprising a heavy chain variable domain derived from SEQ ID NO: 31 and a light chain variable domain derived from SEQ ID NO: 32.

[0408] In certain embodiments, the immune checkpoint inhibitor comprises an antibody comprising: (a) a heavy chain variable region (VH) comprising CDR-1 comprising the amino acid sequence FTFSNFG (SEQ ID NO: 33), a CDR-2 comprising the amino acid sequence ISGGGRDT (SEQ ID NO: 34), and a CDR-3 comprising the amino acid sequence VKWGNIYFDY (SEQ ID NO: 35); and (b) a light chain variable region (VL) comprising CDR-1 comprising the amino acid sequence LSINTF (SEQ ID NO: 36), a CDR-2 comprising the amino acid sequence AAS (SEQ ID NO: 37), and a CDR-3 comprising the amino acid sequence QQSSNTPFT (SEQ ID NO: 38).

[0409] In some embodiments, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.

[0410] In some embodiments, the anti-PD-1 antibody is PDR001 (CAS Registry Number 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PD1 antibody that blocks the binding of PD-L1 and PD-L2 to PD-1.

[0411] In some embodiments, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD1 antibody that is also known as LIBTAYO® and cemiplimab-rwlc.

[0412] In some embodiments, the anti-PD-1 antibody is BGB-108 (BeiGene). In some embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).

[0413] In some embodiments, the anti-PD-1 antibody is JS-001 (Shanghai Junshi). JS-001 is a humanized anti-PD1 antibody.

[0414] In some embodiments, the anti-PD-1 antibody is STI-A1110 (Sorrento). STI-A1110 is a human anti-PD1 antibody.

[0415] In some embodiments, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD1 antibody.

[0416] In some embodiments, the anti-PD-1 antibody is PF-06801591 (Pfizer).

[0417] In some embodiments, the anti-PD-1 antibody is TSR-042 (also known as ANB011; Tesaro / AnaptysBio).

[0418] In some embodiments, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).

[0419] In some embodiments, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits PD-1 function without blocking the binding of PD-L1 to PD-1.

[0420] In some embodiments, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits the binding of PD-L1 to PD-1.

[0421] In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular portion or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. AMP-224 (CAS Registry Number 1422184-00-6; GlaxoSmithKline / MedImmune), also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0422] In some embodiments, the PD-1 binding antagonist is a peptide or a small molecule compound. In some embodiments, the PD-1 binding antagonist is AUNP-12 (PierreFabre / Aurigene). See, e.g., WO 2012 / 168944, WO 2015 / 036927, WO 2015 / 044900, WO 2015 / 033303, WO 2013 / 144704, WO 2013 / 132317, and WO 2011 / 161699.

[0423] In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. A variety of anti-PD-L1 antibodies are contemplated and described herein. In any of the embodiments herein, the isolated anti-PD-L1 antibody is capable of binding to human PD-L1, such as the human PD-L1 set forth in UniProtKB / Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof. In some embodiments, the anti-PD-L1 antibody is capable of inhibiting the binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the anti-PD-L1 antibody is a humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody. Examples of anti-PD-L1 antibodies useful in the methods of the invention, and methods for making them, are described in PCT patent application WO2010 / 077634A1 and U.S. Patent No. 8,217,149, which are incorporated herein by reference.

[0424] Exemplary PD-L1 binding antagonists include, but are not limited to, MEDI4736 (durvalumab; AstraZeneca; see WO 2011 / 066389), MSB-0010718C (see U.S. Patent Application Publication No. 2014 / 0341917), YW243.55.S70 (see SEQ ID NO: 20 in WO 2010 / 077634 and U.S. Patent No. 8,217,149), MIH1 (Affymetrix eBioscience; EP 3 230 319), MDX-1105 (Roche / Genentech; see WO 2013019906 and U.S. Patent No. 8,217,149), STI-1014 (Sorrento; see WO 2013 / 181634), CK-301 (checkpoint therapy), KN035 (3D Med / Alphamab; see Zhang et al., 2017, Cell Discov. 3:17004), atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267; see U.S. Patent No. 9,724,413), BMS-936559 (Bristol Myers Squibb), Squibb; U.S. Patent No. 7,943,743 and International Publication No. WO 2013 / 173223), avelumab (Bavencio; U.S. Patent Application Publication No. 2014 / 0341917), LY3300054 (Eli Lilly Co.), CX-072 (Proclaim-CX-072; also known as CytomX; see WO 2016 / 149201), FAZ053, KN035 (see WO 2017020801 and WO 2017020802), MDX-1105 (see U.S. Patent Application Publication No. 2015 / 0320859), the anti-PD-L1 antibodies disclosed in U.S. Patent No. 7,943,743, including 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, the anti-PD-L1 antibodies disclosed in WO 2010 / 077634, U.S. Patent No. No. 8,217,149, WO 2010 / 036959, WO 2010 / 077634, WO 2011 / 066342, U.S. Pat. No. 8,217,149, U.S. Pat. No. 7,943,743, WO 2010 / 089411, U.S. Pat. No. 7,635,757, U.S. Pat. No. 8,217,149, U.S. Patent No. 2009 / 0317368, WO 2011 / 066389, WO 2017 / 034916, WO 2017 / 020291, WO 2017 / 020858, WO 2017 / 020801, WO 2016 / 111645, WO 2016 / 197367, WO 2016 / 061142, WO 2016 / 149201, WO 2016 / 000619, WO 2016 / 160792, WO These include the anti-PD-L1 antibodies described in WO 2016 / 022630, WO 2016 / 007235, WO 2015 / 179654, WO 2015 / 173267, WO 2015 / 181342, WO 2015 / 109124, WO 2018 / 222711, WO 2015 / 112805, WO 2015 / 061668, WO 2014 / 159562, WO 2014 / 165082, and WO 2014 / 100079.

[0425] In specific embodiments, the anti-PD-L1 antibody comprises atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267), durvalumab (MEDI4736), BMS-936559, avelumab (BAVENCIO), lodapolimab (LY3300054), CX-072 (Proclaim-CX-072), FAZ053, KN035, or MDX-1105.

[0426] PD-1 axis-binding antagonists, such as anti-PD-1 antibodies and anti-PD-L1 antibodies, can be administered in any manner and by any route known in the art. The method and route of administration depend on the type of PD-1 axis-binding antagonist used.

[0427] The PD-1 axis binding antagonist may be administered in the form of any suitable pharmaceutical composition described herein.

[0428] PD-1 axis binding antagonists, such as anti-PD-1 antibodies and anti-PD-L1 antibodies, can be administered in the form of nucleic acids, such as DNA or RNA, encoding the PD-1 axis binding antagonists, such as anti-PD-1 antibodies or anti-PD-L1 antibodies. For example, the antibodies can be delivered encoded by expressing nucleic acids as described herein. The nucleic acid molecules can be delivered by themselves, for example, in the form of a plasmid or mRNA molecule, or can be complexed with a delivery vehicle, such as a liposome, lipoplex, or any other nucleic acid particle, nucleic acid-lipid particle. PD-1 axis binding antagonists, such as anti-PD-1 antibodies and anti-PD-L1 antibodies, can also be administered via oncolytic viruses containing an expression cassette encoding the PD-1 axis binding antagonist.

[0429] immunostimulants An "immunostimulant" is any substance that stimulates the immune system by inducing activation or increasing the activity of any of the components of the immune system, particularly immune effector cells. Immunostimulants can be pro-inflammatory (e.g., when treating infections or cancer) or anti-inflammatory (e.g., when treating autoimmune diseases).

[0430] In one embodiment, the immunostimulatory agent is a cytokine or a variant thereof. Examples of cytokines include interferons, such as interferon-α (IFN-α) or interferon-γ (IFN-γ), interleukins, such as IL2, IL7, IL12, IL15, and IL23, colony-stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor. In another embodiment, the immunostimulatory agent comprises an adjuvant-type immunostimulatory agent, such as an APC Toll-like receptor agonist or a costimulatory / cell adhesion membrane protein. Examples of Toll-like receptor agonists include costimulatory / adhesion proteins, such as CD80, CD86, and ICAM-1.

[0431] The term "cytokine" refers to proteins with a molecular weight of approximately 5 to 60 kDa that are involved in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). In particular, cytokines, when released, affect the behavior of cells in the vicinity of their release. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they typically act at much more variable concentrations than hormones and (ii) they are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons are typically characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that alter and regulate intracellular gene transcription by binding to interferon receptors on the surface of the cells, thereby preventing viral replication within the cells. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factors (TNFs), bone morphogenetic proteins (BMPs), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (INFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), and variants and derivatives thereof.

[0432] According to the present disclosure, cytokine can be naturally occurring cytokine or its functional fragment or variant.Cytokine can be human cytokine and can be derived from any vertebrate, particularly any mammal.One particularly preferred cytokine is interferon-α.

[0433] The immunostimulatory agent can be provided to the subject by administering to the subject the RNA that encodes the immunostimulatory agent in the formulation for selectively delivering RNA to liver or liver tissue.The delivery of RNA to such target organ or tissue is preferred, particularly when it is desired to express a large amount of immunostimulatory agent, and / or when it is desired or required that the immunostimulatory agent is present in a systemic manner, particularly in a significant amount.

[0434] RNA delivery systems have an inherent selectivity for the liver. This is relevant for lipid-based particles, cationic and neutral nanoparticles, especially lipid nanoparticles.

[0435] The example of suitable immunostimulant for targeting liver is the cytokine that is involved in the proliferation and / or maintenance of T cell.Example of suitable cytokine includes IL2 or IL7, its fragment and variant, and the fusion protein of these cytokines, fragment and variant, such as extended PK cytokine.

[0436] In another embodiment, the RNA encoding the immunostimulatory agent can be administered in a formulation for selective delivery of the RNA to the lymphatic system, particularly to secondary lymphoid organs, more particularly to the spleen. Delivery of the immunostimulatory agent to such target tissues is particularly preferred when the presence of the immunostimulatory agent in this organ or tissue is desirable (for example, when the immunostimulatory agent, such as a cytokine, is required to induce an immune response, particularly during T cell priming or for the activation of resident immune cells), but when the immunostimulatory agent is not desirable to be present systemically, especially in significant amounts (for example, because the immunostimulatory agent has systemic toxicity).

[0437] Examples of suitable immunostimulatory agents include cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-α, or IFN-β, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

[0438] interferon Interferons (IFNs) are a group of signaling proteins produced and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, and parasites, as well as tumor cells. In a typical scenario, a virus-infected cell releases interferons to enhance the antiviral defenses of nearby cells.

[0439] Based on the type of receptor through which the interferons signal, interferons are typically divided into three classes: type I interferons, type II interferons, and type III interferons.

[0440] All type I interferons bind to a specific cell surface receptor complex known as the IFN-α / β receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains.

[0441] The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ, and IFNω. Generally, type I interferons are produced when the body recognizes an invading virus. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, resulting in the expression of proteins that prevent the virus from producing and replicating its RNA and DNA.

[0442] IFNα proteins are primarily produced by plasmacytoid dendritic cells (pDCs). They are primarily involved in innate immunity against viral infections. The genes responsible for their synthesis belong to 13 subtypes, designated IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. These genes are found together in a cluster on chromosome 9.

[0443] IFNβ proteins are produced in large amounts by fibroblasts. They have antiviral activity and are primarily involved in the innate immune response. Two types of IFNβ have been described: IFNβ1 and IFNβ3. Both native and recombinant forms of IFNβ1 possess antiviral, antibacterial, and anticancer properties.

[0444] Type II interferons (IFNγ in humans), also known as immune interferons, are activated by IL12. Additionally, type II interferons are released by cytotoxic T cells and T helper cells.

[0445] Type III interferons signal through a receptor complex consisting of IL10R2 (also known as CRF2-4) and IFNLR1 (also known as CRF2-12). Although more recently discovered than type I and type II IFNs, recent information has demonstrated the importance of type III IFNs in several types of viral or fungal infections.

[0446] In general, type I and type II interferons are responsible for regulating and activating the immune response.

[0447] According to the present disclosure, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα.

[0448] According to the present disclosure, the interferon can be a naturally occurring interferon or a functional fragment or variant thereof. The interferon can be a human interferon and can be derived from any vertebrate, particularly any mammal.

[0449] Interleukin Interleukins (ILs) are a group of cytokines (secreted proteins and signaling molecules) that can be divided into four major groups based on prominent structural features. However, their amino acid sequence similarity is fairly weak (typically 15-25% identity). The human genome encodes over 50 interleukins and related proteins.

[0450] According to the present disclosure, the interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. The interleukin may be a human interleukin and may be derived from any vertebrate, particularly any mammal.

[0451] extended PK group The immunostimulatory polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising an immunostimulatory moiety and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory agent). The immunostimulatory agent can be fused to an extended PK group that increases its circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that increase the circulating half-life of immunostimulatory agents, such as cytokines or variants thereof, are also applicable to the present disclosure. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0452] As used herein, the term "PK" is an acronym for "pharmacokinetics" and encompasses, by way of example, the characteristics of a compound, including absorption, distribution, metabolism, and excretion by a subject. As used herein, an "extended PK group" refers to a protein, peptide, or moiety that, when fused to or administered together with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of extended PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (disclosed in U.S. Patent Application Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary extended PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, the entire contents of which are incorporated herein by reference. As used herein, an "extended PK" immunostimulatory agent refers to an immunostimulatory moiety in combination with an extended PK group. In some embodiments, an extended PK immunostimulatory agent is a fusion protein in which the immunostimulatory moiety is linked or fused to the extended PK group.

[0453] In certain embodiments, the serum half-life of the extended PK immunostimulant is increased compared to the immunostimulant alone (i.e., the immunostimulant not fused to an extended PK group). In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold longer than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0454] As used herein, "half-life" refers to the time required for the serum or plasma concentration of a compound, such as a peptide or polypeptide, to decrease by 50% in vivo, for example, due to degradation and / or clearance or sequestration by natural mechanisms. Extended PK immunostimulants suitable for use herein are stabilized in vivo, and their half-life is increased, for example, by fusion to serum albumin (e.g., HSA or MSA), which resists degradation and / or clearance or sequestration. Half-life can be determined by any method known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, generally include the steps of administering an appropriate dose of an amino acid sequence or compound to a subject; collecting blood or other samples from the subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in the blood samples; and calculating, from a plot of the data thus obtained, the time until the level or concentration of the amino acid sequence or compound decreases by 50% compared to the initial level at the time of administration. Further details are provided in standard handbooks such as, for example, Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0455] In certain embodiments, the extended PK group comprises serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are included in the term "albumin" for purposes of this disclosure). The polypeptides described herein can be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Patent Application Publication No. 20070048282.

[0456] As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) with at least one molecule of a protein, such as a Therapeutic protein, particularly an immunostimulant. Albumin fusion proteins can be produced by translation of a nucleic acid in which a polynucleotide encoding a Therapeutic protein is linked in-frame to a polynucleotide encoding albumin. Once part of an albumin fusion protein, the Therapeutic protein and albumin can be referred to as "portions," "regions," or "moieties" of the albumin fusion protein, respectively (e.g., a "Therapeutic protein portion" or an "albumin protein portion"). In highly preferred embodiments, the albumin fusion protein comprises at least one molecule of a Therapeutic protein (including, but not limited to, the mature form of a Therapeutic protein) and at least one molecule of albumin (including, but not limited to, the mature form of albumin). In some embodiments, the albumin fusion protein is processed by host cells, such as hepatocytes, in the target organ of the administered RNA and secreted into the circulation. Processing of the nascent albumin fusion protein in the secretory pathway of the host cell used to express the RNA may include, but is not limited to, signal peptide cleavage; disulfide bond formation; proper folding; carbohydrate addition and processing (e.g., N-linked and O-linked glycosylation); specific proteolytic cleavage; and / or assembly into a multimeric protein. The albumin fusion protein is preferably encoded by the RNA in an unprocessed form, particularly with a signal peptide at its N-terminus, and after secretion by the cell, preferably exists in a processed form, particularly with the signal peptide cleaved. In the most preferred embodiment, the "processed form of the albumin fusion protein" refers to the albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as the "mature albumin fusion protein."

[0457] In preferred embodiments, albumin fusion proteins containing a therapeutic protein have higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the period from when a therapeutic protein is administered in vivo and transported into the bloodstream, until the therapeutic protein is degraded and removed from the bloodstream to organs such as the kidneys or liver, and finally, when the therapeutic protein is removed from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be easily determined by common assays known in the art.

[0458] As used herein, "albumin" collectively refers to an albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, particularly the mature form of human albumin, or albumin or a fragment thereof from another vertebrate, or a variant of these molecules. Albumin can be derived from any vertebrate, particularly any mammal, such as human, bovine, ovine, or porcine. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein can be derived from a different animal than the therapeutic protein portion.

[0459] In certain embodiments, the albumin is human serum albumin (HSA), or a fragment or variant thereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO 2011 / 124718, WO 2013 / 075066, and WO 2011 / 0514789.

[0460] The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and include human serum albumin (and fragments and variants thereof) as well as albumin (and fragments and variants thereof) from other species.

[0461] As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of a Therapeutic protein refers to a fragment of albumin of sufficient length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein, such that the plasma stability of the Therapeutic protein portion of the albumin fusion protein is extended or expanded compared to its plasma stability in the unfused state.

[0462] The albumin portion of the albumin fusion protein may comprise the full length of the albumin sequence, or may comprise one or more fragments thereof that can stabilize or extend therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may comprise approximately 15, 20, 25, 30, 50, or more consecutive amino acids from the albumin sequence, or may comprise part or all of a particular domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0463] Generally speaking, an albumin fragment or variant is at least 100 amino acids in length, preferably at least 150 amino acids in length.

[0464] According to the present disclosure, the albumin can be a naturally occurring albumin or a fragment or variant thereof. The albumin can be human albumin and can be derived from any vertebrate, particularly any mammal.

[0465] Preferably, the albumin fusion protein comprises albumin as the N-terminal moiety and a Therapeutic protein as the C-terminal moiety. Alternatively, albumin fusion proteins comprising albumin as the C-terminal moiety and a Therapeutic protein as the N-terminal moiety may also be used. In other embodiments, the albumin fusion protein has Therapeutic proteins fused to both the N- and C-termini of albumin. In a preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are the same Therapeutic protein. In another preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins. In some embodiments, both different Therapeutic proteins are cytokines.

[0466] In some embodiments, one or more therapeutic proteins are linked to albumin via one or more peptide linkers. A linker peptide between the fusion moieties can provide greater physical separation between the moieties, thus maximizing the accessibility of the therapeutic protein moieties to, for example, bind to their cognate receptors. The linker peptide can be composed of amino acids that are flexible or more rigid. The linker sequence can be protease- or chemically cleavable.

[0467] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of the two heavy chains of the native immunoglobulin. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain in which the Fc domain does not include an Fv domain. In certain embodiments, the Fc domain begins at the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or a portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, an Fc domain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). An Fc domain, as used herein, generally refers to a polypeptide comprising all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides, e.g., comprising only the hinge, CH2, and CH3 domains.The Fc domain can be derived from any species and / or any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The Fc domain encompasses native Fc and Fc variant molecules. As described herein, those skilled in the art will understand that any Fc domain can be modified such that its amino acid sequence differs from that of a native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0468] The Fc domain of the polypeptide described herein can be derived from different immunoglobulin molecules.For example, the Fc domain of the polypeptide can include a CH2 and / or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.In another example, the Fc domain can include a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule.In another example, the Fc domain can include a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0469] In certain embodiments, the extended PK group comprises an Fc domain or a fragment thereof, or a variant of an Fc domain or a fragment thereof (all of which are encompassed by the term "Fc domain" for purposes of this disclosure). The Fc domain does not comprise a variable region that binds to an antigen. Fc domains suitable for use in the present disclosure can be obtained from several different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain can be derived from an immunoglobulin of another mammalian species, including, for example, a rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species.

[0470] Furthermore, the Fc domain (or a fragment or variant thereof) can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0471] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains can be selected, including Fc domain sequences that lack specific effector functions and / or have specific modifications that reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and appropriate Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using techniques widely recognized in the art.

[0472] In certain embodiments, the extended PK group is a serum albumin binding protein, such as those described in U.S. Patent Application Nos. 2005 / 0287153, 2007 / 0003549, 2007 / 0178082, 2007 / 0269422, 2010 / 0113339, WO 2009 / 083804, and WO 2009 / 133208, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is transferrin, as disclosed in U.S. Patent Nos. 7,176,278 and 8,158,579, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is a serum immunoglobulin binding protein, such as those disclosed in US Patent Application No. 2007 / 0178082, US Patent Application No. 2014 / 0220017, and US Patent Application No. 2017 / 0145062, the entire contents of which are incorporated herein by reference. In certain embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US Patent Application No. 2012 / 0094909, the entire contents of which are incorporated herein by reference. A method for producing a fibronectin-based scaffold domain protein is also disclosed in US Patent Application No. 2012 / 0094909. A non-limiting example of an Fn3-based extended PK group is Fn3 (HSA), i.e., an Fn3 protein that binds to human serum albumin.

[0473] In certain embodiments, the extended PK immunostimulatory agent suitable for use according to the present disclosure can use one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that connects two or more domains (e.g., an extended PK portion and an immunostimulatory portion) in the linear amino acid sequence of a polypeptide chain. For example, a peptide linker can be used to connect the immunostimulatory portion to the HSA domain.

[0474] For example, suitable linkers for fusing an extended PK group to an immunostimulatory agent are well known in the art. Exemplary linkers include glycine-serine polypeptide linkers, glycine-proline polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0475] immune effector cells As used herein, immune effector cells can be administered to a subject in need of treatment or can be endogenously present in the subject in need of treatment. Administration of RNA encoding a vaccine antigen and a PD-1 axis binding antagonist to a subject allows for stimulation of immune effector cells. The methods and medicaments described herein are particularly useful for treating diseases characterized by disease cells expressing an antigen against which immune effector cells are directed. In some embodiments, the immune effector cells bear an antigen receptor, such as a T cell receptor (TCR) or chimeric antigen receptor (CAR), with binding specificity for the antigen or its processing product. In some embodiments, the immune effector cells are present in the subject to be treated and express the antigen receptor. In some embodiments, the immune effector cells are present in the subject to be treated and are genetically modified in vivo in the subject to express the antigen receptor. In some embodiments, immune effector cells from either the subject to be treated or a different subject are administered to the subject to be treated. The administered immune effector cells may be genetically modified ex vivo prior to administration, or may be genetically modified in vivo in the subject after administration to express an antigen receptor. In some embodiments, the antigen receptor is endogenous to the immune effector cells.

[0476] In some embodiments, immune effector cells include any cells that are responsive to vaccine antigens. Such responsiveness includes activation, differentiation, proliferation, survival, and / or display of one or more immune effector functions. Cells include, in particular, lytic cells, particularly lymphoid cells, preferably T cells, particularly cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, these cytotoxic lymphocytes each cause target cell destruction. For example, cytotoxic T cells cause target cell destruction by one or both of the following means: First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and trigger a cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between T cells and target cells. The cells used in connection with the present invention are preferably autologous cells, although xenogeneic or allogeneic cells can be used. In some embodiments, the immune effector cells are endogenous to the subject being treated.

[0477] The term "effector function" in the context of the present invention includes any function mediated by a component of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, including, for example, the killing of diseased cells, such as tumor cells, or the inhibition of tumor dissemination and metastasis. Preferably, the effector function in the context of the present invention is a T cell-mediated effector function. Such a function is mediated by helper T cells (CD4 + T cells), cytokine release and / or CD8 + It involves the activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, the elimination of cells, i.e., cells characterized by expression of the antigen, e.g., via apoptosis or perforin-mediated cytolysis, the production of cytokines such as IFN-γ and TNF-α, and the specific cytolytic killing of target cells expressing the antigen.

[0478] The term "immune effector cell" or "immunoreactive cell" in the context of the present invention relates to a cell that exerts an effector function during an immune response. In some embodiments, an "immune effector cell" can bind to an antigen, such as an antigen presented in association with MHC on a cell or expressed on the surface of a cell, and mediate an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, an "immune effector cell" is a T cell, preferably a CD4 + and / or CD8 + T cells, most preferably CD8 + According to the present invention, the term "immune effector cells" also includes cells that can mature into immune cells (such as T cells, especially T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells are CD34 + They include hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells resembles the clonal selection of the immune system upon exposure to antigen.

[0479] Preferably, "immune effector cells" recognize antigens with some degree of specificity, especially when presented in the context of MHC or present on the surface of diseased cells such as cancer cells. Preferably, such recognition enables the cells that recognize the antigen to be responsive or reactive. The cells are called helper T cells (CD4 + T cells), such responsiveness or reactivity may be mediated by the release of cytokines and / or CD8 +The activation of lymphocytes (CTLs) and / or B cells may be included. If the cells are CTLs, such responsiveness or reactivity may include the elimination of cells, i.e., cells characterized by the expression of an antigen, via, for example, apoptosis or perforin-mediated cytolysis. According to the present disclosure, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing the antigen. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness. Such CTLs that recognize and are responsive or reactive to an antigen are also referred to herein as "antigen-responsive CTLs."

[0480] In some embodiments, the genetically modified immune effector cells are CAR-expressing immune effector cells. In some embodiments, the genetically modified immune effector cells are TCR-expressing immune effector cells.

[0481] Immune effector cells may express an endogenous antigen receptor, such as a T cell receptor or a B cell receptor, or may lack expression of an endogenous antigen receptor.

[0482] "Lymphoid cells" are cells or precursors of such cells that can generate immune responses, such as cellular immune responses, optionally after appropriate modification, for example, after introduction of antigen receptors such as TCRs or CARs, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells can be immune effector cells as described herein. Preferred lymphoid cells are T cells, which can be modified to express antigen receptors on their cell surface. In some embodiments, lymphoid cells lack endogenous expression of T cell receptors.

[0483] antigen receptor The immune effector cells described herein express an antigen receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR)-binding antigen, or its processing product, particularly when present on or presented by a target cell, such as an antigen-presenting cell or a diseased cell. The cells may naturally express the antigen receptor or may be modified to express the antigen receptor. In some embodiments, the immune effector cells are genetically modified to express the antigen receptor ex vivo / in vitro or in vivo in the subject to be treated. In some embodiments, the modification to express the antigen receptor is performed ex vivo / in vitro. The modified cells can then be administered to the patient. In some embodiments, the modification to express the antigen receptor is performed in vivo. The cells may be endogenous cells of the patient or may have been administered to the patient.

[0484] Chimeric Antigen Receptor Adoptive cell transfer therapy using CAR-engineered T cells expressing chimeric antigen receptors is a promising anticancer treatment because CAR-modified T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells can be genetically engineered (modified) to express a CAR that specifically targets an antigen on the patient's tumor cells, and then infused back into the patient.

[0485] According to the present invention, the term "CAR" (or "chimeric antigen receptor") is synonymous with the terms "chimeric T cell receptor" and "artificial T cell receptor" and relates to an artificial receptor comprising a single molecule or complex of molecules that can recognize, i.e., bind to, a target structure (e.g., an antigen) on a target cell, such as a cancer cell (e.g., by binding of an antigen-binding domain to an antigen expressed on the surface of the target cell), and confer specificity to an immune effector cell, such as a T cell, that expresses the CAR on its cell surface. Such cells do not necessarily require antigen processing and presentation for target cell recognition, but rather can preferably specifically recognize any antigen present on the target cell. Preferably, recognition of the target structure by a CAR results in activation of the immune effector cell that expresses the CAR. A CAR can comprise one or more protein units comprising one or more domains described herein. The term "CAR" does not include T cell receptors.

[0486] CARs generally contain a target-specific binding element, also referred to as an antigen-binding portion or antigen-binding domain, which is part of the extracellular domain of the CAR. The antigen-binding domain recognizes a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Specifically, the CARs of the present invention target antigens, such as tumor antigens, on diseased cells, such as tumor cells.

[0487] In some embodiments, the binding domain of the CAR specifically binds to an antigen. In some embodiments, the antigen to which the binding domain in the CAR binds is expressed on cancer cells (tumor antigens). In some embodiments, the antigen is expressed on the surface of cancer cells. In some embodiments, the binding domain binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In some embodiments, the binding domain binds to a natural epitope of the antigen present on the surface of a living cell.

[0488] In some embodiments of the present invention, the antigen-binding domain comprises a variable region (VH) of an immunoglobulin heavy chain having specificity for an antigen and a variable region (VL) of an immunoglobulin light chain having specificity for an antigen. In some embodiments, the immunoglobulin is an antibody. In some embodiments, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are connected via a peptide linker. Preferably, part of the antigen-binding portion of the CAR is an scFv.

[0489] CAR is designed to include a transmembrane domain fused to the extracellular domain of CAR. In some embodiments, the transmembrane domain is not naturally associated with one of the domains in CAR. In some embodiments, the transmembrane domain is naturally associated with one of the domains in CAR. In some embodiments, the transmembrane domain is modified by amino acid substitution to prevent such domain from binding to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex. The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domains particularly useful in the present invention can be derived from (i.e., comprise at least one or more of) the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it contains primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0490] In some cases, the CAR comprises a hinge domain that forms a link between the transmembrane domain and the extracellular domain.

[0491] The cytoplasmic domain or other intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0492] It is known that signals generated solely through the TCR are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0493] In some embodiments, the CAR comprises a primary cytoplasmic signaling sequence derived from CD3 zeta. Additionally, the cytoplasmic domain of the CAR may comprise a CD3 zeta signaling domain combined with a costimulatory signaling region.

[0494] The identity of the costimulatory domain is limited only by its ability to enhance cell proliferation and survival upon binding of the targeting moiety by the CAR. Suitable costimulatory domains include CD28, CD137 (4-1BB), a member of the tumor necrosis factor receptor (TNFR) superfamily, CD134 (OX40), a member of the TNFR superfamily of receptors, and CD278 (ICOS), a CD28 superfamily costimulatory molecule expressed on activated T cells. Those skilled in the art will understand that sequence variants of these described costimulatory domains can be used without adversely affecting the present invention if they have the same or similar activity as the domain they are modeled after. Such variants have at least about 80% sequence identity with the amino acid sequence of the domain from which they are derived. In some embodiments of the present invention, the CAR construct comprises two costimulatory domains. Specific combinations include all possible variations of the four described domains, with specific examples including CD28+CD137(4-1BB) and CD28+CD134(OX40).

[0495] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in random or specified order. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0496] In some embodiments, the CAR comprises a signal peptide that directs the nascent protein into the endoplasmic reticulum. In some embodiments, the signal peptide precedes the antigen-binding domain. In some embodiments, the signal peptide is derived from an immunoglobulin, such as IgG.

[0497] According to the present disclosure, a CAR recognizes an antigen, such as on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, such that when present on a T cell, the T cell is stimulated and / or expanded or exerts an effector function, as described above.

[0498] Genetic modification of immune effector cells CD8 + Particles functionalized for specific targeting of immune effector cells, such as T cells, can be used ex vivo / in vitro or in vivo to deliver nucleic acids encoding antigen receptors to immune effector cells, such as T cells, to produce cells genetically modified to express the antigen receptor. Such genetic modifications include non-viral DNA transfection, non-viral RNA transfection (e.g., mRNA transfection), transposon-based systems, and viral-based systems. Non-viral DNA transfection has a lower risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids without integration elements. Viral-based systems include the use of gamma-retrovirus and lentiviral vectors. Gamma-retroviruses are relatively easy to generate and efficiently and persistently transduce T cells, and have been preliminarily proven to be safe in terms of integration in primary human T cells. Lentiviral vectors also efficiently and persistently transduce T cells, but are more expensive to manufacture. They are also potentially safer than retroviral-based systems.

[0499] In some embodiments of all aspects of the invention, T cells or T cell precursors are transfected with a nucleic acid encoding an antigen receptor either ex vivo or in vivo. In some embodiments, a combination of ex vivo and in vivo transfection may be used. In some embodiments of all aspects of the invention, the T cells or T cell precursors are derived from a subject to be treated. In some embodiments of all aspects of the invention, the T cells or T cell precursors are derived from a subject different from the subject to be treated.

[0500] In one embodiment of the present invention, CAR T cells can be generated in vivo, thus almost instantly, using particles such as nanoparticles that target T cells. For example, lipid and / or polymer-based nanoparticles can be coupled to CD8-specific targeting moieties to bind to CD8 on T cells. Upon binding to T cells, these nanoparticles are endocytosed. Their contents, such as nucleic acids encoding antigen receptors, such as plasmid DNA encoding anti-tumor antigen CARs, can be directed to the T cell nucleus because they contain, for example, peptides containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS). The inclusion of a transposon flanking the nucleic acid encoding the antigen receptor, such as a CAR gene expression cassette, and a separate nucleic acid, such as a plasmid, encoding a hyperactive transposase can enable efficient integration of the nucleic acid encoding the antigen receptor, such as a CAR vector, into the chromosome.

[0501] Another possibility is to use CRISPR / Cas9 technology to deliberately place an antigen receptor coding sequence, such as a CAR coding sequence, at a specific genetic locus, for example, knocking out an existing T cell receptor (TCR) while knocking in the CAR and placing it under the dynamic regulatory control of an endogenous promoter that would otherwise silence TCR expression.

[0502] Therefore, in addition to nucleic acids encoding antigen receptors, the particles described herein can also deliver gene editing tools such as CRISPR / Cas9 (or related) or transposon systems such as Sleeping Beauty or Piggybag as cargo. Such tools for genome integration / editing (e.g., transposases, gene editing tools such as CRISPR / Cas9) can be delivered as proteins or encoding nucleic acids (DNA or RNA). Nevertheless, delivery of mRNA is also an option for inducing transient expression of antigen receptors such as CARs or T cell receptors (TCRs).

[0503] In some embodiments of all aspects of the invention, cells genetically modified to express an antigen receptor are stably or transiently transfected with a nucleic acid encoding the antigen receptor, and thus the nucleic acid encoding the antigen receptor may or may not be integrated into the genome of the cell.

[0504] In some embodiments of all aspects of the invention, the cells genetically modified to express an antigen receptor are inactivated with respect to expression of endogenous T cell receptors and / or endogenous HLA.

[0505] In some embodiments of all aspects of the present invention, the cells described herein can be autologous, allogeneic, or syngeneic to the subject being treated. In some embodiments, the present disclosure contemplates the removal of cells from the patient and subsequent re-delivery of the cells to the patient. In some embodiments, the present disclosure does not contemplate the removal of cells from the patient. In the latter case, all steps of genetic modification of the cells are performed in vivo.

[0506] The term "autologous" is used to refer to something derived from the same subject. For example, "autologous transplantation" refers to the transplantation of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome immunological barriers that would otherwise result in rejection.

[0507] The term "allogeneic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical.

[0508] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, i.e., derived from identical twins or the same inbred strain of animals, or tissues thereof.

[0509] The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. As an example, transferring bone marrow from one individual to another constitutes a xenogeneic transplant. A xenogeneic gene is a gene that originates from a source other than the subject.

[0510] particle The nucleic acid described herein, such as RNA, particularly mRNA, can be present in particles comprising (i) nucleic acid and (ii) at least one cationic or cationic ionizable compound, such as polymer or lipid, that complexes nucleic acid.The electrostatic interaction between positively charged molecules, such as polymer and lipid, and negatively charged nucleic acid is involved in particle formation.This leads to the complexation and spontaneous formation of nucleic acid particles.

[0511] Various types of RNA-containing particles have previously been described as suitable for delivering RNA in a microparticulate form (see, e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). In the case of non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA can physically protect the RNA from degradation and, depending on the specific chemical properties, can aid in cellular uptake and endosomal escape.

[0512] In the context of the present disclosure, the term "particle" refers to a structured entity formed by a molecule or molecular complex, particularly a particle-forming compound. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellae) made of one or more amphiphilic substances (e.g., amphiphilic lipids). In this context, the term "amphiphilic substance" means that the substance has both hydrophilic and lipophilic properties. The envelope may also contain additional substances (e.g., additional lipids) that do not necessarily have amphiphilic properties. Thus, the particle may be a monolamellar or multilamellar structure in which the substance constituting one or more layers or lamellae contains one or more amphiphilic substances (e.g., selected from the group consisting of amphiphilic lipids), optionally in combination with additional substances (e.g., additional lipids) that do not necessarily have amphiphilic properties. In some embodiments, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0513] "RNA particles" can be used to deliver RNA to a desired target site (e.g., cells, tissues, organs, etc.). RNA particles can be formed from lipids containing at least one cationic or cationic ionizable lipid or lipid-like substance. Without intending to be bound by any theory, it is believed that the cationic or cationic ionizable lipid or lipid-like substance binds together with RNA to form aggregates, and this aggregation results in colloidally stable particles.

[0514] The RNA particles described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0515] Generally, lipoplexes (LPX) can be obtained by mixing two aqueous phases: one containing RNA and one containing a lipid dispersion. In some embodiments, the lipid phase comprises liposomes.

[0516] In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles, the lamellae of which comprise a lipid bilayer and the enclosed lumen of which comprises an aqueous phase. A prerequisite for using liposomes to form nanoparticles is that they can form a lamellar (bilayer) phase in an aqueous environment to which the lipids in the mixture are applied as needed.

[0517] In some embodiments, liposomes comprise a single or multiple phospholipid bilayer surrounding an aqueous core (also referred to herein as an aqueous lumen). They can be prepared from materials with polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids used to formulate liposomes designed for nucleic acid delivery are amphiphilic in nature, consisting of a positively charged (cationic) amine head group linked via glycerol to a hydrocarbon chain or cholesterol derivative.

[0518] In some embodiments, the lipoplexes are multilamellar liposome-based formulations formed upon electrostatic interaction between cationic liposomes and RNA. In some embodiments, the formed lipoplexes have a different internal arrangement of molecules resulting from the conversion of the liposomal structure to a compact RNA-lipoplex. In some embodiments, these formulations are characterized by insufficient RNA encapsulation and incomplete RNA capture.

[0519] In some embodiments, the LPX particles comprise amphiphilic lipids, particularly cationic or cationic ionizable amphiphilic lipids, and RNA (particularly mRNA) as described herein. In some embodiments, electrostatic interactions between positively charged liposomes (made from one or more amphiphilic lipids, particularly cationic or cationic ionizable amphiphilic lipids) and negatively charged nucleic acids (particularly mRNA) result in complexation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes can generally be synthesized using cationic or cationic ionizable amphiphilic lipids, such as DOTMA and / or DODMA, and additional lipids, such as DOPE. In some embodiments, the RNA (particularly mRNA) lipoplex particles are nanoparticles.

[0520] Generally, lipid nanoparticles (LNPs) can be obtained by directly mixing RNA in an aqueous phase with lipids in a phase containing an organic solvent such as ethanol. In this case, lipids or lipid mixtures that do not form a lamellar (bilayer) phase in water can be used for particle formation.

[0521] In some embodiments, the LNPs comprise or consist of cationic / ionizable lipids and helper lipids, such as phospholipids, cholesterol, and / or polyethylene glycol (PEG) lipids. In some embodiments, in the RNA LNPs described herein, the mRNA is bound by an ionizable lipid that occupies the central core of the LNP. In some embodiments, the PEG lipids, together with the phospholipids, form the surface of the LNP. In some embodiments, the surface comprises a bilayer. In some embodiments, charged and uncharged forms of cholesterol and ionizable lipids can be distributed throughout the LNP.

[0522] In some embodiments, RNA (e.g., mRNA) may be non-covalently associated with a particle described herein. In embodiments, the RNA (particularly mRNA) may be attached to the outer surface of the particle (surface RNA (particularly surface mRNA)) and / or may be contained within the particle (encapsulated RNA (particularly encapsulated mRNA)).

[0523] In some embodiments, the particles described herein (e.g., LNP and LPX) have a particle size in the range of about 10 to about 2000 nm, e.g., at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most 1900 nm (e.g., at most about 1900 nm, at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), for example, about 20 to about 1500 nm, for example, about 30 to about 1200 nm, about 40 to about 1100 nm , about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to 800 nm, about 80 to 700 nm, about 90 to 600 nm, or about 50 to 500 nm or about 100 to 500 nm, for example, 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, or 70 to 150 nm.

[0524] In some embodiments, the particles described herein (e.g., LNP and LPX) may be from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 30 0nm, about 50nm to about 250nm, about 50nm to about 200nm, about 100nm to about 1000nm, about 100nm to about 800nm, about 100nm to about 700nm, about 100nm to about 6 00nm, approximately 100nm to approximately 500nm, approximately 100nm to approximately 450nm, approximately 100nm to approximately 400nm, approximately 100nm to approximately 350nm, approximately 100nm to approximately 300nm, approximately 100nm to approximately 250nm, about 100nm to about 200nm, about 150nm to about 1000nm, about 150nm to about 800nm, about 150nm to about 700nm, about 150nm to about 600nm, about 150n m ~ about 500nm, about 150nm - about 450nm, about 150nm - about 400nm, about 150nm - about 350nm, about 150nm - about 300nm, about 150nm - about 250nm, about 150 The average diameter ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, or from about 200 nm to about 250 nm.

[0525] In some embodiments, the particle described herein is nanoparticle.The term " nanoparticle " refers to the nano-sized particle that comprises nucleic acid (particularly mRNA) described herein and at least one cationic or cationic ionizable lipid, and the three external dimensions of the particle are all nanoscale, that is, at least about 1 nm and less than about 1000 nm.Preferably, the size of the particle is its diameter.

[0526] The nucleic acid particles (particularly mRNA particles) described herein can exhibit a polydispersity index (PDI) of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the nucleic acid particles can exhibit a polydispersity index ranging from about 0.01 to about 0.4 or from about 0.1 to about 0.3.

[0527] The N / P ratio gives the ratio of the number of nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. This correlates with the charge ratio, since nitrogen atoms (depending on the pH) are usually positively charged and phosphate groups are negatively charged. If charge balance exists, the N / P ratio is pH dependent. Because positively charged nanoparticles are thought to favor transfection, lipid formulations are often formed with an N / P ratio greater than 4 and up to 12. In that case, the RNA is considered fully bound to the nanoparticles.

[0528] The nucleic acid particles described herein (particularly RNA particles such as mRNA particles) can be prepared using a wide variety of methods, which may include obtaining a colloid from at least one cationic or cationic ionizable lipid and mixing the colloid with nucleic acid to obtain the nucleic acid particle.

[0529] The term "colloid," as used herein, refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic, with particle sizes ranging from 1 to 1000 nanometers. The mixture may be called a colloid or a colloidal suspension. The term "colloid" may refer only to the particles in the mixture and not to the suspension as a whole.

[0530] For the preparation of colloids containing at least one cationic or cationic-ionizable lipid, suitably adapted methods conventionally used to prepare liposome vesicles are applicable herein. The most commonly used methods for preparing liposome vesicles share the following basic steps: (i) dissolving the lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipid (using various aqueous media).

[0531] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of a flask. The resulting lipid film is hydrated with a suitable aqueous medium to obtain a liposome dispersion. Further miniaturization steps may also be included.

[0532] Reverse phase evaporation is an alternative method to membrane hydration for preparing liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of this mixture is necessary to homogenize the system. Removal of the organic phase under reduced pressure results in a milky gel that then transforms into a liposome suspension.

[0533] The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. Generally, the RNA (especially mRNA) lipoplex particles described herein can be obtained by adding RNA (especially mRNA) to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersions are formed, in some embodiments, as follows: an ethanol solution containing lipids, such as cationic or cationic ionizable lipids like DOTMA and / or DODMA, and additional lipids, is injected into an aqueous solution under stirring. In some embodiments, the RNA (especially mRNA) lipoplex particles described herein can be obtained without an extrusion step.

[0534] The term "extrusion" or "extrusion" refers to the creation of particles having a fixed cross-sectional profile. In particular, it refers to the compaction of particles by forcing them through a filter with defined pores.

[0535] Other methods that have organic solvent-free properties may also be used in accordance with the present disclosure to prepare colloids.

[0536] In some embodiments, LNPs comprise four components: an ionizable cationic lipid, a neutral lipid such as a phospholipid, a steroid such as cholesterol, and a polymer-conjugated lipid. In some embodiments, LNPs can be prepared by rapidly mixing lipids dissolved in ethanol with RNA in an aqueous buffer. While the RNA particles described herein can comprise a polymer-conjugated lipid such as a PEG-lipid, the present specification also provides RNA particles that do not comprise a polymer-conjugated lipid such as a PEG-lipid.

[0537] In some embodiments, LNPs comprising RNA and at least one cationic or cationic ionizable lipid described herein are prepared by the following steps: (a) preparing an RNA solution containing water and a buffer system; (b) preparing an ethanol solution containing cationic or cationic ionizable lipid, and, if present, one or more additional lipids; and (c) mixing the RNA solution prepared in (a) with the ethanol solution prepared in (b), thereby preparing a formulation comprising LNPs. Step (c) can be followed by one or more steps selected from dilution and filtration, such as tangential flow filtration.

[0538] In some embodiments, LNPs comprising RNA and at least one cationic or cationic ionizable lipid described herein are prepared by the following steps: (a') preparing a liposome or colloid preparation of the cationic or cationic ionizable lipid, and, if present, one or more additional lipids in an aqueous phase; (b') preparing an RNA solution containing water and a buffer system; and (c') mixing the liposome or colloid preparation prepared in (a') with the RNA solution prepared in (b'). Step (c') can be followed by one or more steps selected from dilution and filtration, such as tangential flow filtration.

[0539] The present disclosure describes particles that comprise RNA (especially mRNA) and at least one cationic or cationic ionizable lipid that associates with RNA to form RNA particles, as well as compositions that comprise such particles.RNA particles can comprise RNA that is complexed with particles in various forms through non-covalent interactions.Particles described herein are not virus particles, particularly infectious virus particles, that is, they cannot infect cells with viruses.

[0540] Suitable cationic or cationic ionizable lipids form nucleic acid particles and are included in the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that associates with nucleic acid to form nucleic acid particles. Such components include any component that can be part of a nucleic acid particle.

[0541] In some embodiments, an RNA particle (particularly an mRNA particle) comprises multiple types of RNA molecules, where the molecular parameters of the RNA molecules may be similar or different from each other, such as with regard to molar mass or basic structural elements such as molecular structure, capping, coding regions or other features. In particle formulations, each RNA species can be formulated separately as an individual particle formulation. In that case, each individual particle formulation contains one RNA species. The individual particle formulations can exist as separate entities, for example, in separate containers. Such formulations can be obtained by providing each RNA species separately (typically in the form of an RNA-containing solution) together with a particle-forming agent, thereby allowing particles to form. Each particle contains only the specific RNA species provided when the particle is formed (individual particle formulation). In some embodiments, a composition, such as a pharmaceutical composition, contains multiple individual particle formulations. Each pharmaceutical composition is referred to as a mixed particle formulation. A mixed particle formulation according to the present invention can be obtained by separately forming individual particle formulations and then mixing the individual particle formulations. The mixing step can result in a formulation containing a mixed population of RNA-containing particles. The individual particle populations can be present together in one container containing a mixed population of individual particle formulations. Alternatively, all RNA species of the pharmaceutical composition can be formulated together as a combined particle formulation. Such a formulation can be obtained by providing a combined formulation (typically a combined solution) of all RNA species together with a particle-forming agent, thereby allowing particles to form. In contrast to mixed particle formulations, combination particle formulations typically contain particles that contain multiple RNA species. In combination particle compositions, different RNA species are typically present together in a single particle.

[0542] polymer Polymers are commonly used materials for nanoparticle-based delivery, given their high chemical flexibility. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines that change protonation state in the pH range of 5.5 to 7.5, leading to an ionic imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters), in particular, are widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0543] As used herein, the term "polymer" is given its usual meaning, i.e., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units may all be identical, or in some cases, there may be two or more types of repeating units in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties, may also be present in the polymer.

[0544] When two or more types of repeating units are present in a polymer, the polymer is said to be a "copolymer." It should be understood that a polymer as used herein may be a copolymer. The repeating units forming a copolymer may be arranged in any manner. For example, the repeating units may be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., containing one or more regions each containing a first repeating unit (e.g., a first block), and one or more regions each containing a second repeating unit (e.g., a second block), etc. A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

[0545] In certain embodiments, the polymer is biocompatible. A biocompatible polymer is typically a polymer that does not cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer can be chemically and / or biologically degraded in a physiological environment, such as within the body.

[0546] In certain embodiments, the polymer may be protamine or a polyalkyleneimine.

[0547] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, particularly in association with DNA. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate with heat, and produces primarily arginine upon hydrolysis. In purified form, they are used to neutralize the anticoagulant effect of heparin in long-acting formulations of insulin.

[0548] In accordance with the present disclosure, the term "protamine" as used herein is intended to include any protamine amino acid sequence and fragments thereof obtained or derived from natural or biological sources, and multimeric forms of said amino acid sequence or fragments thereof, as well as artificial, specifically designed for a particular purpose (synthetic) polypeptides that cannot be isolated from natural or biological sources.

[0549] In some embodiments, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75×10 2 ~10 7 Da, preferably 1000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0550] According to the present disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

[0551] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In some embodiments, cationic polymers contemplated for use herein include any cationic polymer with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is entrapped or encapsulated.

[0552] The particles described herein can also include polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0553] lipids The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are typically insoluble or poorly soluble in water, but are soluble in many organic solvents. In aqueous environments, their amphiphilic nature allows them to self-assemble into organized structures and various phases. One of these phases consists of lipid bilayers when they exist in aqueous environments as vesicles, multilamellar / unilamellar liposomes, or membranes. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can include polar and / or charged groups, including carbohydrates, phosphate groups, carboxylate groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0554] As used herein, the term "hydrophobic" refers to any molecule, moiety, or group that is substantially immiscible or insoluble in a...

Claims

1. 1. A method for inducing an immune response in a subject, comprising: (i) administering to the subject a non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope for inducing an immune response in the subject against an antigen; (ii) providing to said subject a PD-1 axis binding antagonist; A method comprising:

2. 10. The method of claim 1, wherein the subject has a disease, disorder, or condition associated with expression or elevated expression of an antigen.

3. 1. A method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: (i) administering to the subject a non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope for inducing an immune response in the subject against the antigen; (ii) providing to said subject a PD-1 axis binding antagonist; A method comprising:

4. The method of any one of claims 1 to 3, wherein the immune response is a T cell-mediated immune response.

5. The method of claim 1 or 3, wherein the immune response comprises the generation of antigen-specific T cells.

6. The method of claim 1 or 3, wherein the antigen is a tumor-associated antigen.

7. 4. The method of claim 2 or 3, wherein the disease, disorder or condition is cancer.

8. (i) the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope; and (ii) a PD-1 axis binding antagonist or an RNA encoding a PD-1 axis binding antagonist 4. The method of claim 1 or 3, comprising administering to the subject

9. (i) the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope; and (ii) PD-1 axis binding antagonist 4. The method of claim 1 or 3, comprising administering to the subject

10. 10. The method of claim 1 or 3, wherein the non-immunogenic RNA, when administered, results in reduced dendritic cell activation, T cell activation and / or IFN-α secretion compared to standard RNA.

11. 10. The method of claim 1 or 3, wherein the non-immunogenic RNA is rendered non-immunogenic by incorporation of modified nucleosides and / or elimination of double-stranded RNA (dsRNA).

12. 12. The method of claim 11, wherein the modified nucleoside inhibits RNA-mediated activation of an innate immune receptor.

13. 12. The method of claim 11, wherein the modified nucleoside comprises a substitution of one or more uridines with a nucleoside comprising a modified nucleobase.

14. 14. The method of claim 13, wherein the modified nucleobase is a modified uracil.

15. The nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-hydroxymethyl-uridine (mcm5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (mnm5U), 1-taurinomethyl-pseudouridine Lysine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O- 14. The method of claim 13, wherein the uridine is selected from the group consisting of methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine].

16. The nucleoside containing a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m １ ψ) or 5-methyl-uridine (m ５ U).

17. 14. The method of claim 13, wherein the nucleoside comprising a modified nucleobase is 1-methyl-pseudouridine.

18. The method of claim 1 or 3, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is mRNA.

19. 10. The method of claim 1 or 3, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is an in vitro transcribed RNA.

20. 4. The method of claim 1 or 3, wherein the non-immunogenic RNA encoding a peptide or polypeptide comprising the epitope is administered in a formulation for targeting the lymphatic system, such as a secondary lymphoid organ, in particular the spleen.

21. 10. The method of claim 1 or 3, wherein the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope is administered in a formulation for targeting dendritic cells.

22. 22. The method of claim 21, wherein the dendritic cells are immature dendritic cells.

23. 10. The method of claim 1 or 3, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is administered in a formulation comprising lipoplex (LPX) particles.

24. 10. The method of claim 1 or 3, wherein the PD-1 axis binding antagonist comprises a PD-1 binding antagonist.

25. 25. The method of claim 24, wherein the PD-1 binding antagonist comprises an anti-PD-1 antibody.

26. 26. The method of claim 25, wherein the anti-PD-1 antibody comprises nivolumab or pembrolizumab.

27. The method of claim 1 or 3, wherein the PD-1 axis binding antagonist comprises a PD-L1 binding antagonist.

28. 28. The method of claim 27, wherein the PD-L1 binding antagonist comprises an anti-PD-L1 antibody.

29. 29. The method of claim 28, wherein the anti-PD-L1 antibody comprises atezolizumab, avelumab, or durvalumab.

30. The method of claim 1 or 3, which does not include administering an immunostimulant or RNA encoding an immunostimulant.

31. 31. The method of claim 30, wherein the immunostimulant is a pro-inflammatory or anti-inflammatory immunostimulant.

32. 31. The method of claim 30, wherein the immunostimulant comprises a cytokine or a variant thereof.

33. 33. The method of claim 32, wherein the cytokine comprises a type I interferon or a variant thereof.

34. 34. The method of claim 33, wherein the type I interferon comprises interferon-α or a variant thereof.

35. 33. The method of claim 32, wherein the cytokine comprises an interleukin or a variant thereof.

36. 33. The method of claim 32, wherein the cytokine supports T cell priming.

37. 33. The method of claim 32, wherein the cytokine comprises IL12, IL15, or a variant thereof.

38. 33. The method of claim 32, wherein the cytokine supports the proliferation and / or maintenance of T cells.

39. 33. The method of claim 32, wherein the cytokine comprises IL2, IL7, or a variant thereof.

40. 9. The method of claim 8, wherein the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope and the PD-1 axis binding antagonist or RNA encoding a PD-1 axis binding antagonist are administered in a common formulation or in separate formulations.

41. The method of claim 1 or 3, which is a method for treating or preventing cancer in a subject.

42. 10. The method of claim 1 or 3, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is transiently expressed in the subject's cells.

43. The method of claim 1 or 3, wherein the subject is a human.

44. (i) a non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope for inducing an immune response to an antigen in a subject; and (ii) a PD-1 axis binding antagonist or an RNA encoding a PD-1 axis binding antagonist 10. A pharmaceutical formulation comprising:

45. 45. The pharmaceutical formulation of claim 44 for treating a disease, disorder or condition associated with expression or elevated expression of an antigen.

46. 46. ​​The pharmaceutical preparation of claim 44 or 45, wherein the immune response is a T cell-mediated immune response.

47. 46. ​​The pharmaceutical preparation of claim 44 or 45, wherein the immune response comprises the generation of antigen-specific T cells.

48. 46. ​​The pharmaceutical preparation of claim 44 or 45, wherein the antigen is a tumor-associated antigen.

49. 46. ​​The pharmaceutical preparation of claim 45 or 45, wherein the disease, disorder or condition is cancer.

50. (i) the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope; and (ii) PD-1 axis binding antagonist 46. ​​The pharmaceutical formulation of claim 44 or 45, comprising:

51. 46. ​​The pharmaceutical formulation of claim 44 or 45, wherein the non-immunogenic RNA, when administered, results in reduced dendritic cell activation, T cell activation and / or IFN-α secretion compared to standard RNA.

52. 46. ​​The pharmaceutical formulation of claim 44 or 45, wherein the non-immunogenic RNA is rendered non-immunogenic by incorporation of modified nucleosides and / or removal of dsRNA.

53. 53. The pharmaceutical preparation of claim 52, wherein the modified nucleoside inhibits RNA-mediated activation of an innate immune receptor.

54. 53. The pharmaceutical formulation of claim 52, wherein the modified nucleoside comprises a substitution of one or more uridines with a nucleoside comprising a modified nucleobase.

55. 55. The pharmaceutical formulation of claim 54, wherein the modified nucleobase is a modified uracil.

56. The nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-hydroxymethyl-uridine (mcm5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5 U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (mnm5U), 1-taurinomethyl-pseudouridine Lysine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl 55. The pharmaceutical formulation of claim 54, wherein the uridine is selected from the group consisting of thyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine].

57. The nucleoside containing a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m １ ψ) or 5-methyl-uridine (m ５ 55. The pharmaceutical formulation of claim 54, wherein:

58. 55. The pharmaceutical preparation of claim 54, wherein the nucleoside comprising a modified nucleobase is 1-methyl-pseudouridine.

59. 46. ​​The pharmaceutical preparation of claim 44 or 45, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is mRNA.

60. 46. ​​The pharmaceutical preparation of claim 44 or 45, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is an in vitro transcribed RNA.

61. 46. ​​The pharmaceutical formulation of claim 44 or 45, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is present in a formulation for targeting the lymphatic system, such as a secondary lymphoid organ, in particular the spleen.

62. 46. ​​The pharmaceutical formulation of claim 44 or 45, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is present in a formulation for targeting dendritic cells.

63. 63. The pharmaceutical preparation of claim 62, wherein the dendritic cells are immature dendritic cells.

64. 45. The pharmaceutical formulation of claim 44, wherein the non-immunogenic RNA encoding an epitope-containing peptide or polypeptide is administered in a formulation comprising lipoplex (LPX) particles.

65. 46. ​​The pharmaceutical formulation of claim 44 or 45, wherein the PD-1 axis binding antagonist comprises a PD-1 binding antagonist.

66. 66. The pharmaceutical formulation of claim 65, wherein the PD-1 binding antagonist comprises an anti-PD-1 antibody.

67. 67. The pharmaceutical preparation of claim 66, wherein the anti-PD-1 antibody comprises nivolumab or pembrolizumab.

68. 46. ​​The pharmaceutical formulation of claim 44 or 45, wherein the PD-1 axis binding antagonist comprises a PD-L1 binding antagonist.

69. 69. The pharmaceutical formulation of claim 68, wherein the PD-L1 binding antagonist comprises an anti-PD-L1 antibody.

70. 70. The pharmaceutical preparation of claim 69, wherein the anti-PD-L1 antibody comprises atezolizumab, avelumab, or durvalumab.

71. 46. ​​The pharmaceutical formulation of claim 44 or 45, which does not contain an immunostimulant or RNA encoding an immunostimulant.

72. 72. The pharmaceutical preparation of claim 71, wherein the immunostimulant is a pro-inflammatory or anti-inflammatory immunostimulant.

73. 72. The pharmaceutical preparation of claim 71, wherein the immunostimulant comprises a cytokine or a variant thereof.

74. 74. The pharmaceutical preparation of claim 73, wherein the cytokine comprises a type I interferon or a variant thereof.

75. 75. The pharmaceutical formulation of claim 74, wherein the type I interferon comprises interferon-α or a variant thereof.

76. 74. The pharmaceutical preparation of claim 73, wherein the cytokine comprises an interleukin or a variant thereof.

77. 74. The pharmaceutical preparation of claim 73, wherein the cytokine supports T cell priming.

78. 74. The pharmaceutical preparation of claim 73, wherein the cytokine comprises IL12, IL15, or a variant thereof.

79. 74. The pharmaceutical preparation of claim 73, wherein the cytokine supports the proliferation and / or maintenance of T cells.

80. 74. The pharmaceutical preparation of claim 73, wherein the cytokine comprises IL2, IL7, or a variant thereof.

81. 46. ​​The pharmaceutical formulation of claim 44 or 45, wherein the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope and the PD-1 axis binding antagonist or RNA encoding a PD-1 axis binding antagonist are present in a common formulation or in separate formulations.

82. 46. ​​The pharmaceutical preparation of claim 44 or 45, which is a kit.

83. The pharmaceutical formulation of claim 44 or 45, wherein the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope and the PD-1 axis binding antagonist or RNA encoding a PD-1 axis binding antagonist are contained in a pharmaceutical composition.

84. The pharmaceutical formulation of claim 44 or 45, wherein the non-immunogenic RNA encoding a peptide or polypeptide comprising an epitope and the PD-1 axis binding antagonist or RNA encoding a PD-1 axis binding antagonist are contained in separate containers.

85. 46. ​​The pharmaceutical formulation of claim 44 or 45, further comprising instructions for using said pharmaceutical formulation.

86. 46. ​​The pharmaceutical preparation of claim 44 or 45, which is a pharmaceutical composition.

87. 46. ​​A pharmaceutical formulation according to claim 44 or 45 for pharmaceutical use.

88. 88. The pharmaceutical preparation of claim 87, wherein the pharmaceutical use comprises the therapeutic or prophylactic treatment of a disease or disorder.

89. 89. The pharmaceutical preparation of claim 88, wherein the disease or disorder is cancer.

90. 46. ​​A pharmaceutical formulation according to claim 44 or 45 for use in the method of claim 1 or 3.

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