Antigen-specific physiological dendritic cells for therapeutic use

Abstract

The present invention relates to a composition comprising physiological dendritic cells and at least one mRNA. The present invention further relates to a composition comprising physiological dendritic cells and at least one mRNA for use in therapeutic treatments.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to the field of therapeutic treatments for diseases based on physiological dendritic cells. The present invention further relates to therapeutic compositions comprising physiological dendritic cells and at least one mRNA, and therapeutic compositions for use in therapeutic treatments for infectious diseases and hyperproliferative diseases such as cancer or tumors. [Background technology]

[0002] Background of the Invention Dendritic cells (DCs) have the ability to take up and process antigens and present them to T cells and B cells on their cell surface. Because DCs activate naive, effector, and memory immune cells, they are promising therapeutic agents for diseases such as cancer or infectious diseases. The potent activation of de novo T cell and B cell responses also suggests that dendritic cells are promising agents for preventative purposes.

[0003] However, dendritic cells in the human body make up only about 0.3% of total circulating white blood cells and constitute a heterogeneous population with varying levels of maturation and the ability to stimulate tolerance-induced or stimulative immune responses.

[0004] Techniques for isolating and maturing dendritic cells are described in numerous documents and include various methods, such as contacting monocytes with hematopoietic growth factors and cytokines like IFN-α, GM-CSF, TNF-α, IL-3, or combinations thereof (see, for example, European Patent No. 922,758 or European Patent No. 663,930, WO95 / 28479).

[0005] In vitro photoferrosis (ECP) has been successfully used to treat cutaneous T-cell lymphoma (CTCL) in some patients. In classical ECP, patient peripheral blood mononuclear cells (PBMCs) are isolated, passed through a clear plastic plate while exposed to 8-methoxypsoralen and UVA (8-MOP UVA), and then returned to the patient. The underlying mechanism of action is presumed to involve dendritic cells taking up antigens released by malignant T cells exposed to 8-MOP UVA and presenting them to the patient's immune system. Depending on the mode of action, these DCs are called immunostimulatory DCs. In the course of investigating the principle of ECP, it has been found that ECP leads to the conversion of passaged blood monocytes into DCs.

[0006] However, it remains unclear which antigens are precisely taken up and presented during this process. Therefore, it is still necessary to selectively provide immunostimulatory DCs that also possess predefined antigens in order to elicit the desired and targeted immunostimulatory response. [Overview of the project] [Problems that the invention aims to solve]

[0007] Objective and Summary of the Invention One object of the present invention is to provide a therapeutic composition comprising phDCs and at least one mRNA. Another object of the present invention is to provide a therapeutic composition comprising antigen-specific phDCs and at least one mRNA. A further object of the present invention is to provide a therapeutic composition comprising antigen-specific monocytes and at least one mRNA. Another object of the present invention is to use the therapeutic composition for therapeutic treatment.

[0008] Another objective is to provide therapeutic compositions for use in methods of treating diseases in a given area.

[0009] Finally, it is an object of the present invention to provide a kit comprising phDC and at least one mRNA encoding at least one antigen protein. [Means for solving the problem]

[0010] These and other objectives are addressed by the subject matter of the independent claims, as they will become apparent from the subsequent description below. Some preferred embodiments of the invention form the subject matter of the dependent claims. Further embodiments of the invention can be interpreted from the subsequent description below.

[0011] The present invention, as described below illustratively, can be adequately practiced in the absence of any one or more elements or limitations not specifically disclosed herein. While the present invention is described below with respect to specific embodiments and with reference to certain figures, it is not limited thereto and is limited only by the claims.

[0012] This invention is based to some extent on the data and experiments presented below, which lead to the insight that dendritic cells carrying selected disease antigens can be effectively used in the therapeutic treatment of diseases such as cancer or infectious diseases. As a primary prerequisite for acquiring antigen-specific DCs, dendritic cells must (i) be acquired and (ii) possess disease-specific antigens. With respect to (i), so-called physiological dendritic cells (phDCs) can be used. phDCs can be produced in vitro, among other things, by applying shear force to monocytes (e.g., from blood samples). See, for example, Ventura et al., 2018, "Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity," WO2014 / 106629, WO2016 / 001405, or Hanlon et al., 2020, "Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy." PhDCs can be characterized by the absence of marker expression, including markers such as HLA-DR, CD83, CD86, ICAM, or PLAUR, and / or increased GILZ expression (see, for example, Ventura et al., 2018, "Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity" or WO2014 / 106629). Since monocytes do not need to be supplied with cytokines to induce differentiation into dendritic cells, it is presumed that dendritic cells acquired by subjecting monocytes to physical force closely resemble the characteristics of naturally occurring dendritic cells, and for this reason, dendritic cells acquired by subjecting monocytes to physical force will be referred to below as physiological dendritic cells (phDCs).

[0013] As previously described (Ventura et al., 2018, "Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity" or WO2014 / 106629), physical forces to induce differentiation of monocytes into phDCs can be applied by passing monocytes through a flow chamber that may take the form of, for example, a plate or a bag (e.g., a flexible bag or a plastic bag). Such bags are described, for example, in Buechler et al., 2004, "Generation of Dendritic Cells Using Cell Culture Bags - Description of a Method and Review of Literature". It is hypothesized that passing monocytes in this manner, for example, through a plate, or placing them in, for example, a flexible bag, or a combination thereof (e.g., a hybrid of a bag and a chamber disclosed herein), exposes monocytes to shear stress and induces differentiation into dendritic cells.

[0014] The presence of platelets can enhance the maturation process. As described, monocytes can mature into dendritic cells using this method without the need to add expensive cytokine cocktails. Also as described, the above processes of passing monocytes, for example, through a plate, or placing them, for example, in a bag, or a chamber-bag hybrid, mimic some aspects of what is presumed to occur in vivo, and for this reason, dendritic cells produced by passing through a plate (or placement and movement in a bag) are referred to throughout this disclosure as “physiological dendritic cells” (phDCs). Although phDCs have been found to function particularly well in carrying disease-specific antigens, in principle, any population of DCs can be used to generate antigen-specific DCs of the present invention, such as DCs acquired after incubation of blood-derived monocytes with cytokines. Furthermore, as previously shown (Ventura et al., 2018, "Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity" or WO2016 / 001405), phDCs produced preferably in the absence of apoptotic signals and subsequently loaded with antigens would be effective in stimulating an immune response. It is important to understand that transfection (e.g., using at least one mRNA encoding at least one antigen protein) can occur even at the monocyte stage when monocytes are subjected to physical forces. Thus, monocytes can be combined with disease-specific antigens (e.g., in the form of at least one mRNA encoding at least one antigen protein) and subjected to physical forces such as shear forces. As described in WO2016 / 001405, monocytes activated by physical forces (so-called globally activated monocytes) can be identified, among other things, by increased expression of at least HLA-DR, PLAUR, or ICAM-1.

[0015] Regarding (ii), the inventors have found that phDCs can internalize antigens and can present antigens on the surface of phDCs. Antigens can be provided in various forms, including in the form of mRNA-containing lipid nanoparticles (LNPs). PhDCs can express antigens encoded by disease-related proteins or peptides. Supported antigen-specific phDCs (e.g., therapeutic compositions comprising phDCs and at least one mRNA) are useful in targeted immunotherapy for various diseases, such as cancer or infectious diseases. The antigen-specific phDCs of the present invention can elicit a therapeutic immune response through their intrinsic ability to present specific antigens on MHC molecules, resulting in T cell activation, proliferation, and T cell differentiation into armed effector T cells. One important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells, which together constitute cell-mediated immunity, as well as the activation of B cells by both Th2 and Th1 cells, which produce various classes of antibodies and thus drive humoral immune responses. Importantly, T cells (e.g., cytotoxic T cells) can be transmitted to the brain, leading to superior therapeutic effects compared to the administration of antibodies, for example, which generally cannot cross the blood-brain barrier. The direct use of antigen-specific phDCs (e.g., therapeutic compositions comprising phDCs and at least one mRNA) can also be considered a safer therapeutic approach compared to other therapies, which offer a greater chance of randomly spreading into the systemic circulation, missing target cells, and producing unwanted side effects, as the immune system is activated by a highly defined set of cells. Because the antigen-specific phDCs of the present invention (e.g., therapeutic compositions comprising phDCs and at least one mRNA) are highly efficient, the present invention also offers the possibility of significant dose savings. Yet another advantage of antigen-specific phDCs (e.g., therapeutic compositions comprising phDCs and at least one mRNA) is the rapid onset of action after administration. Antigen-specific phDCs can directly induce clonal proliferation of existing target T cells into effector cytotoxic T cells. Unlike naive T cells, memory T cells can induce cytotoxic function immediately upon encountering an antigen.Therefore, therapeutic treatment using antigen-specific phDCs leads to an efficient and rapid cell-mediated immune response, supported by their additional efficacy in inducing a subsequent humoral immune response.

[0016] First aspect: Therapeutic composition In a first aspect, the present invention relates to a therapeutic composition comprising a pharmaceutically effective amount of phDC and at least one RNA having a coding sequence encoding at least one antigen protein.

[0017] In one embodiment, RNA is single-stranded RNA, mRNA, circular RNA, auto-amplified RNA, and / or synthetic RNA. In a preferred embodiment, RNA is auto-amplified mRNA. In a preferred embodiment, RNA is mRNA. Thus, the present invention relates to a therapeutic composition comprising a pharmaceutically effective amount of phDC and at least one mRNA (e.g., one mRNA molecule or more than one mRNA molecule) containing a coding sequence encoding at least one antigen protein.

[0018] In one embodiment, RNA (e.g., mRNA) contains at least one chemical modification. If more than one RNA (e.g., mRNA) is used, each RNA (e.g., mRNA) independently contains at least one chemical modification (i.e., the chemical modifications may differ from RNA to RNA). For the purposes of this disclosure, chemical modification means that one of the four naturally occurring standard nucleosides present in RNA (adenosine (A), guanosine (G), uridine (U), and cytidine (C)) is replaced by the modified form, and the modification affects the base portion within the nucleoside. The modified nucleoside may be naturally occurring or not naturally occurring. Naturally occurring modified nucleosides are preferred.

[0019] Such replacement of A, C, U, and G with naturally occurring or non-naturally occurring modified nucleosides is hypothesized to reduce the Toll-like receptor (TLR)-mediated immune response of recipient dendritic cells and / or increase the expression of antigens encoded by RNA (e.g., mRNA).

[0020] In one embodiment, the chemical modification is the replacement of one or more nucleosides of RNA (e.g., mRNA) with one or more modified nucleosides.

[0021] Naturally occurring modified nucleosides include 1-methyladenosine (m 1 A), N 6 -methyladenosine (m 6 A), 2'-O-methyladenosine (Am), 5-methylcytidine (m 5 C), 2'-O-methylcytidine (Cm), 2-thiocytidine (s 2 C), N 4 -acetylcytidine (ac 4 C), 5-formylcytidine (f 5 C), 2'-O-methylguanosine (Gm), inosine (I), pseudouridine (Ψ), 5-methyluridine (m 5 U), 2'-O-methyluridine (Um), 1-methylpseudouridine (m1Ψ), 2-thiouridine (s2U), 4-thiouridine (s4U), 5-methoxyuridine (mo5U), and 3-methyluridine (m3U).

[0022] It is preferable to replace at least one or more uridine nucleosides of the RNA (e.g., mRNA) with naturally occurring modified uridine nucleosides from among the four nucleosides A, U, C, and G. The aforementioned effects on the TLR-mediated immune response and / or the expression level of the antigen encoded by the RNA (e.g., mRNA) are presumed to depend on the extent of uridine replacement by naturally occurring modified uridine nucleosides. In preferred embodiments, therefore, all uridine nucleosides in the antigen-coding RNA (e.g., mRNA) are replaced with naturally occurring modified uridine nucleosides. However, the present invention also considers cases where less than 90%, less than 80%, less than 70%, less than 60%, or less than 50% of the uridine nucleosides present in the antigen-coding RNA (e.g., mRNA) are replaced with naturally occurring modified uridine nucleosides. When uridine nucleosides are replaced by their naturally occurring modified forms, pseudouridine is preferred, and N1-methylpseudridine or N1-ethylpseudridine is more preferred. N1-methylpseudridine is most preferred. Therefore, the present invention considers antigen-coding RNA (e.g., mRNA) in which all uridines are replaced by N1-methylpseudridine as a particularly preferred embodiment.

[0023] The present invention also considers replacing uridine nucleosides, preferably in addition to uridine nucleosides, with A, C, and / or G nucleosides in their modified forms. For such replacements, it is preferable to use naturally occurring modified forms of A, C, and / or G. When such additional replacements are considered, it is preferable that all of A, C, and / or G nucleosides are replaced with their modified forms.

[0024] In one embodiment, the RNA (e.g., mRNA) comprises structural elements including a 5' untranslated region (UTR), a 3'UTR, a 5' cap, and / or a poly(A) tail. In one embodiment, the RNA (e.g., mRNA) contains all of these elements. In one embodiment, the 5' cap is a cap1 structure or an m7GpppG cap. Preferably, the 5' cap is a cap1 structure. In one embodiment, the sequence of the RNA (e.g., mRNA) is optimized. In one embodiment, the sequences of the 5'UTR, 3'UTR, and / or coding sequence for the antigen protein are optimized. In one embodiment, the sequence of the RNA (e.g., mRNA) has optimized codon usage or optimized G / C content. In one embodiment, the codon usage, G / C content, and structural elements are optimized. Optimization of the sequence of structural elements 3' and / or 5'UTR may additionally include using one or more of heterologous UTRs, Kozak sequences, FI elements, removed Aures elements, and enzymatically added tails (e.g., poly(A) tails). In one embodiment, a poly(A) tail is enzymatically added.

[0025] At least one type of RNA (e.g., mRNA) may be included in the nanoparticles. The nanoparticles include lipid nanoparticles, poly(amine-co-ester) particles (PACE), poly-beta-amino-ester particles, PACE polyplex particles, lipoplex, and poly(N,N-cystamine bis(acrylamide)-co-4-amino-1-butanol) (pABOL) particles. In one embodiment, the nanoparticles are lipid nanoparticles (LNP).

[0026] In one more preferred embodiment, RNA (e.g., mRNA) is contained in the lipid nanoparticles (LNPs). The lipid nanoparticles may include cationic lipids, PEG-modified lipids, cholesterol, DSPE-PEG-maleimide, DSPN-PEG-azide, and / or non-cationic lipids. In one embodiment, the lipid nanoparticles include cationic lipids, PEG-modified lipids, cholesterol, and / or non-cationic lipids. In one embodiment, the lipid nanoparticles include cationic lipids, PEG-modified lipids, cholesterol, and non-cationic lipids. The cationic lipids include cKK-E12, cKK-E14, LP01, SM102, Lipid 5, etc. In one embodiment, the cationic lipid is cKK-E12 lipid. In one embodiment, the cationic lipid is SM102 lipid. In one embodiment, the cationic lipid is MC3 lipid (DLin-MC3-DMA). In one embodiment, the lipid nanoparticles contain cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE. In one embodiment, the lipid nanoparticles contain cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 30-40:41-51:1.0-4.0:12-21. In one embodiment, the lipid nanoparticles contain cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 33-37:44-48:2.0-3.0:14-18. In one embodiment, the lipid nanoparticles contain cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 34-36:45-47:2.2-2.8:15-17. In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16.

[0027] In one embodiment, the lipid nanoparticles include SM-102, cholesterol, C14-PEG 2000-PE, and DOPE. In one embodiment, the lipid nanoparticles include SM-102, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 30-40:41-51:1.0-4.0:12-21. In one embodiment, the lipid nanoparticles include SM-102, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 33-37:44-48:2.0-3.0:14-18. In one embodiment, the lipid nanoparticles include SM-102, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 34-36:45-47:2.2-2.8:15-17. In one embodiment, the lipid nanoparticles contain SM-102, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16. In some embodiments, the lipid nanoparticles contain SM102, cholesterol, DMG-PEG-2K, and DSPC. In one embodiment, the lipid nanoparticles contain SM102, cholesterol, DMG-PEG-2K, and DSPC in a ratio of 30-50:35-45:1.0-4.0:10-20.

[0028] In one embodiment, the lipid nanoparticles contain MC3, cholesterol, C14-PEG 2000-PE, and DOPE. In one embodiment, the lipid nanoparticles contain MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 30-40:41-51:1.0-4.0:12-21. In one embodiment, the lipid nanoparticles contain MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 33-37:44-48:2.0-3.0:14-18. In one embodiment, the lipid nanoparticles contain MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 34-36:45-47:2.2-2.8:15-17. In one embodiment, the lipid nanoparticles comprise MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16.

[0029] In one embodiment, at least one mRNA encoding at least one antigen protein is heterologous or exogenous to phDC. In one embodiment, the antigen protein is exogenous to phDC. As used herein, the terms "heterologous" or "exogenous" may refer to nucleic acid sequences, e.g., mRNA, or proteins originating from a specified cell type, e.g., a source other than phDC, in relation to a specific cell type, e.g., phDC. Thus, heterologous mRNA or exogenous mRNA to phDC refers to mRNA that is not endogenous to phDC but has been introduced into phDC, e.g., by transfection or other means.

[0030] In one embodiment, the antigen protein is an infectious disease-related antigen or a tumor-related antigen. In one embodiment, the antigen protein is an infectious disease-related antigen. Therefore, the therapeutic composition comprises phDC and at least one mRNA containing a coding sequence encoding at least one infectious disease-related antigen protein. In one embodiment, the infectious disease-related antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen. In one embodiment, the infectious disease-related antigen is a bacterial antigen. In one embodiment, the bacterial antigen is derived from a Borrelia or Mycobacteria species. In another embodiment, the infectious disease-related antigen is a fungal antigen. In one embodiment, the fungal antigen is derived from Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans. In another embodiment, the infectious disease-related antigen is a parasitic antigen. In one embodiment, the parasitic antigen is derived from Plasmodium malariae.

[0031] In one embodiment, the infectious disease-associated antigen is a viral antigen. Therefore, the therapeutic composition comprises phDC and at least one mRNA containing a coding sequence encoding at least one viral antigen protein. In one embodiment, the viral antigen is a coronavirus antigen protein or an HIV antigen protein. In one embodiment, the viral antigen is a coronavirus antigen protein. In one embodiment, the viral antigen is a beta-coronavirus antigen protein. In another embodiment, the viral antigen is a SARS-CoV-2 antigen protein. In one embodiment, the coronavirus antigen protein or beta-coronavirus antigen protein is the antigen protein of the spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or a fragment of any of the aforementioned. In one embodiment, the SARS-CoV-2 antigen is the antigen protein of the spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or a fragment of any of the aforementioned. In one embodiment, the SARS-CoV-2 antigen is the antigen protein of the spike protein or a fragment of the same. The fragment contains at least 10, at least 50, at least 100, at least 200, at least 400, or at least 800 amino acid residues.

[0032] In one embodiment, RNA (e.g., mRNA) includes a sequence or a portion of a sequence selected from the group including SEQ ID NO: 1 (spike protein transcript), SEQ ID NO: 2 (envelope protein transcript), SEQ ID NO: 3 (nucleocapsid protein transcript), SEQ ID NO: 4 (membrane protein transcript), and / or SEQ ID NO: 5 (Orf1ab polyprotein transcript), which is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100% identical to SEQ ID NO: 1. In one embodiment, the antigen protein encoded by RNA (e.g., mRNA) contains 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 1000, at least 1200, at least 2000, or at least 3000 amino acids. In one embodiment, the antigen protein encoded by RNA (e.g., mRNA) contains at least 50 amino acids. In one embodiment, the antigen protein encoded by RNA (e.g., mRNA) contains at least 500 amino acids. In one embodiment, the antigen protein encoded by RNA (e.g., mRNA) contains at least 1000 amino acids.

[0033] In one embodiment, the SARS-CoV-2 antigen is a protein or peptide comprising a sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to a sequence or portion of a sequence selected from the group comprising SEQ ID NO: 6 (spike amino acid sequence), SEQ ID NO: 7 (envelope amino acid sequence), SEQ ID NO: 8 (nucleocapsid amino acid sequence), SEQ ID NO: 9 (membrane amino acid sequence), and / or SEQ ID NO: 10 (Orf1ab amino acid sequence). In one embodiment, the protein or peptide comprises a sequence or portion of a sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to SEQ ID NO: 6.

[0034] In one embodiment, at least one antigen protein is the SARS-CoV-2 hexaprospike protein (the RNA sequence shown in bold and underlined in Table 3) or the RNA encoding it (e.g., mRNA), having a foldon domain in its extracellular domain immediately above its transmembrane domain. The corresponding RNA (e.g., mRNA) may be modified with one or more of the following: N1-methylpseudridine, a cap-1 structure, and a poly-A tail. In one embodiment, the RNA (e.g., mRNA) includes N1-methylpseudridine (e.g., each uridine is replaced by N1-methylpseudridine), a cap-1 structure, and a poly-A tail. In one embodiment, the codon may be optimized to GC-enrich the RNA (e.g., mRNA), and optimized 5' and 3' UTRs may be used. In one embodiment, the RNA (e.g., mRNA) includes a sequence or a portion of a sequence corresponding to SEQ ID NO: 19, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 19; and a poly-A tail (e.g., about 200-400 nucleotides).

[0035] In one embodiment, the viral antigen is derived from HIV. In one embodiment, the HIV antigen is derived from an envelope protein (env), a group antigen polyprotein (gag), a reverse transcriptase (pol), and / or a negative factor protein (nef). In one embodiment, the RNA encoding the antigen protein (e.g., mRNA) includes a sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to a sequence or portion of a sequence selected from the group including SEQ ID NO: 11 (env protein transcript), SEQ ID NO: 12 (gag protein transcript), SEQ ID NO: 13 (pol protein transcript), and / or SEQ ID NO: 14 (nef protein transcript).

[0036] In one embodiment, the HIV antigen is a protein or peptide containing a sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to a sequence or portion of a sequence selected from the group including SEQ ID NO: 15 (env amino acid sequence), SEQ ID NO: 16 (gag amino acid sequence), SEQ ID NO: 17 (pol amino acid sequence), and / or SEQ ID NO: 18 (nef amino acid sequence).

[0037] The therapeutic composition of the present invention may comprise a pharmaceutically effective amount of physiological DCs (phDCs) and at least one mRNA containing a coding sequence encoding at least one antigen protein, wherein the mRNA may be at least partially incorporated into the phDCs. In some embodiments, substantially all of the mRNA is incorporated into the phDCs. mRNA not incorporated into the phDCs may be removed by washing the phDCs or by other means known in the art. Thus, the resulting therapeutic composition may comprise phDCs in which the phDCs have incorporated mRNA encoding at least one antigen protein. The mRNA can be translated into the antigen protein. In some embodiments, the phDCs express the antigen protein. The antigen protein may be exogenous or heterologous to the phDCs. In some embodiments, free, i.e., unincorporated mRNA is not removed from the therapeutic composition, and therefore the composition contains unincorporated mRNA. In another embodiment, the therapeutic composition of the present invention may comprise a pharmaceutically effective amount of physiological DCs (phDCs) and at least one mRNA containing a coding sequence encoding at least one antigen protein, wherein the mRNA is at least partially incorporated into the phDCs. In some embodiments, only a small portion of the mRNA contained in the therapeutic composition is taken up by phDCs present in the composition. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the mRNA is not incorporated into the phDCs. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the mRNA contained in the therapeutic composition of the present invention is incorporated into the phDCs.

[0038] In one embodiment, at least one antigen protein is a tumor-associated antigen. Therefore, the therapeutic composition comprises phDC and at least one mRNA containing a coding sequence encoding at least one tumor-associated antigen protein. In one embodiment, the tumor-associated antigen is a leukemia antigen, melanoma antigen, lymphoma antigen, endometrial cancer antigen, kidney cancer antigen, brain tumor antigen, cervical cancer antigen, liver cancer antigen, head and neck cancer antigen, gastrointestinal cancer antigen, lymph node cancer antigen, pancreatic cancer antigen, otolaryngological (ENT) cancer antigen, breast cancer antigen, prostate cancer antigen, ovarian cancer antigen, or lung cancer antigen. In one embodiment, at least one antigen protein is a hematological cancer-associated antigen. The hematological cancer antigen protein may be a leukemia antigen protein, a lymphoma antigen protein, or a myeloma antigen protein.

[0039] In one embodiment, phDCs can be obtained by subjecting monocytes (e.g., acquired from a donor) to physical force. Physical force can be applied to the monocytes by passing them through a flow chamber. The flow chamber may be a plate, bag, or flow chamber of a device, such as a large ECP device, such as a clinical ECP device (e.g., THERAKOS® CELLEX® device; a combination of Terumo Spectra Optia and UVA PIT; a combination of Fresenius apheresis device and Macopharma Macogenix; a single-needle option for the Amicus® in vitro photopheresis protocol), or a small ECP device, such as a transimmunization plate described in WO2017 / 005700 A1, or a combination thereof (e.g., a hybrid of a bag and a chamber disclosed herein). In one embodiment of the present invention, phDCs can be obtained by passing monocytes through a plate using a process derived from in vitro photopheresis (ECP). Thus, in one embodiment, the flow chamber is a plate. Monocytes (e.g., obtained from a donor) are passed through a plate, thereby exposing the monocytes to shear force. Preferably, platelets, which may originate from or be provided separately from a donor blood sample or a fraction thereof, are present on the plate. Additionally or alternatively, plasma components, which may originate from or be provided separately from a donor blood sample or a fraction thereof, may be present on the plate. The process of preparing phDCs, and the term itself, have been previously described, for example, in Hanlon et al., 2020 ("Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy"). In one embodiment, all method steps are performed in vitro.

[0040] In another embodiment, the flow chamber is a bag. Optionally, the bag is a flexible bag or a plastic bag. In one embodiment, the flow chamber is a flexible bag. In one embodiment, the bag is a plastic bag. In one embodiment, the material of the flow chamber (e.g., a plate, a flexible bag, or a plastic bag) is plastic. In one embodiment, the material of the flow chamber is non-plastic, such as glass, ceramic, or silicone. If plastic materials are considered, acrylic, polycarbonate, polyetherimide, polysulfone, polyphenylsulfone, styrene, polyurethane, polyethylene, Teflon®, or any other suitable medical-grade plastic may be used. In a preferred embodiment of the present invention, the flow chamber is made from acrylic plastic. If a bag (e.g., a flexible bag) is considered, the material may be plastic, rubber, or silicone. In a preferred embodiment, the material is plastic. Plastic materials include polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymer, ethylene vinyl alcohol, polyvinylidene fluoride, and / or other plastics including materials approved for medical use.

[0041] In some embodiments, the flow chamber includes or consists of a plate. The plate may be made from a variety of materials, including but not limited to plastic materials. In one embodiment, the material of the plate is plastic. In one embodiment, the material of the plate is non-plastic, such as glass, ceramic, or silicone. Non-limiting examples of materials for the plate include acrylic, polycarbonate, polyetherimide, polysulfone, polyphenylsulfone, styrene, polyurethane, polyethylene, Teflon, or any other suitable medical-grade plastic. The plate may be rigid or flexible. In some embodiments, the material of the plate may include or consist of plastic, rubber, or silicone. In some embodiments, the plate is elastic, i.e., made from an elastic material. Elastic materials may include cyclic olefin copolymers (COC), polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymer, ethylene vinyl alcohol, polyvinylidene fluoride, polydimethylsiloxane (PDMS), dimethicone, and / or other plastics, including materials approved for medical use. In one embodiment, the plate is made from PDMS, for example, PDMS RTV-615 or PDMS Sylgard 184. In another preferred embodiment of the present invention, the plate is made from acrylic plastic.

[0042] In some embodiments, the flow chamber is a hybrid flow chamber. A hybrid flow chamber may include a chamber and a bag, a chamber and a plate, or a bag and a plate. The individual components of the hybrid flow chamber, namely the bag, plate, or chamber, are as defined herein.

[0043] In one embodiment, human AB serum, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS is added to the composition. In one embodiment, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS is added to the composition. In one embodiment, mouse serum, mouse plasma, or FBS is added to the composition. Alternatively, before adding monocytes, the flow chamber, bag, plate, or hybrid flow chamber may be coated with human AB serum, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS. In one embodiment, the flow chamber, bag, plate, or hybrid flow chamber is coated with autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS. In another embodiment, the flow chamber, bag, plate, or hybrid flow chamber is coated with mouse serum, mouse plasma, or FBS.

[0044] In one embodiment, phDCs are incubated with at least one antigen protein, or at least one mRNA encoding at least one antigen protein. Incubation may be carried out in a standard medium such as RPMI-1640 medium under standard conditions for human cell culture, for example, at 37°C and 5% CO2. Alternatively or additionally, the incubation step may also be inserted after the generation of phDCs from monocytes. Incubation may be carried out for 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, or 24 hours. In another embodiment, the incubation step may be carried out for at least 0.5 hours, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 12 hours, or at least 20 hours. In one embodiment, incubation is carried out for 6 hours. In one embodiment, incubation is carried out for 12 hours. In one embodiment, incubation is carried out for 20 hours. In one embodiment, incubation is carried out for 6 to 20 hours. In another embodiment, incubation is carried out for 8 to 20 hours. Generally, phDC has a higher transfection capacity compared to other DCs. The incubation step increases transfection efficiency.

[0045] Monocytes can be obtained, for example, from a blood sample or a fraction thereof obtained from a donor by any suitable means. The blood sample or a fraction thereof may be, for example, pia mater containing leukocytes and platelets. Alternatively, the blood sample or a fraction thereof may be isolated peripheral blood mononuclear cells (PMBCs). In one embodiment, the monocytes are autologous.

[0046] In one embodiment, the therapeutic composition further comprises human AB serum, autologous (e.g., human) serum, autologous (e.g., human) plasma, allogeneic (e.g., human) serum, allogeneic (e.g., human) plasma, mouse serum, mouse plasma, or FBS. In one embodiment, the therapeutic composition further comprises autologous serum, autologous plasma, allogeneic serum, allogeneic plasma, mouse serum, mouse plasma, or FBS. In one embodiment, the therapeutic composition further comprises mouse serum, mouse plasma, or FBS.

[0047] In one embodiment, the present invention relates to a therapeutic composition comprising a phDC and at least one mRNA containing a coding sequence encoding at least one infectious disease-associated antigen protein or at least one tumor-associated antigen protein.

[0048] In one embodiment, the present invention relates to a therapeutic composition comprising a phDC and at least one mRNA comprising a coding sequence that encodes at least one viral antigen protein, optionally a coronavirus (e.g., beta-coronavirus) antigen protein, or an HIV antigen protein.

[0049] In one embodiment, the present invention relates to a therapeutic composition comprising a phDC and at least one mRNA containing a coding sequence encoding at least one SARS-CoV-2 antigen protein.

[0050] In one embodiment, the present invention relates to a therapeutic composition comprising a phDC and at least one mRNA comprising a coding sequence that encodes at least one tumor-associated antigen protein, and optionally a blood cancer antigen protein.

[0051] In one embodiment, the present invention relates to a therapeutic composition comprising a phDC and at least one mRNA having a coding sequence encoding at least one infectious disease-associated antigen protein or at least one tumor-associated antigen protein, wherein the at least one mRNA is contained in nanoparticles and the at least one mRNA is optionally modified with one or more naturally occurring modified nucleosides.

[0052] In one embodiment, the present invention relates to a therapeutic composition comprising a phDC and at least one mRNA having a coding sequence encoding at least one infectious disease-associated antigen protein or at least one tumor-associated antigen protein, wherein the at least one mRNA is contained in lipid nanoparticles and the at least one mRNA is optionally modified with one or more naturally occurring modified nucleosides.

[0053] In one embodiment, the present invention relates to a therapeutic composition comprising phDC and at least one viral antigen protein, and optionally at least one mRNA comprising a coding sequence encoding a SARS-CoV-2 antigen protein, wherein at least one mRNA is contained in lipid nanoparticles, and at least one mRNA is optionally modified with one or more naturally occurring modified nucleosides. In a preferred embodiment, at least one mRNA encodes a SARS-CoV-2 spike protein or a fragment thereof. In one embodiment, the fragment has a length of at least 100, at least 200, at least 400, at least 600, or at least 800 amino acids. In one embodiment, the fragment has a length of 400 to 1200 amino acids, 600 to 1200 amino acids, or 800 to 1200 amino acids.

[0054] In a preferred embodiment, at least one mRNA encoding the SARS-CoV-2 antigen protein includes the sequence of SEQ ID NO: 19, or a sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to SEQ ID NO: 19.

[0055] In one embodiment, the present invention relates to a therapeutic composition comprising a phDC and at least one mRNA comprising a coding sequence encoding at least one blood cancer antigen protein, wherein the at least one mRNA is contained in lipid nanoparticles and the at least one mRNA is optionally modified with one or more naturally occurring modified nucleosides.

[0056] With respect to the embodiments described above, the embodiments described above and below apply mutatis mutandis to the phDC, lipid nanoparticles, mRNA, and at least one antigen protein. For example, the lipid nanoparticles may include cationic lipids, polyethylene glycol (PEG)-modified lipids, cholesterol-based lipids, and / or non-cationic lipids. Optionally, the cationic lipids may be present in a molar ratio of 30% to 40%, the PEG-modified lipids in a molar ratio of 1.5% to 4.0%, the cholesterol-based lipids in a molar ratio of 40% to 52%, and the non-cationic lipids in a molar ratio of 11% to 21%, all molar ratios relative to the total lipid content of the LNP. Furthermore, at least one mRNA, optionally modified with one or more naturally occurring modified nucleosides, may be completely modified with N1-methyl-pseudouridine instead of any uridine. In one embodiment, the therapeutic composition comprises an antigen-specific phDC (e.g., at least a portion thereof).

[0057] The antigen-specific phDC can be obtained by combining the phDC with at least one mRNA encoding at least one antigen protein, or by combining a monocyte with at least one mRNA encoding at least one antigen protein, and then subjecting the mixture of the monocyte and at least one mRNA encoding at least one antigen protein to physical force.

[0058] The present invention further relates to a therapeutic composition comprising antigen-specific monocytes and at least one mRNA encoding at least one antigen protein. Antigen-specific monocytes are - Combining a monocyte (acquired as described above) with at least one mRNA encoding at least one antigen protein; - A monocyte and at least one mRNA encoding at least one antigen protein are subjected to physical force. It can be obtained by doing so.

[0059] In another embodiment, the present invention relates to a therapeutic composition comprising antigen-specific monocytes, phDCs, and at least one mRNA encoding at least one antigen protein. A physical force (e.g., shear force) may be applied by passing the monocytes through a flow chamber which may take the form of, for example, a plate or a bag (e.g., a flexible bag or a plastic bag), or a combination thereof (e.g., a hybrid of a plate and a bag). The at least one mRNA may be contained in nanoparticles, particularly lipid nanoparticles. With regard to, for example, the at least one mRNA encoding at least one antigen protein, or, for example, nanoparticles (particularly lipid nanoparticles), all embodiments disclosed herein apply mutatis mutandis.

[0060] Second aspect: A therapeutic composition comprising phDC for use in therapeutic procedures. A therapeutic composition according to the first embodiment may be used in a therapeutic treatment. Thus, in one embodiment, the present invention relates to a therapeutic composition for use in a method of treating a disease in a subject, the method comprising administering the therapeutic composition to the subject. In one embodiment, the subject has been previously diagnosed with a disease.

[0061] The therapeutic compositions of the present invention are suitable for use in the treatment of diseases caused by the pathogens listed in the first embodiment, or in the treatment of tumors / cancers listed in the first embodiment. Furthermore, the therapeutic compositions of the present invention are suitable for use in the treatment of diseases caused by the pathogens listed in the "Pathogens" section of the detailed description, or in the treatment of tumors / cancers listed in the "Tumors / Cancers" section of the detailed description. For example, depending on the at least one antigen or at least one mRNA (encoding at least one antigenic protein) present in the composition, the therapeutic composition is suitable for use in the treatment of AIDS, hepatitis A, hepatitis B, hepatitis C, polio, gastroenteritis, dengue fever, yellow fever, encephalitis, meningitis, or meningoencephalitis caused by West Nile virus, influenza, Ebola, rabies, mumps, measles, herpes, syphilis, Middle East respiratory syndrome, severe acute respiratory syndrome, Covid-19, borreliosis, pseudomoniasis, Burkholderiasis, tuberculosis, and / or malaria. In one embodiment, the therapeutic composition of the present invention is for use in the treatment of AIDS and / or Covid-19. In one embodiment, the therapeutic composition of the present invention is for use in the treatment of Covid-19. In one embodiment, the therapeutic composition of the present invention is for use in the treatment of Covid-19 and is administered intranasally.

[0062] In one embodiment, the subject and donor are the same. In this embodiment, the subject is treated with autologous (antigen-specific) phDCs. In another embodiment, the subject and donor are related by blood. In yet another embodiment, the subject and donor are different (i.e., not related by blood). In this embodiment, the subject is treated with allogeneic (antigen-specific) phDCs.

[0063] The therapeutic composition of the present invention may be administered subcutaneously, intravenously, intramuscularly, by inhalation, intra-articularly, intra-bursally, intrasternally, intrathecally, intrahepatically, intra-lesionally, intracranially, percutaneously, intradermally, intrapulmonaryly, intraperitoneally, intracardiacly, intra-arterially, intranasally, or sublingually. In one embodiment, administration is by inhalation, intravenously, intradermally, intranasally, or intramuscularly. In one embodiment, administration is intradermally or intramuscularly. In one embodiment, administration is intranasally. In one embodiment, administration is intravenously. In some embodiments, the therapeutic composition of the present invention is administered more than once, for example, at least two times, at least three times, at least four times, or at least five times.

[0064] The therapeutic compositions of the present invention can stimulate an immune response to disease-related antigens or antigenic proteins in a subject. In one embodiment, the therapeutic compositions of the present invention can stimulate an immune response to infectious disease-related antigens or infectious disease-related antigenic proteins. In one embodiment, the therapeutic compositions of the present invention can stimulate an immune response to tumor-related antigens or tumor-related antigenic proteins.

[0065] In one embodiment, the disease is caused by disease-causing particles that optionally include viruses, bacteria, fungi, parasites, and / or tumor cells. In one embodiment, the disease-causing particles include pathogens such as viruses, bacteria, fungi, and parasites. In one embodiment, the disease-causing particles include viruses. In one embodiment, the disease-causing particles include tumor cells (or cancer cells).

[0066] In some embodiments, the therapeutic compositions provided herein may be characterized in that a subject treated with such a composition (e.g., using at least one dose, at least two doses, etc.) may show a reduced and / or more transient presence of disease-causing particles at the relevant site (e.g., the nose and / or lungs, and / or any other tissue susceptible to the corresponding disease or disorder) compared to a suitable control (e.g., a comparable subject or population that is untreated and exposed to disease-causing particles; or a comparable subject or population treated with a different therapeutic agent such as an RNA therapeutic agent that does not contain dendritic cells).

[0067] In one embodiment, the concentration of disease-causing particles is lower compared to the concentration of disease-causing particles in a second subject previously diagnosed with the same disease but not administered the therapeutic composition of the present invention; or compared to the concentration of disease-causing particles in a second subject previously diagnosed with the same disease but treated with a different therapeutic agent, preferably a dendritic cell-free therapeutic agent; more preferably a dendritic cell-free RNA therapeutic agent, for the same disease-causing particles.

[0068] In one embodiment, the concentration of disease-causing particles is effectively lower after tumor development in a subject compared to the concentration of disease-causing particles in a second subject previously diagnosed with the same tumor and not administered the therapeutic composition of the present invention; or compared to the concentration of disease-causing particles in a second subject previously diagnosed with the same tumor and treated with a different therapeutic agent, preferably a dendritic cell-free therapeutic agent; more preferably a dendritic cell-free RNA therapeutic agent, for the same tumor-causing particles.

[0069] In one embodiment, the anti-antigen antibody titer in a subject is lower after treatment with the therapeutic composition of the present invention compared to the anti-antigen antibody titer in a second subject treated with the same disease-causing particles with a different therapeutic agent, preferably a dendritic cell-free therapeutic agent; more preferably a dendritic cell-free RNA therapeutic agent. Importantly, subjects treated with the therapeutic composition of the present invention are treated equally well or even better than a second subject exhibiting a higher anti-antigen antibody titer (e.g., showing less severe symptoms, a more transient presence of disease-causing particles, and / or a shorter course of the disease). This effect may be attributed to the antigen-specific phDCs of the present invention supporting T-cell-based cellular immunity, while other therapeutic agents, particularly RNA-based therapeutic agents, may lead to a stronger antibody response.

[0070] In one embodiment, the concentration of disease-causing particles is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% lower in the subject. In one embodiment, the duration of the subject exhibiting symptoms of the disease is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%. In one embodiment, the concentration of disease-causing particles is lower systemically in the subject. In one embodiment, the concentration of disease-causing particles is lower locally. In one embodiment, the concentration of disease-causing particles is lower in the brain. In one embodiment, the concentration of disease-causing particles is lower in mucosal tissue (i.e., the therapeutic composition of the present invention induces mucosal immunity). The reduction may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Comparisons may be made with a suitable control, for example, compared to before the onset of the disease; compared to the second subject described above; or compared to a subject that has not been treated with the therapeutic composition of the present invention or has been treated with a different therapeutic agent, such as an RNA therapeutic agent that does not contain dendritic cells.

[0071] In one embodiment, after treatment with the therapeutic composition of the present invention, the subject shows an increase in the proportion of central memory T cells (Tcm) and / or stem-like T cells that are specific to at least one disease-related antigen (or antigen protein). In one embodiment, after treatment with the therapeutic composition of the present invention, the subject shows a decrease in the proportion of effector-dominant T cells (Teff) that are specific to at least one disease-related antigen (or antigen protein). In one embodiment, after treatment with the therapeutic composition of the present invention, the subject shows an increase in the proportion of natural killer cells (NK). In one embodiment, after treatment with the therapeutic composition of the present invention, the subject shows an increase in IFN-γ levels. In one embodiment, after treatment with the therapeutic composition of the present invention, the subject shows a decrease in the proportion of exhausted effector T cells. These effects may be observed after a certain time after the first dose (or after the second, third, fourth, or fifth dose), for example, at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks after the administration of each dose. Comparisons may be made with a suitable control, for example, compared to before the onset of the disease; or compared to the levels or percentages in subjects treated with different therapeutic agents, such as RNA therapeutics that have not been treated with the therapeutic composition of the present invention or that do not contain dendritic cells. The increase in the percentage of central memory T cells and / or stem-like T cells and / or natural killer cells may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The reduction in the proportion of effector-dominant T cells and / or exhausted effector T cells may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.The increase in IFN-γ secretion levels may be at least 2-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 5000-fold. The proportion of Tcms can be determined by measuring markers associated with the central memory T cell phenotype, such as CD44 / CD62L. Teffs show decreased CD62L expression compared to Tcms. The proportion of stem-like T cells can be determined by measuring markers associated with the stem-like T cell phenotype, such as IL7Ra / SCA-1. Expression of both markers increases in relation to stem-like T cells. NK cells can be determined, for example, by positive selection using the NK1.1 cell isolation method. The proportion of exhausted effector T cells can be determined by measuring expression markers associated with the exhausted state, such as PD1. Treatment may refer to a single dose or multiple doses, such as at least two, at least three, at least four, or at least five doses.

[0072] In one embodiment, the therapeutic composition of the present invention is administered to a subject previously diagnosed with an infectious disease. The subject may have previously been diagnosed with a viral infectious disease. In one embodiment, the subject may have previously been diagnosed with coronavirus disease 2019 (Covid-19). In another embodiment, the subject may have previously been diagnosed with cancer or a tumor. In some embodiments, the cancer (or tumor) can be classified as stage I, II, III, or IV according to the Tumor Nodule Metastasis (TNM) Anatomical / Prognostic Classification System of the American Joint Committee on Cancer. In some embodiments, the cancer (or tumor) can be classified as stage I according to the Tumor Nodule Metastasis (TNM) Anatomical / Prognostic Classification System of the American Joint Committee on Cancer. In some embodiments, the cancer (or tumor) is classifiable as Stage II according to the Tumor Nodule Metastasis (TNM) Anatomical / Prognostic Classification System of the Joint Commission on Cancer (JCCR) Cancer Staging System. In some embodiments, the cancer (or tumor) is classifiable as Stage III according to the Tumor Nodule Metastasis (TNM) Anatomical / Prognostic Classification System of the JCCR Cancer Staging System. In some embodiments, the cancer (or tumor) is classifiable as Stage IV according to the Tumor Nodule Metastasis (TNM) Anatomical / Prognostic Classification System of the JCCR Cancer Staging System. In some embodiments, the subject is elderly, pregnant, an infant, has a chronic medical condition, has recently undergone cancer treatment, has a lung disease, or is immunocompromised. In some embodiments, the subject is over 60 years of age and under 18 years of age, is immunocompromised, is pregnant, has a genetic disorder affecting the immune system, has been previously diagnosed with an infectious disease, has an underlying medical condition, is overweight or obese, and / or has a past diagnosis of COVID-19. In one embodiment, administration of the therapeutic composition of the present invention additionally provides a vaccine effect in the subject.

[0073] In one embodiment, the target to be treated with the therapeutic composition of the present invention is an elderly person (used synonymously with "elderly," for example, a person older than 60, 65, 70, 75, 80, 85 years old, for example, a person between 65 and 85 years old). In some embodiments, the target to be treated with the therapeutic composition of the present invention is 18 years old or younger. In some embodiments, the target to be treated with the therapeutic composition of the present invention is 12 years old or younger. In some embodiments, the target to be treated with the therapeutic composition of the present invention is 10 years old or younger. In some embodiments, the target to be treated with the therapeutic composition of the present invention is an infant, for example, under 1 year old. In some embodiments, the target to be treated with the therapeutic composition of the present invention is a pregnant woman. In one embodiment, the subjects to be treated with the therapeutic composition of the present invention are immunocompromised subjects (e.g., persons with HIV / AIDS; cancer and transplant patients taking certain immunosuppressants; autoimmune diseases or other physiological conditions expected to require immunosuppressive therapy (e.g., within 3 months, 6 months, or longer); and persons with genetic disorders affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency)). In one embodiment, the subjects to be treated with the therapeutic composition of the present invention are persons previously diagnosed with an infectious disease (e.g., persons infected with human immunodeficiency virus (HIV) and / or hepatitis viruses (e.g., HBV, HCV)). In one embodiment, the subject to be treated with the therapeutic composition of the present invention is an underlying medical condition (e.g., hypertension, cardiovascular disease, diabetes, chronic respiratory disease, such as chronic lung disease, asthma, etc., cancer, and other chronic diseases, such as lupus, rheumatoid arthritis, chronic liver disease, chronic kidney disease (e.g., in some embodiments, 60 mL / min / 1.73 m) 2 This includes individuals with a glomerular filtration rate (GFR) of less than 30%, or those with stage 3 or more severe disease.

[0074] In one embodiment, the target to be treated with the therapeutic composition of the present invention is, for example, about 30 kg / m³. 2The subjects are overweight or obese, specifically including those with a body mass index (BMI) exceeding a certain threshold. In one embodiment, the subjects to be treated with the therapeutic composition of the present invention have a past diagnosis of COVID-19 or evidence of current or past SARS-CoV-2 infection, for example, based on serology or nasal swab. In some embodiments, the therapeutic composition of the present invention is administered to one or more of the above risk groups.

[0075] In one embodiment, the present invention relates to a therapeutic composition of a first aspect for use in a method of treating a disease in a subject, wherein the subject has been previously diagnosed with a disease, and the method comprises administering the therapeutic composition to the subject.

[0076] In one embodiment, the therapeutic composition of the present invention is for use in a method of treating a disease in a subject, the subject having been previously diagnosed with a disease, and the method comprises administering the therapeutic composition to the subject, the disease being an infectious disease or a tumor.

[0077] In one embodiment, the therapeutic composition of the present invention is for use in a method of treating a viral disease in a subject, the subject having been previously diagnosed with a viral disease, and the method comprises administering the therapeutic composition to the subject.

[0078] In one embodiment, the therapeutic composition of the present invention is for use in a method of treating a Covid-19 disease in a subject, the subject having been previously diagnosed with Covid-19, and the method comprises administering the therapeutic composition to the subject.

[0079] In one embodiment, the therapeutic composition of the present invention is for use in a method of treating a blood cancer in a subject, the subject having been previously diagnosed with a blood cancer, and the method comprises administering the therapeutic composition to the subject.

[0080] In one embodiment, the therapeutic composition of the present invention is for use in a method of treating a solid tumor in a subject, the subject having been previously diagnosed with having a solid tumor, and the method comprises administering the therapeutic composition to the subject.

[0081] Third aspect: Method for treating a disease in a subject In a third aspect, the present invention relates to a method for treating a disease in a subject, wherein the subject has been previously diagnosed with the disease, and the method comprises administering a therapeutic composition of the first aspect (including all embodiments of the first and second aspects described above) to the subject.

[0082] Fourth aspect: Use of therapeutic compositions for therapeutic treatment In a fourth aspect, the present invention relates to the use of a therapeutic composition for therapeutic treatment, wherein the therapeutic composition is the composition of the first aspect. Thus, with respect to the therapeutic composition, phDC, nanoparticles, RNA (e.g., mRNA), and at least one antigenic protein, the respective embodiments of the first, second, and third aspects apply mutatis mutandis.

[0083] Fifth aspect: Kit containing phDC and mRNA In a fifth aspect, the present invention relates to a kit comprising a phDC and at least one RNA (e.g., mRNA) encoding at least one antigen protein. The kit may further comprise nanoparticles (e.g., lipid nanoparticles). If the kit comprises lipid nanoparticles, the nanoparticles comprise RNA (e.g., mRNA) encoding at least one antigen protein. With respect to the phDC, nanoparticles (e.g., lipid nanoparticles), RNA (e.g., mRNA), and at least one antigen protein, the respective embodiments of the first aspect apply mutatis mutandis.

[0084] The present invention also relates to a kit comprising monocytes and at least one RNA (e.g., mRNA) encoding at least one antigen protein. The kit may further comprise nanoparticles (e.g., lipid nanoparticles). If the kit comprises nanoparticles, the nanoparticles comprise RNA (e.g., mRNA) encoding at least one antigen protein. With respect to monocytes, nanoparticles (e.g., lipid nanoparticles), RNA (e.g., mRNA), and at least one antigen protein, the respective embodiments of the first embodiment apply mutatis mutandis. [Brief explanation of the drawing]

[0085] [Figure 1] This figure shows that murid phDCs specifically internalize and express the spike protein encoded by mRNA-containing LNPs. A and B are FACS plots showing spike protein positivity in CD11b-positive phDCs with and without LNP transfection (A and B). C and D are confocal microscopy analyses (projected Z-stack images) using the same samples showing spike protein expression in CD11b+Ly6G-phDCs. E. Z-planar slice image of LNP-transfected phDCs. [Figure 2A] This figure shows C57BL / 6 mice treated intravenously with OVA mRNA LNP-transduced phDCs or via the im pathway with OVA mRNA LNP (without phDCs) two days after EG7-OVA subcutaneous tumor transplantation (3 × 10⁶ cells per mouse). Approximately 10 ng of LNP was transduced into phDCs (per mouse), and the same amount was injected via im per mouse (0.5 ug / kg of LNP, equivalent to the dose of the COVID-19 Pfizer mRNA LNP vaccine in the current human vaccine setting). Treatment was administered twice a week, with each mouse receiving a total of five treatments. At the start of treatment, the tumors were visible and palpable (>50 mm³). [Figure 2B] This figure shows the results of monitoring EG7-OVA tumor growth through the experiment. [Figure 3-1] Figure A shows that phDC[ova] or IM[ova] mice were treated with a total of five doses of EG7-OVA at a dose of 10 ng[ova] per mouse, starting two days after subcutaneous tumor inoculation using 3*10⁶ cells per mouse (same experiment as Figure 3). Figures B-C show the cumulative tumor growth per experimental group (B) and the tumor growth curves for individual mice (C), monitoring tumor growth throughout the duration of the experiment. [Figure 3-2] Images D-E show CD8+ spleen T cells isolated from treated mice on day 30 and immediately subjected to an IFN-g Elispot assay at a rate of 1*10⁵ cells per well for 18 hours, either in the presence or absence of 10 ug / mL SIINFEKL peptide. Representative Elispot well images (D) and IFNg spot quantifications (E) are provided for each experimental group. *[ova] = LNP-mRNA encoding ovalbumin. [Figure 4A-1] Figure A shows the results of a total of five treatments (same experiment as Figure 3) in which phDC[ova] or IM[ova] mice were treated with EG7-OVA subcutaneous tumor inoculation using 3*10⁶ cells per mouse, starting two days after inoculation, at a dose of 10 ng[ova] per mouse. Splenocytes from the treated mice were collected at the end of a 28-day observation period for T cell analysis and characterization by flow cytometry for A (antigen (SIINFEKL)-specific CD8 T cells by dextramer analysis; Tem (effector) / Tcm (central memory) type phenotypic analysis by CD44 / CD62L expression; and detection of stem-like T cells by IL7Ra / SCA-1 expression), and for B (evaluation of T cell exhaustion markers by PD-1 expression). *[ova] = LNP-mRNA encoding ovalbumin. [Figure 4A-2]Figure A shows the results of a total of five treatments (same experiment as Figure 3) in which phDC[ova] or IM[ova] mice were treated with EG7-OVA subcutaneous tumor inoculation using 3*10⁶ cells per mouse, starting two days after inoculation, at a dose of 10 ng[ova] per mouse. Splenocytes from the treated mice were collected at the end of a 28-day observation period for T cell analysis and characterization by flow cytometry for A (antigen (SIINFEKL)-specific CD8 T cells by dextramer analysis; Tem (effector) / Tcm (central memory) type phenotypic analysis by CD44 / CD62L expression; and detection of stem-like T cells by IL7Ra / SCA-1 expression), and for B (evaluation of T cell exhaustion markers by PD-1 expression). *[ova] = LNP-mRNA encoding ovalbumin. [Figure 4B] Figure A shows the results of a total of five treatments (same experiment as Figure 3) in which phDC[ova] or IM[ova] mice were treated with EG7-OVA subcutaneous tumor inoculation using 3*10⁶ cells per mouse, starting two days after inoculation, at a dose of 10 ng[ova] per mouse. Splenocytes from the treated mice were collected at the end of a 28-day observation period for T cell analysis and characterization by flow cytometry for A (antigen (SIINFEKL)-specific CD8 T cells by dextramer analysis; Tem (effector) / Tcm (central memory) type phenotypic analysis by CD44 / CD62L expression; and detection of stem-like T cells by IL7Ra / SCA-1 expression), and for B (evaluation of T cell exhaustion markers by PD-1 expression). *[ova] = LNP-mRNA encoding ovalbumin. [Figure 4]Figure A shows the results of a total of five treatments (same experiment as Figure 3) in which phDC[ova] or IM[ova] mice were treated with EG7-OVA subcutaneous tumor inoculation using 3*10⁶ cells per mouse, starting two days after inoculation, at a dose of 10 ng[ova] per mouse. Splenocytes from the treated mice were collected at the end of a 28-day observation period for T cell analysis and characterization by flow cytometry for A (antigen (SIINFEKL)-specific CD8 T cells by dextramer analysis; Tem (effector) / Tcm (central memory) type phenotypic analysis by CD44 / CD62L expression; and detection of stem-like T cells by IL7Ra / SCA-1 expression), and for B (evaluation of T cell exhaustion markers by PD-1 expression). *[ova] = LNP-mRNA encoding ovalbumin. [Figure 5] This figure shows the results of vaccinating phDC[ova] or IM[ova] mice on day -7 and day 0 (prime / boost), respectively. Undifferentiated splenocytes were collected after vaccination and immediately placed in 18-hour IFN-g Elispots without additional antigen stimulation (A, B, and C). To identify cells involved in spleen IFN-g, isolated CD8+ or NK1.1+ splenocytes were collected from phDC[ova] mice after vaccination and assessed for IFN-g spots (D). *[ova] = LNP-mRNA encoding ovalbumin. [Figure 6] Figure A shows C57BL / 6 mice that were intravenously treated with 1 ug / mL of OVA mRNA LNP-transduced phDC or 14 and 7 days prior to EG7-OVA subcutaneous tumor transplantation (1 × 10⁶ cells per mouse) or intravenously treated with soluble OVA protein (50 ug / mouse, without phDC). Figure B shows the results of monitoring EG7-OVA tumor growth throughout the experiment. [Figure 7]This figure shows the results of staining for intracellular OVA proteins after transducing mouse PhDCs with 1 ug / ml OVA mRNA LNP (CKK) and culturing overnight. PhDCs transduced with SP LNP (1 ug / ml) were used as a control. (n=3) OVA detection using Rockland OVA ab (FITC conjugate). [Figure 8A] Mouse phDCs were incubated overnight with various antigenic forms of OVA, including soluble, expressed in tumor cells, or OVA-coding mRNA-containing LNPs. Cells were then harvested and stained surface for CD11b, Ly6G, and 25.D1 (ab against SIINFEKL-binding MHC I in H-2Kb strains). The results are shown in the figure. Ly6G-positive cells were selected and removed to exclude neutrophils. FACs plot showing 25.D1 positivity in CD11b+ phDCs. [Figure 8B] Mouse phDCs were incubated overnight with various antigenic forms of OVA, including soluble, expressed in tumor cells, or OVA-coding mRNA-containing LNPs. Cells were then harvested and stained surface for CD11b, Ly6G, and 25.D1 (ab against SIINFEKL-binding MHC I in H-2Kb strains). The results are shown in the figure. Ly6G-positive cells were selected and removed to exclude neutrophils. FACs plot showing 25.D1 positivity in CD11b+ phDCs. [Figure 8C] Mouse phDCs were incubated overnight with various antigenic forms of OVA, including soluble, expressed in tumor cells, or OVA-coding mRNA-containing LNPs. Cells were then harvested and stained surface for CD11b, Ly6G, and 25.D1 (ab against SIINFEKL-binding MHC I in H-2Kb strains). The results are shown in the figure. Ly6G-positive cells were selected and removed to exclude neutrophils. FACs plot showing 25.D1 positivity in CD11b+ phDCs. [Figure 8D]Mouse phDCs were incubated overnight with various antigenic forms of OVA, including soluble, expressed in tumor cells, or OVA-coding mRNA-containing LNPs. Cells were then harvested and stained surface for CD11b, Ly6G, and 25.D1 (ab against SIINFEKL-binding MHC I in H-2Kb strains). The results are shown in the figure. Ly6G-positive cells were selected and removed to exclude neutrophils. FACs plot showing 25.D1 positivity in CD11b+ phDCs. [Figure 9A] In a standard overnight culture protocol, PhDCs (PP PBMCs derived from B6 mice) were transduced with OVA mRNA LNP (1 ug / ml). Cells were harvested, washed, and fixed (using an intraprep kit) at various time points, and the results of staining for surface CD11b and SIINFEKL-MHC I complex (25.D1 ab) are shown in the figure. The 25.D1 expression levels in CD11b+ cells at various time points are shown in the FACs plot (upper panel). The percentage of 25.D1-positive cells and the MFI level of 25.D1 in the CD11b+ subset are shown in the bar graph (lower). [Figure 9B] In a standard overnight culture protocol, PhDCs (PP PBMCs derived from B6 mice) were transduced with OVA mRNA LNP (1 ug / ml). Cells were harvested, washed, and fixed (using an intraprep kit) at various time points, and the results of staining for surface CD11b and SIINFEKL-MHC I complex (25.D1 ab) are shown in the figure. The 25.D1 expression levels in CD11b+ cells at various time points are shown in the FACs plot (upper panel). The percentage of 25.D1-positive cells and the MFI level of 25.D1 in the CD11b+ subset are shown in the bar graph (lower). [Figure 9C]In a standard overnight culture protocol, PhDCs (PP PBMCs derived from B6 mice) were transduced with OVA mRNA LNP (1 ug / ml). Cells were harvested, washed, and fixed (using an intraprep kit) at various time points, and the results of staining for surface CD11b and SIINFEKL-MHC I complex (25.D1 ab) are shown in the figure. The 25.D1 expression levels in CD11b+ cells at various time points are shown in the FACs plot (upper panel). The percentage of 25.D1-positive cells and the MFI level of 25.D1 in the CD11b+ subset are shown in the bar graph (lower). [Figure 9D] In a standard overnight culture protocol, PhDCs (PP PBMCs derived from B6 mice) were transduced with OVA mRNA LNP (1 ug / ml). Cells were harvested, washed, and fixed (using an intraprep kit) at various time points, and the results of staining for surface CD11b and SIINFEKL-MHC I complex (25.D1 ab) are shown in the figure. The 25.D1 expression levels in CD11b+ cells at various time points are shown in the FACs plot (upper panel). The percentage of 25.D1-positive cells and the MFI level of 25.D1 in the CD11b+ subset are shown in the bar graph (lower). [Figure 10A] Enriched phDCs (monocytes purified from plate-passing PBMCs derived from mice) or BMDCs (cytokine-inducible DCs derived from mouse bone marrow) were incubated overnight in a standard overnight protocol with or without LNPs containing 0.1 ug / ml of OVA mRNA. Cells were collected, and the results of staining for surface CD11b and 25.D1 (ab against SIINFEKL-bound MHC I in H-2Kb strains) for phDC samples, and for CD11c and 25.D1 for BMDC samples are shown. FACs plots showing 25.D1 positivity in CD11b+ phDCs and CD11c+ BMDCs are also shown. [Figure 10B]Enriched phDCs (monocytes purified from plate-passing PBMCs derived from mice) or BMDCs (cytokine-inducible DCs derived from mouse bone marrow) were incubated overnight in a standard overnight protocol with or without LNPs containing 0.1 ug / ml of OVA mRNA. Cells were collected, and the results of staining for surface CD11b and 25.D1 (ab against SIINFEKL-bound MHC I in H-2Kb strains) for phDC samples, and for CD11c and 25.D1 for BMDC samples are shown. FACs plots showing 25.D1 positivity in CD11b+ phDCs and CD11c+ BMDCs are also shown. [Figure 11A] Mouse phDCs were transduced with either SIINFEKL peptide mRNA LNP or OVA protein mRNA LNP. After overnight incubation, cells were harvested and stained with 25.D1 ab for detection of the SIINFEKL MHC I complex signal. The figure shows the results. FACS plot showing the 25.D1 signal in the CD11b+ phDC subset. [Figure 11B] Mouse phDCs were transduced with either SIINFEKL peptide mRNA LNP or OVA protein mRNA LNP. After overnight incubation, cells were harvested and stained with 25.D1 ab for detection of the SIINFEKL MHC I complex signal. The figure shows the results. FACS plot showing the 25.D1 signal in the CD11b+ phDC subset. [Figure 11C] Mouse phDCs were transduced with either SIINFEKL peptide mRNA LNP or OVA protein mRNA LNP. After overnight incubation, cells were harvested and stained with 25.D1 ab for detection of the SIINFEKL MHC I complex signal. The figure shows the results. FACS plot showing the 25.D1 signal in the CD11b+ phDC subset. [Figure 12A]Mouse-derived phDCs were pulsed with various concentrations of OVA mRNA LNP and then cultured with OT1 T cells. The proliferation of OT1 CD8 T cells (CFSE-labeled) was evaluated as a decrease in CFSE signaling, as shown in the figure. [Figure 12B] Mouse-derived phDCs were pulsed with various concentrations of OVA mRNA LNP and then cultured with OT1 T cells. The proliferation of OT1 CD8 T cells (CFSE-labeled) was evaluated as a decrease in CFSE signaling, as shown in the figure. [Figure 13A] Mouse (B6) phDCs were pulsed with 1 ug / ml OVA mRNA and then cultured with 100,000 OT1 T cells at various phDC counts (96-well U-bottom plate). The figure shows the results of evaluating the proliferation of OT1 CD8 T cells (CFSE labeled) as a readout value. [Figure 13B] Mouse (B6) phDCs were pulsed with 1 ug / ml OVA mRNA and then cultured with 100,000 OT1 T cells at various phDC counts (96-well U-bottom plate). The figure shows the results of evaluating the proliferation of OT1 CD8 T cells (CFSE labeled) as a readout value. [Figure 14] This figure shows C57BL / 6 mice treated IV with OVA mRNA LNP*-transduced phDCs, or injected with mock (SP) mRNA LNP* or OVA mRNA LNP (without phDCs) via the im* pathway, four days after EG7 tumor transplantation (3 × 10⁶ cells per mouse). Treatment was administered twice weekly (Tx on days 4, 8, 11, 15, 18, and 25). At the start of treatment, the tumors were visible and palpable (>50 mm³). N=10 for each group. [Figure 15A](a) This figure shows the results of analyzing mouse-derived splenocytes from Experiment 21-1008 (therapeutic experiment, HB) for SIINFEKL tetramer positivity at the end of the in vivo tumor monitoring period (day 32). H-2Kb OVA SIINFEKL tetramer positivity in the CD8+ subset is shown in the FACs plot. Splenocytes from 10 mice were pooled before analysis. The final treatment was administered one week prior to analysis. [Figure 15B] (b) This figure shows the results of transferring T cells isolated from splenocytes of three experimental groups into mice with EG7 tumors (15M T cells / mouse) and monitoring tumor development over a 19-day period. [Figure 16A] Figure 16A: An example of phDC ELISpot using PBMCs isolated from a single human donor 4 weeks after SARS-CoV-2 infection. Compared to negative controls, IFNγ release was significantly increased in the presence of phDC[spike]. This demonstrates the detection of human spike-specific T cells via mRNA-transduced phDC in Covid convalescent donors. [Figure 16B] Figure 16B: An example of phDC ELISpot dose response using single-human donor PBMCs 4 weeks after SARS-CoV-2 infection. When LNP [spike] antigen was added at 62.5 ng / well, IFNγ release increased to levels exceeding 150 SFU / million. T cell activation further improved at higher doses, but showed evidence of a response plateau at 250 ng / well. This demonstrates a detectable phDC LNP spike dose response even at low levels of 62.5 ng / well. [Figure 17A]Figure 17A: Eighteen previously vaccinated human donors were screened for SARS-CoV-2 spike antigen in phDC ELISpot. Donors were divided into two cohorts based on whether they had previously been infected with SARS-CoV-2 (black) or not (blue). Statistical analysis was performed using the unpaired two-sided Mann-Whitney U test. This demonstrates that human phDCs transduced with LNP [spike] can distinguish between T-cell responses associated with innate immunity and responses associated solely with vaccination. [Figure 17B] Figure 17B: Eleven previously vaccinated and convalescent human donors were screened for SARS-CoV-2 spike antigen using phDC ELISpot. The dotted line represents the standard threshold cutoff (50 SFU / million cells) for a positive response. This demonstrates that 1) response intensity generally correlates negatively with convalescence duration; and 2) the phDC-induced IFNγ response is persistent and detectable up to one year after Covid infection. [Figure 17C] Figure 17C: Either CD8 or CD4 T cells were depleted from plate-passed PBMCs of vaccinated and convalescent donors before incorporation into a standard 18-hour phDC[spike]ELISpot IFNγ assay. CD8 T cell depletion eliminated most of the phDC[spike] response, while CD4 depletion had minimal effect. This demonstrates that the convalescent phDC[spike]IFNγ response is primarily driven by CD8+ T cells. [Figure 17D] Figure 17D: An example of phDC ELISpot using PBMCs isolated from a single human donor, before and 6 weeks after SARS-CoV-2 infection. Post-infection, IFNγ release increased beyond the positive threshold of 50 SFU / million cells. This demonstrates an increase in phDC-induced IFNγ T cell response after spontaneous Covid infection, thus showing the feasibility and practicality of long-term immune monitoring. [Modes for carrying out the invention]

[0086] Detailed explanation Exemplary preparation of I. phDC In one example, phDCs may be prepared from monocytes (e.g., acquired from a donor). For example, phDCs may be generated by subjecting monocytes (e.g., acquired from a donor) to a physical force. The physical force may be generated, for example, by passing or moving the monocytes through a flow chamber. In one embodiment of the present invention, phDCs are obtained by passing monocytes through a plate using a process derived from extracorporeal photoferrosis (ECP). Thus, the flow chamber may be a plate in one embodiment. Plates for preparing phDCs have been previously described in the literature; see, for example, Durazzo et al., 2014 ("Induction of Monocyte-to-Dendritic Cell Maturation by Extracorporeal Photochemotherapy: Initiation via Direct Platelet Signaling") or Ventura et al., 2018 ("Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity"). Methods and devices for extracorporeal activation of monocytes and generation of dendritic cells therefrom are described in WO2014 / 106629 A1, WO2014 / 106631 A1, WO2016 / 001405 A1, and WO2017 / 005700 A1, each of which is incorporated herein by reference as a whole. ECP describes a process of exposing monocytes derived from a blood sample or a fraction thereof to mechanical stress (e.g., shear force) and plasma components (e.g., platelets) or derivatives or mimetic bodies thereof, thereby activating the monocytes and differentiating them into healthy physiological dendritic cells, also referred herein as phDCs. However, simply providing monocytes and subjecting them to physical force is sufficient for monocyte activation and differentiation into phDCs.ECP and ECP-derived processes, including the differentiation of monocytes into phDCs, may be carried out in a flow chamber (e.g., a plate; a large-scale ECP device, such as a clinical ECP device (e.g., a THERAKOS® CELLEX® device); or a small-scale ECP device, such as a transimmunization plate as described in WO2017 / 005700 A1; or a flow chamber in a bag (e.g., a flexible bag or plastic bag); or any combination thereof).

[0087] The bag may be made from any material that does not leak liquid, such as rubber, silicone, or plastic. Preferably, the material is easily bendable without tearing.

[0088] Optionally, the bags or plates are made from plastic materials. Suitable plastic materials include polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymer, ethylene vinyl alcohol, polyvinylidene fluoride, and / or other plastics, including materials approved for medical use. A preferred plastic material is ethylene-vinyl acetate copolymer. The bags or plates may be made from materials that provide a degree of transparency that allows visible or UV light to be irradiated onto the sample or cell mixture.

[0089] In one embodiment, the flow chamber may be a hybrid flow chamber or a combination of a chamber and a bag, each of which is described herein.

[0090] The inventors have found that phDCs obtained by the method described above are advantageous compared to DCs obtained by other methods, such as incubation of blood monocytes with cytokines or direct isolation from donors, because phDCs are generated physiologically (without the need for chemicals such as cytokines and / or apoptotic agents) with higher reproducibility and controllability under precise in vitro laboratory conditions. The advantages of the above method for generating phDCs compared to other methods, such as incubation with cytokines, include one or more of the following: higher yield, a faster process, better intracellular antigen treatment, and more effective priming of disease-specific cytotoxic T cells.

[0091] Donor phDCs are obtained by passing a blood sample (obtained from the donor) or a fraction thereof through a flow chamber, thereby subjecting the monocytes contained in the blood sample to shear force. Preferably, platelets, which may originate from or be provided separately from the donor's blood sample or a fraction thereof, are present in the flow chamber. Additionally or alternatively, plasma components, which may originate from or be provided separately from the donor's blood sample or a fraction thereof, may be present in the flow chamber. After the phDCs are obtained, they may be stored cryopreserved until further use, for example, in combination with at least one mRNA encoding at least one disease-associated antigen or at least one antigenic protein.

[0092] Donor monocytes can be obtained, for example, from a blood sample or a fraction thereof by any suitable means. A fraction of a blood sample may be, for example, pia mater containing leukocytes and platelets. Alternatively, a fraction of a blood sample may be isolated peripheral blood mononuclear cells (PMBCs). PMBCs can be isolated from a blood sample, for example, by centrifugation on a Ficoll-Hypaque gradient (Isolymph, CTL Scientific). In another example, a fraction of a blood sample may be a purified or enriched monocyte preparation. Monocytes can be enriched from PBMCs using, for example, plastic bonding; CD14 magnetic bead positive selection (e.g., from Miltenyi Biotec); and Monocyte Isolation Kit II (Miltenyi Biotec).

[0093] Any appropriate volume of blood may be used. The blood sample (e.g., the blood sample from which the fraction is derived) may be about 1 μL to about 500 mL, for example, about 1 μL to about 10 mL, about 1 μL to about 5 mL, about 1 μL to about 1 mL, about 1 μL to about 750 μL, about 1 μL to about 500 μL, about 1 μL to about 250 μL, about 10 mL to about 450 mL, about 20 mL to about 400 mL, about 30 mL to about 350 mL, about 40 mL to about 300 mL, about 50 mL to about 200 mL, or about 50 mL to about 100 mL. In some embodiments, the blood sample or a fraction thereof, or an additional blood sample or a fraction thereof, is about 100 mL or less (e.g., about 50 mL to about 100 mL). In one embodiment, the monocytes are derived from a blood sample obtained from a donor. However, monocytes may originate from other sources in the donor, such as bone marrow, spleen, or other peripheral tissues. In one embodiment, monocytes are derived from peripheral blood mononuclear cells (PBMCs) obtained from the donor.

[0094] Those skilled in the art are familiar with methods for distinguishing dendritic cells, including phDCs, from monocytes, for example, by assessing gene expression. For example, CD80, CD83, and CD86 are markers expressed at higher levels by mature dendritic cells compared to monocytes. In one embodiment, the donor is a mammal. Mammals include, but are not limited to, humans, non-human primates, pigs, dogs, cats, and rodents. In a preferred embodiment, the donor is human.

[0095] II. Antigen Proteins PhDCs are combined with at least one mRNA encoding at least one antigen protein. The antigen protein may be a disease-related antigen or a fragment thereof. When it is referred to as a “disease-related antigen” or “antigen protein,” the meaning includes “its fragment.” As soon as a combination of a phDC and a disease-related antigen is established, the phDCs can take up the antigen, process it, and present the antigen on their surface. This process is also called “carrying.” Such carried phDCs can elicit a potent anti-disease immune response, such as an anti-infective disease or anti-tumor response.

[0096] At least one disease-related antigen can be carried as the antigen itself (e.g., a protein, peptide, epitope, cell, cell or tissue lysate, virus, viral particle, etc., including fragments of each of the aforementioned) or as a nucleic acid encoding the antigen (the phDC is carried in the sense that it takes up the antigen or antigen protein). Therefore, in one embodiment, the phDC includes a nucleic acid encoding an antigen protein or disease-related antigen, preferably RNA, more preferably mRNA. In one embodiment, at least one disease-related antigen is carried as a nucleic acid encoding the antigen. At least one disease-related antigen can be provided directly or stored in a frozen or lyophilized form until use. Methods for carrying antigens on dendritic cells are known to those skilled in the art. In one example, the phDC is carried by a combination of the phDC and at least one disease-related antigen, e.g., incubation. In some embodiments, various antigens can be carried on the phDC to produce a multivalent antigen-specific phDC. In other examples, phDCs are supported by electroporation, polymer or lipid-based nanoparticles, or cell compression. In one embodiment, phDCs are supported by encapsulating at least one disease-related antigen (e.g., in the form of mRNA) within lipid nanoparticles, and then combining the phDC with the encapsulated at least one disease-related antigen. If the at least one antigen is encoded by RNA, particularly mRNA, the at least one disease-related antigen is referred to as an antigenic protein for the purposes of this invention.

[0097] Regarding the cell compression method, phDCs are passed through a solution containing at least one disease-related antigen or a fragment thereof. For loading, the phDCs are compressed by passing them through a microfluidic construct smaller than the diameter of the cell. This causes transient breakdown of the cell membrane, allowing at least one disease-related antigen to enter the cytosol of the phDCs.

[0098] Once phDCs are obtained and combined with at least one disease-related antigen, the mixture can be incubated, for example, under standard conditions. Culturing can be carried out under standard conditions at 37°C and 5% CO2 in standard culture media for human cells, such as RPMI-1640 medium (e.g., available from GIBCO) supplemented with 15% AB serum (e.g., available from Gemini Bio-Products). An incubation step may be added after the combination of phDCs with at least one disease-related antigen. Alternatively or additionally, an incubation step may also be inserted after the generation of phDCs from monocytes. The incubation step may be carried out for 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 20 hours, or 24 hours. In another embodiment, the incubation step may be performed for at least 0.5 hours, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 20 hours, or at least 24 hours. It should be understood that all method steps are performed in vitro.

[0099] II.A Disease-causing particles At least one disease-related antigen or at least one antigenic protein may originate from disease-causing particles, including pathogens and tumor cells. Pathogens include viruses, bacteria, fungi, prions, and parasites. Thus, in one embodiment, disease-causing particles include viruses, bacteria, fungi, prions, parasites, and tumor cells. In the following sections relating to pathogens and tumors / cancer, the term “antigen” is used interchangeably with the term “antigenic protein.” For example, when an infectious disease antigen or tumor antigen is described, it is also referred to as an infectious disease-related antigenic protein or tumor-related antigenic protein. When a viral antigen is described, it is also referred to as a viral antigenic protein. When an HIV or SARS-CoV-2 antigen is described, it is also referred to as an HIV or SARS-CoV-2 antigenic protein.

[0100] II.A.1 PathogensIn some embodiments, the disease-associated antigen is an infectious disease-associated antigen. In some embodiments, the disease-associated antigen is a viral antigen. Examples of viruses from which the disease-associated antigen may originate include: Retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1 (also known as HTLV-III, LAV, or HTLV-III / LAV) or HIV-III; and other isolates, e.g., HIV-LP, HIV-2, etc.); Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., , dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., Hantan virus, Bunga virus, Phlebovirus, and Nairovirus); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus);This also includes unclassified viruses (e.g., the infectious agent of spongiform encephalopathy, the virulence factor of hepatitis delta (considered to be a defective satellite of hepatitis B virus), the virulence factors of non-hepatitis A and non-hepatitis B (enteral transmission; parenteral transmission (i.e., hepatitis C)); Norwalk and related viruses, and astroviruses).

[0101] Preferably, the disease-associated antigen is a viral antigen, more preferably a Retroviridae or Coronaviridae antigen, and most preferably an HIV or SARS-CoV-2 antigen. In a more preferred embodiment, the disease-associated antigen is derived from SARS-CoV-2.

[0102] In some embodiments, the disease-associated antigen is a bacterial antigen. Examples of bacteria from which disease-related antigens may originate include Helicobacter pyloris, Borrelia species (e.g., Borrelia burgdorferi), Legionella pneumophilia, Mycobacteria species (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (Viridance group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, and Bacillus. anthracis, Corynebacterium diphtheriae, Corynebacterium species, Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides species, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pertenue, Leptospira and Actinomyces israelii, Burkholderia pseudomallei, Burkholderia mallei, Pseudomonas aeruginosa.

[0103] Preferably, the bacterial antigen is a Borrelia species or Mycobacteria species antigen.

[0104] In some embodiments, the disease-associated antigen is a fungal antigen. Examples of fungi from which the disease-associated antigen may originate include Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

[0105] In some embodiments, the disease-associated antigen is a parasitic antigen. Parasitic antigens may originate from protozoa, parasitic helminths, or ectoparasites. Examples of parasites from which disease-associated antigens may originate include Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Leishmania species, Trypanosome species (Africa and America), Cryptosporidium, Isospora species, Naegleria fowleri, Acanthamoeba species, Balamuthia mandrillaris, Toxoplasma gondii, and Pneumocystis carinii.

[0106] II.A.2 Tumor / Cancer In some embodiments, disease-related antigens are tumor or cancer antigens, such as tumor-related antigens (TAAs) or tumor-specific antigens (TSAs). Unless otherwise described for the purposes of this disclosure, TAAs include the group of TSAs. Tumor or cancer antigens may be found on tumors or cancer cells. Tumor or cancer antigens may originate from solid tumors or hematological malignancies.

[0107] Examples of tumors or cancers from which disease-associated antigens may originate include leukemia, melanoma, lymphoma, endometrial cancer, kidney cancer, brain tumor, cervical cancer, liver cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, pancreatic cancer, ear, nose, and throat (ENT) cancer, breast cancer, prostate cancer, ovarian cancer, or lung cancer.

[0108] In certain embodiments, disease-related antigens are tumor-related antigens. Tumor-related antigens include Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B-cell maturation antigen (BCMA), ERK5, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-related antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, and glial fibrillary acidic protein. This includes (GFAP), overt cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein. In certain embodiments, tumor-associated antigens are CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostain, TARP (T cell receptor gamma surrogate leading frame protein), Trp-p8, or STEAP1 (six-transmembrane prostatic epithelial antigen 1). In certain embodiments, TAA is cancer / testicular (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE. In certain other embodiments, TAA is a carbohydrate or ganglioside, e.g., fuc-GMI, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, etc. In certain other embodiments, TAA is alpha-actinin-4, Bage-1,BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27, 29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gpi00(Pmel 17), tyrosinase, TRP-1, TRP 2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-i, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, 13-catenin, Mum-1, p16, TAGE, PSMA (prostate-specific membrane antigen), B-cell maturation antigen (BCMA), CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KPi, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, integrin cv3 (CD61), galactin, K Ras (V-Ki-ras2 Carsten rat sarcoma virus oncogene), or Ral-B.

[0109] Other tumor-associated antigens are known to those skilled in the art and may be present in the compositions disclosed herein, or may be combined with the phDCs provided herein to produce their respective antigen-specific phDCs.

[0110] II.B Means for providing disease-related antigens II.B.1 Antigens derived from disease-causing particles Disease-related antigens may originate from disease-causing particles, such as cells, biopsies, tissue lysates, or other particles containing disease-causing particles. In one embodiment, the disease-related antigen may originate from tumor or cancer cells being treated. For example, a sample of tumor cells may be taken from a subject with cancer, e.g., a biopsy, or a sample may be taken from a subject with an infectious disease. The sample may be further treated with an active agent suitable for releasing the antigen, such as a photoactivator combined with light (particularly UV light), such as a combination of 8-MOP and UVA. The treated sample may then be combined with phDC.

[0111] In another example, the pathogen may be cultured in a culture medium. The associated antigen may be released into the culture medium by the pathogen and then collected for combination with phDC. Those skilled in the art will be familiar with various and further methods known in the art for providing antigens derived from disease-causing particles.

[0112] II.B.2 Antigens expressed from nucleic acids A phDC can be supported with nucleic acids (including their functional fragments) encoding at least one disease-related antigen. In one embodiment of the present invention, a phDC is supported with RNA (including its functional fragments) encoding at least one disease-related antigen. In this embodiment, the at least one disease-related antigen corresponds to at least one antigen protein. Thus, the phDC is directly combined with RNA (e.g., mRNA). After entering the phDC, the RNA (e.g., mRNA) can be translated into the respective disease-related antigen (i.e., antigen protein). The RNA may be single-stranded RNA, mRNA, self-amplified RNA, circular RNA, and / or synthetic RNA. In a preferred embodiment, the RNA is mRNA. Optionally, the mRNA is self-amplified mRNA. The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3'UTR, a 5' cap, and a poly-A tail.

[0113] Therefore, in some embodiments, at least one disease-related antigen (i.e., an antigenic protein) is encoded by mRNA present in the composition together with the phDC (first embodiment). In other words, therapeutic compositions comprising a phDC and at least one mRNA encoding (including a coding sequence) at least one antigenic protein are provided herein.

[0114] If a polyvalent composition is desired, the phDC may be present in the composition with multiple mRNAs encoding one or more antigen proteins, such as two, three, four, five, six, seven, eight, nine, or ten types of antigen proteins derived from the same or different pathogens or tumors. Alternatively or additionally, polycistronic mRNAs that can be translated into one or more antigen proteins may be designed.

[0115] Code region In one embodiment, the mRNA encodes one or more antigenic proteins of disease-causing particles listed above, or the mRNA encodes one or more antigenic proteins of tumor or infectious disease-associated antigens listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of pathogens listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of viruses, bacteria, fungi, prions, or parasites listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of viruses, optionally coronaviruses, listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of disease-causing particles listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of tumors or cancers listed above. In one embodiment, the mRNA encodes one or more antigenic proteins of TAAs listed above. In one embodiment, at least one mRNA encodes one antigenic protein.

[0116] SARS-CoV-2 sequence In one embodiment, at least one mRNA encodes one or more antigen proteins derived from SARS-CoV, optionally SARS-CoV-2. Antigen proteins derived from SARS-CoV or SARS-CoV-2 include spike proteins, envelope proteins, nucleocapsid proteins, membrane proteins, and / or Orf1ab polyproteins. Thus, in one embodiment, the antigen protein is a fragment of a spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or any of the aforementioned. In one embodiment, the fragment contains at least 10, at least 50, at least 100, at least 200, at least 400, or at least 800 amino acid residues. Due to their surface expression characteristics, RNA polynucleotides encoding structural proteins are considered to have preferred immunogenic activity and are therefore most suitable for the compositions of the present invention. In preferred embodiments, the mRNA encodes one or more antigen proteins derived from the spike protein or nucleocapsid protein. More preferably, the mRNA encodes one or more antigen proteins derived from the spike protein. The mRNA may encode antigen proteins derived from the S1 subunit or S2 subunit of the spike protein. Variants of the aforementioned SARS-CoV-2 proteins are also explicitly included. For example, the first-generation spike protein variant is referred to as "S-2P" (Pallesen et al., 2017) and contains two proline substitutions at positions 986 and 987 (see, e.g., Polack et al., 2020; Bos et al., 2020; Corbett et al., 2020; Wrapp et al., 2020). The second-generation spike construct, referred to as "HexaPro," contains four additional proline substitutions at positions 817, 892, 899, and 942. HexaPro is expressed at higher levels than wild-type spike protein or S-2P and exhibits improved stability compared to S-2P under low-temperature storage and multiple freeze-thaw cycles (Edwards et al., 2020).

[0117] In some embodiments, the mRNA includes a sequence or a portion of a sequence selected from the group including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5.

[0118] In some embodiments, the mRNA includes a sequence or a portion of a sequence selected from the group including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the selected sequence. In one embodiment, the mRNA includes a sequence or a portion of a sequence selected from the group including SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the selected sequence, and the selected RNA sequence is modified with one or more (e.g., all) naturally occurring modified nucleosides as described in the section “Modification of RNA”. In one embodiment, the naturally occurring modified nucleoside is 1-methylpseudridine.

[0119] The sequence shown above is based on the reference genome from NCBI (accession number NC_045512.2) and is also presented in Table 1 below. The corresponding amino acid sequence is given in Table 2 below.

[0120] In some embodiments, the mRNA includes a sequence or a portion of a sequence corresponding to SEQ ID NO: 19, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO: 19 (Table 3).

[0121] Those skilled in the art will further understand, upon reading this disclosure, that it describes various mRNA constructs containing sequences encoding the full-length SARS-CoV-2 spike protein (including, for example, embodiments in which the thus encoded SARS-CoV-2 spike protein may contain at least one or more amino acid substitutions, such as the proline substitutions described herein, and / or embodiments in which the mRNA sequence is optimized for, for example, mammalian, for example, human subjects, and / or embodiments in which the mRNA contains one or more chemical modifications).

[0122] HIV sequence In one embodiment, the mRNA encodes one or more antigen proteins derived from HIV. Antigen proteins derived from HIV include envelope proteins (env), group antigen polyproteins (gag), reverse transcriptases (pol), and / or negative factor proteins (nef). Due to their surface expression characteristics, RNA polynucleotides encoding structural proteins are considered to have preferred immunogenic activity and are therefore most suitable for the compositions of the present invention. Variants of the aforementioned HIV proteins are also explicitly included.

[0123] In some embodiments, the mRNA includes a sequence or a portion of a sequence selected from the group including SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and / or SEQ ID NO: 14.

[0124] In some embodiments, the mRNA includes a sequence or a portion of a sequence selected from the group including SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and / or SEQ ID NO: 14, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the selected sequence.

[0125] The sequence shown above is based on the reference genome from NCBI (accession number NC_AF033819, HIV-1) and is also presented in Table 1 below. The corresponding amino acid sequence is given in Table 2 below.

[0126] Those skilled in the art will further understand, upon reading this disclosure, that it describes a variety of mRNA constructs containing sequences encoding the full-length HIV structural protein (including, for example, embodiments in which the thus encoded HIV structural protein may contain at least one or more amino acid substitutions, and / or embodiments in which the mRNA sequence is optimized for, for example, mammalian, e.g., human subjects, and / or embodiments in which the mRNA contains one or more chemical modifications).

[0127] [Table 1-1]

[0128] [Table 1-2]

[0129] [Table 1-3]

[0130] [Table 1-4]

[0131] [Table 1-5]

[0132] [Table 1-6]

[0133] [Table 1-7]

[0134] [Table 1-8]

[0135] Table 1-9

[0136] Table 1-10

[0137] Table 1-11

[0138] Table 1-12

[0139] Table 1-13

[0140] Table 2-1

[0141] Table 2-2

[0142] Table 2-3

[0143] Table 2-4

[0144] Table 3-1

[0145] [Table 3-2]

[0146] Tumor-associated antigen sequences and tumor-specific antigen sequences In one embodiment, at least one mRNA encodes one or more antigen proteins derived from a tumor or cancer. In one embodiment, the mRNA encodes the amino acid sequence of a TAA polypeptide, for example, TAA. TAA is an amino acid sequence of Her2, prostate stem cell antigen (PSCA), PSMA (prostate-specific membrane antigen), B cell maturation antigen (BCMA), ERK5, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (G This includes FAP), overt cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein. In certain embodiments, TAA is CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostain, TARP (T cell receptor gamma surrogate leading frame protein), Trp-p8, or STEAP1 (six-transmembrane prostatic epithelial antigen 1). In certain embodiments, TAA is cancer / testicular (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE. In certain other embodiments, TAA or TSA is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein,Beta-catenin, CA 125, CA 15-3 (CA 27, 29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gpi00(Pmel 17), tyrosinase, TRP-1, TRP 2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-i, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, 13-catenin, Mum-1, p16, TAGE, PSMA (prostate-specific membrane antigen), B-cell maturation antigen (BCMA), CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KPi, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, integrin cv3 (CD61), galactin, K Ras (V-Ki-ras2 Carsten rat sarcoma virus oncogene), or Ral-B.

[0147] The mRNA encoding the amino acid sequence of TAA may be a sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the RNA sequence of the selected TAA.

[0148] RNA modification In one embodiment, the RNA (e.g., mRNA) is modified RNA, particularly stabilized mRNA. In some embodiments, the RNA (e.g., mRNA) may be modified for maximum potency, for example, to improve intracellular lifespan, transcriptional potency, non-immunogenic properties (e.g., no Toll receptor induction), and / or structural mRNA stability.

[0149] In one embodiment, RNA (e.g., mRNA) independently contains at least one chemical modification. The chemical modification may be, for example, a modified nucleoside. In one embodiment, the chemical modification includes a naturally occurring modified nucleoside. A naturally occurring nucleoside is 1-methyladenosine (m 1 A), N 6 -Methyladenosine (m 6 A) 2'-O-methyladenosine (Am), 5-methylcytidine (m 5 C), 2'-O-methylcytidine (Cm), 2-thiocytidine (s 2 C), N 4 -acetylcytidine (ac 4 C), 5-formylcytidine (f 5 C), 2'-O-methylguanosine (Gm), inosine (I), pseudouridine (Ψ), 5-methyluridine (m 5The modifiers include U), 2'-O-methyluridine (Um), 1-methylpseudridine (m1Ψ), 2-thiouridine (s2U), 4-thiouridine (s4U), 5-methoxyuridine (mo5U), and 3-methyluridine (m3U). In one embodiment, RNA (e.g., mRNA) contains a modified nucleoside in place of at least one uridine. In one embodiment, RNA contains a modified nucleoside in place of each uridine. In one embodiment, the modified nucleoside is independently selected from pseudouridine, N1-methylpseudridine, 5-methyluridine, and N1-ethylpseudridine. In one embodiment, the modified nucleoside is N1-methylpseudridine modification or N1-ethylpseudridine modification. In one embodiment, the modified nucleoside is N1-methylpseudridine. For example, N1-methyl-pseudouridine has been found to be superior in terms of translational ability to several other nucleoside modifications and their combinations. In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine nucleoside in the mRNA is chemically modified.

[0150] Alternatively or in addition to chemical modifications, RNA sequences, particularly mRNA sequences, can be optimized. RNA sequence optimization includes, among other things, codon optimization, G / C content optimization, and optimization of structural elements (e.g., 5' cap, 5' UTR, 3' UTR, and poly(A)-tail).

[0151] In some embodiments, the amino acid sequence of at least one disease-associated antigen (or antigen protein) is encoded by a coding sequence that is codon-optimized and / or has an increased G / C content compared to the 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 regions of the wild-type coding sequence. In one embodiment, the codon optimization and / or increased G / C content preferably do not alter the sequence of the encoded amino acid sequence. In some embodiments, the G / C content of the coding region of RNA is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or more compared to the G / C content of the coding region of wild-type RNA.

[0152] In some embodiments, RNA (e.g., mRNA) may contain one or more optimized structural elements. These structural elements include a 5' cap, 5' UTR, 3' UTR, and a poly(A) tail. Thus, in one embodiment, RNA includes a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and / or a poly(A) tail. In one embodiment, RNA (e.g., mRNA) contains all of these elements. In some embodiments, RNA (e.g., mRNA) includes a 5'-UTR and / or a 3'-UTR. In one embodiment, RNA (e.g., mRNA) includes a cap. In some embodiments, RNA (e.g., mRNA) includes a 3'-poly(A) sequence. In one embodiment, the cap is a cap1 structure or an m7GpppG cap. In one embodiment, the sequences of the 5' UTR and / or 3' UTR are optimized. In some embodiments, the mRNA includes a 5' or 3' UTR derived from a gene different from the sequence encoding at least one disease-associated antigen (or antigen protein), i.e., the UTR is heterologous. In some embodiments, the 5' and / or 3' UTR sequences may be derived from stable mRNA (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzyme) to increase mRNA stability. For example, a 5'-UTR sequence of human alpha-globin mRNA may be used, optionally having an optimized "Kozak sequence" to increase translation efficiency. Alternatively, a 5'-UTR sequence of human cytochrome mRNA, e.g., human cytochrome b-245 alpha mRNA or cytochrome p4502E1 mRNA, may be used. As the 3'-UTR sequence, a combination of two sequence elements (FI elements) can be used, derived from a "split amino-terminal enhancer" (AES) mRNA (referred to as F) positioned between the coding sequence and the poly(A)-tail, and mitochondrial-encoded 12S ribosomal RNA (referred to as I). Alternatively, the 3'-UTR can be two repeating 3'-UTRs of human beta-globin mRNA.

[0153] In one embodiment, the poly(A) sequence contains at least 100 nucleotides. In one embodiment, the poly(A) sequence contains at least 150 nucleotides. In one embodiment, the poly(A) sequence contains at least 250 nucleotides. Furthermore, a 110-nucleotide poly(A)-tail may be used, consisting of a sequence of 30 adenosine residues followed by a 10-nucleotide linker sequence (of random nucleotides) and another 70 adenosine residues. This poly(A)-tail sequence enhances RNA stability and translation efficiency. In one embodiment, the poly(A)-tail is 300 to 800 nucleotides long.

[0154] Furthermore, the secretory signal peptide (sec) may be fused to the antigen-coding region, preferably so that the sec is translated as an N-terminal tag. A sequence encoding a short linker peptide, mainly consisting of the amino acids glycine (G) and serine (S), commonly used in fusion proteins, may be used as a GS-linker (glycine-serine linker). In other embodiments, RNA (e.g., mRNA) may have one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES, are destabilizing sequences found in the 3'UTR. AURES may be removed from RNA (e.g., mRNA). Alternatively, AURES may remain in RNA (e.g., mRNA).

[0155] Lipid nanoparticles At least one disease-related antigen (e.g., at least one mRNA encoding an antigen protein) can be encapsulated within lipid nanoparticles (LNPs). For example, if the at least one disease-related antigen is an antigen protein encoded by RNA (e.g., mRNA), the RNA can be encapsulated within lipid nanoparticles for delivery to the phDC of the present invention. The encapsulated RNA (e.g., mRNA) is combined with the phDC.

[0156] LNPs may contain three, four, or five classes of lipids. LNPs with three classes of lipids include (1) ionizable lipids, (2) PEGylated lipids, and (3) cholesterol-based lipids. LNPs with four classes of lipids include (1) ionizable lipids, (2) PEGylated lipids, (3) cholesterol-based lipids, and (4) helper lipids. LNPs with five classes of lipids include (1) ionizable lipids, (2) PEGylated lipids, (3) cholesterol-based lipids, (4) helper lipids, and (5) DSPE-PEG-maleimide or DSPN-PEG-azide. DSPE-PEG-maleimide or DSPN-PEG-azide allows for the attachment of ligands for targeted delivery.

[0157] (1) Ionizable lipids Ionizable lipids facilitate mRNA encapsulation and may be cationic lipids. Cationic lipids provide a positively charged environment at low pH, facilitating the efficient encapsulation of negatively charged mRNA. In one embodiment, the cationic lipid is cKK-E12((3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione); see Dong et al., PNAS (2014) 111(11):3955~60). Other cationic lipids that may be used include those described in Dong et al., 2014.

[0158] (2)PEG-modified lipid PEG-modified lipid components provide control over the particle size and stability of nanoparticles. The addition of such components may prevent complex aggregation, increase survival time, and provide a means to increase LNP delivery to phDCs.

[0159] The intended PEGylated lipids include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length, covalently attached to lipids having alkyl chains of C6-C20 length (e.g., C8, C10, C12, C14, C16, or C18), such as derivatized ceramides (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, the PEGylated lipids are 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearyl-rac-glycero-polyethylene glycol (DSG-PEG). In particular exemplary embodiments, PEG has a high molecular weight, for example, 2000-2400 g / mol. In some embodiments, PEG is PEG2000, also known as PEG-2K. In some embodiments, the PEG-modified lipid is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000.

[0160] (3) Cholesterol-based lipids Cholesterol components provide stability to the lipid bilayer structure within the nanoparticles. In some embodiments, LNPs contain one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, N,N-dimethyl-N-ethylcarboxamide-cholesterol, 1,4-bis(3-N-oleylaminopropyl)piperazine, imidazole cholesterol esters, β-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in LNPs is cholesterol.

[0161] (4) Helper lipids Helper lipids enhance the structural stability of LNPs and assist LNPs in endosomal escape. They improve the uptake and release of RNA (e.g., mRNA) carriers. The cross-presentation ability of DCs can be limited by nonspecific degradation during endosomal maturation. Therefore, LNPs that provide better endosomal escape may be useful in some embodiments (e.g., when the antigen is in the form of, for example, a protein, peptide, or mRNA). In one embodiment, the helper lipid is a noncationic lipid. In some embodiments, the helper lipid is an amphoteric lipid with fusion properties to enhance carrier uptake and release. Examples of helper lipids include 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); 1,2-dieridoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-distearoylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).

[0162] In one embodiment, the lipid nanoparticles include cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE. In some embodiments, various antigens can be loaded onto phDCs to produce multivalent antigen-specific phDCs. For example, if the antigen protein is encoded by mRNA and encapsulated within the LNP, the LNP may carry mRNA encoding one or more antigen proteins, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more types of antigen proteins from the same or different pathogens or from the same or different tumors. For example, the LNP may carry multiple mRNA molecules, each encoding a different antigen protein. The LNP may also carry polycistronic mRNA that can be translated into one or more antigen proteins. When the LNP carries various mRNA molecules, typically there will be multiple copies of each mRNA molecule.

[0163] Molar ratio of lipid components The specific molar ratios of the above components may be important for the effectiveness of the LNP. The molar ratio of cationic lipids, PEGylated lipids, cholesterol-based lipids, and helper lipids is A:B:C:D, where A+B+C+D=100%. In some embodiments, the molar ratio of cationic lipids in the LNP to total lipids (i.e., A) is 35-50%, optionally 35-45%. In some embodiments, the molar ratio of PEGylated lipid components to total lipids (i.e., B) is 0.25-2.75%, optionally about 1.5%. In some embodiments, the molar ratio of cholesterol-based lipids to total lipids (i.e., C) is 20-46.5%. In some embodiments, the molar ratio of cholesterol-based lipids to total lipids (i.e., C) is about 46.5%. In another embodiment, the molar ratio of cholesterol-based lipids to total lipids (i.e., C) is about 38.5%. In some embodiments, the molar ratio of helper lipids to total lipids (i.e., D) is approximately 10–35%, for example 10–25% or 16–35%, etc. (e.g., 16% or 16–32%). In some embodiments, the molar ratio of helper lipids to total lipids (i.e., D) is approximately 10%. In another embodiment, the molar ratio of helper lipids to total lipids (i.e., D) is approximately 16%. In one embodiment, the ratio of components is 35:2.5:46:16 (A:B:C:D). In another embodiment, the ratio of components is 50:38.5:1.5:10 (A:B:C:D).

[0164] In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE. In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16.

[0165] In some embodiments, the lipid nanoparticles include SM102, cholesterol, DMG-PEG-2K, and DSPC. In one embodiment, the lipid nanoparticles include SM102, cholesterol, DMG-PEG-2K, and DSPC in a ratio of 50:38.5:1.5:10.

[0166] In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the helper lipid. In some embodiments, the LNP contains a molar ratio of cationic lipid to helper lipid greater than 1. To calculate the actual amount of each lipid to be incorporated into the LNP formulation, first, the molar amount of cationic lipid is determined based on the desired N / P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the RNA (e.g., mRNA) to be transported by the LNP. Next, based on the molar amount of cationic lipid and the selected molar ratio, the respective molar amounts of the other lipids are calculated. Then, these molar amounts are converted to weight using the molecular weight of each lipid.

[0167] Lipid nanoparticles containing an antigen (e.g., RNA encoding at least one antigen protein, particularly in mRNA form) may be supplied frozen. This may be useful, for example, if the lipid nanoparticles containing phDC and the antigen (e.g., RNA encoding at least one antigen protein, particularly in mRNA form) are supplied as a kit.

[0168] Size and quantity of lipid nanoparticles Suitable LNPs can be fabricated in a variety of sizes. In some embodiments, the majority of the purified LNPs, i.e., over 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the LNPs, have a size of approximately 50–200 nm (e.g., approximately 145 nm, 140 nm, 135 nm, 130 nm, 125 nm, 120 nm, 115 nm, 110 nm, 105 nm, 100 nm, 95 nm, 90 nm, 85 nm, or 80 nm). In some embodiments, substantially all (e.g., more than 80 or 90%) of the purified lipid nanoparticles have a size of about 70–200 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, more than about 70%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% of the LNPs in the composition have a size ranging from about 85–100 nm (e.g., about 45–85 nm, about 50–80 nm, about 55–75 nm, about 60–70 nm) or about 50–70 nm (e.g., 55–65 nm). Smaller sizes are particularly suitable for inhalation delivery by spraying. In some embodiments, phDC is combined with at least about 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 10 μg, or 20 μg of encapsulated RNA, particularly mRNA. In some embodiments, phDC is combined with about 10 μg of encapsulated RNA, particularly mRNA. In some embodiments, phDC is combined with about 20 μg of encapsulated RNA, particularly mRNA.

[0169] Lipoplex At least one disease-related antigen (e.g., at least one mRNA encoding at least one antigen protein) may be included in the lipoplex. Mixing RNA (e.g., mRNA) with positively charged liposomes results in the formation of lipoplex particles by spontaneous self-assembly. Liposomes typically contain at least two components: cationic lipids and neutral lipids. Lipoplexes are widely described in the art; see, for example, Nanomedicine: Nanotechnology, Biology and Medicine, 2009. In one embodiment, the neutral lipid is a helper lipid as defined above. In an exemplary embodiment, the cationic lipid is DOTMA and the neutral lipid is DOPE.

[0170] In some embodiments, the molar ratio of at least one cationic lipid to at least one neutral lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In exemplary embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1. The RNA lipoplex particles described herein have, in one embodiment, an average diameter ranging from about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In specific embodiments, RNA lipoplex particles have an average diameter of approximately 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, or 700 nm. In one embodiment, RNA lipoplex particles have an average diameter ranging from approximately 250 nm to approximately 700 nm. In another embodiment, RNA lipoplex particles have an average diameter ranging from approximately 300 nm to approximately 500 nm. In an exemplary embodiment, RNA lipoplex particles have an average diameter of approximately 400 nm.

[0171] In one embodiment, the (mRNA) lipoplex particles include at least one cationic lipid and at least one neutral lipid. In one embodiment, the at least one cationic lipid includes 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, the at least one neutral lipid includes 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and at least one neutral lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). RNA lipoplex particles targeting the spleen are described in WO2013 / 143683, which is incorporated herein by reference. It has been found that lipoplex particles having a net negative charge can preferentially target splenic tissue or splenic cells, particularly antigen-presenting cells, such as dendritic cells.

[0172] Oligomers, polymers, or lipidoids containing an oligo(alkyleneamine) moiety. At least one antigen (e.g., at least one mRNA encoding at least one antigen) may be contained in oligomers, polymers, or lipidoids containing an oligo(alkyleneamine) moiety, e.g., a characteristic oligo(alkyleneamine) moiety described in PCT / EP2014 / 063756. In particular, at least one antigen (e.g., at least one mRNA encoding at least one antigen) may be contained in oligomers, polymers, or lipidoids described in PCT / EP2014 / 063756. One key feature of oligomers, polymers, or lipidoids containing an oligo(alkyleneamine) moiety is that they contain the following common structural entity of formula (I):

[0173] [ka]

[0174] Such oligomers, polymers, or lipidoids containing an oligo(alkyleneamine) moiety may be selected from the following: a) Oligomers or polymers containing multiple groups of formula (II) as side chains and / or terminal groups:

[0175] [ka]

[0176] In the formula, the variables a, b, p, m, n, and R 2 ~R 6 For multiple such groups, the following is defined independently for each group of formula (II): a is 1 and b is an integer between 2 and 4; or a is an integer between 2 and 4 and b is 1. p is either 1 or 2. m is either 1 or 2; n is either 0 or 1, and m+n is greater than or equal to 2; R 2 ~R 5 These are, independently of each other, hydrogen; group -CH2-CH(OH)-R 7 ,-CH(R7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 -CH2-CH2-(C=O)-NH-R 7 , or -CH2-R 7 (In the formula, R 7 (Selected from C3-C18 alkyl or C3-C18 alkenyl groups having one CC double bond); protecting group for amino group; -C(NH)-NH2; selected from poly(ethylene glycol) chains; R 6 is hydrogen; group -CH2-CH(OH)-R 7 ,-CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 -CH2-CH2-(C=O)-NH-R 7 , or -CH2-R 7 (In the formula, R 7 The group is selected from a C3-C18 alkyl or C3-C18 alkenyl having one CC double bond; a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and an acceptor ligand. One or more nitrogen atoms in formula (II) may be protonated to provide the cationic group of formula (II); b) Oligomers or polymers containing multiple groups of formula (III) as repeating units:

[0177] [ka]

[0178] In the formula, the variables a, b, p, m, n, and R 2 ~R 5 In multiple such groups, the following is defined independently for each group of formula (III): a is 1 and b is an integer between 2 and 4; or a is an integer between 2 and 4 and b is 1. p is either 1 or 2. m is either 1 or 2; n is either 0 or 1, and m+n is greater than or equal to 2; R 2~R 5 These are, independently of each other, hydrogen; group -CH2-CH(OH)-R 7 ,-CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 -CH2-CH2-(C=O)-NH-R 7 , or -CH2-R 7 (In the formula, R 7 The group is selected from a C3-C18 alkyl or C3-C18 alkenyl having one CC double bond; a protecting group for the amino group; -C(NH)-NH2; and a poly(ethylene glycol) chain; One or more nitrogen atoms in formula (III) may be protonated to provide the cationic group of formula (III); and c) Lipidoids having the structure of formula (IV):

[0179] [ka]

[0180] In the formula, the variables a, b, p, m, n, and R 2 ~R 6 It is defined as follows: a is 1 and b is an integer between 2 and 4; or a is an integer between 2 and 4 and b is 1. p is either 1 or 2. m is either 1 or 2; n is either 0 or 1, and m+n is greater than or equal to 2; R 1 ~R 6 These are, independently of each other, hydrogen; group -CH2-CH(OH)-R 7 ,-CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 -CH2-CH2-(C=O)-NH-R 7 , or -CH2-R 7 (In the formula, R 7The group is selected from a C3-C18 alkyl or C3-C18 alkenyl having one CC double bond; a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain; and an acceptor ligand. However, R 1 ~R 6 At least two of these residues are the -CH2-CH(OH)-R group. 7 ,-CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 -CH2-CH2-(C=O)-NH-R 7 , or -CH2-R 7 And in the formula, R 7 It is selected from C3-C18 alkyl or C3-C18 alkenyl groups having one CC double bond; One or more nitrogen atoms in formula (IV) can be protonated to provide the cationic lipidoid of formula (IV).

[0181] Preferably, the oligomer, polymer, or lipidoid containing the oligo(alkyleneamine) moiety is selected from a) and b), a) is an oligomer or polymer containing multiple groups of formula (IIa) as side chains and / or terminal groups:

[0182] [ka]

[0183] In the formula, a, b, m, n, and R 2 ~R 6 It is defined as described above, One or more nitrogen atoms in formula (IIa) may be protonated to provide a cationic oligomer or polymer structure; b) is an oligomer or polymer containing multiple groups of formula (IIIa) as repeating units:

[0184] [ka]

[0185] wherein, a, b, m, n, and R 2 ~R 5 are defined as described above, one or more of the nitrogen atoms shown in formula (IIIa) may be protonated to provide a cationic oligomer or polymer structure.

[0186] Furthermore, the lipidoid containing an oligo(alkyleneamine) moiety may be selected from lipidoids having the structure of formula (IVa):

[0187]

Chemical formula

[0188] wherein, a, b, m, n, and R 1 ~R 6 are defined as described above, one or more of the nitrogen atoms shown in formula (IVa) may be protonated to provide a cationic lipidoid.

[0189] For such oligomers, polymers, or lipidoids containing an oligo(alkyleneamine) moiety, in formula (II), (IIa), (III), (IIIa), (IV), or (IVa), n may be 1; or m may be 1, and n may be 1.

[0190] Furthermore, for such oligomers, polymers, or lipidoids containing an oligo(alkyleneamine) moiety, in formula (II), (IIa), (III), (IIIa), (IV), or (IVa), a may be 1, and b may be 2; or a may be 2, and b may be 1.

[0191] One non-limiting example of such an oligomer, polymer, or lipidoid containing an oligo(alkyleneamine) moiety is a cationic lipid prepared by mixing 100 mg of N,N'-bis(2-aminoethyl)-1,3-propanediamine (0.623 mmol) with 575.07 mg of 1,2-epoxydodecane (3.12 mmol, (N-1) equivalent, where N is 2 × amounts of primary amine + 1 × amount of secondary amine per oligo(alkyleneamine)) and mixed at 80°C for 96 hours under constant shaking. Such an oligomer, polymer, or lipidoid is also referred to as the lipidoid "C12-(2-3-2)". Oligomers, polymers, or lipidoids, especially polymers, containing an oligo(alkyleneamine) moiety may be copolymers, particularly statistical copolymers. Such copolymers may contain a statistical / random arrangement of alkyleneamine repeat units of alternating lengths (in contrast to less desirable copolymers containing, for example, similar arrangements of alkyleneamine repeat units of non-alternating lengths). The copolymer may be a cationic (e.g., protonated) copolymer. The copolymers to be employed are known in the art and are described, for example, in EP14 19 9439.2, WO01 / 00708, EP-A1 1 198 489, and CA-A1 2,377,207.

[0192] In particular, the copolymer is a plurality of repeating units (a) independently selected from the repeating units of the following formulas (a1) and (a2):

[0193] [ka]

[0194] Furthermore, (b) a plurality of iteration units (b) selected independently of the iteration units of the following equations (b1) to (b4):

[0195] [ka]

[0196] The copolymer may be a statistical copolymer containing the total repeating unit (a) to the total repeating unit (b), with the molar ratio of the total repeating unit (a) being in the range of 0.7 / 1.0 to 1.0 / 0.7, and one or more nitrogen atoms of the repeating units (a) and / or (b) contained in the copolymer may be protonated to provide a cationic copolymer.

[0197] A copolymer can be a statistical copolymer in which arbitrary repeating units (a) and arbitrary repeating units (b) are statistically distributed within the copolymer polymer. It is typically obtained by copolymerization of a mixture of monomers that produce repeating units (a) during polymerization and monomers that produce repeating units (b) during polymerization. Preferably, the copolymer is a random copolymer in which arbitrary repeating units (a) and arbitrary repeating units (b) are randomly distributed within the polymer polymer. Such copolymers can be linear, branched, or dendritic copolymers. As will be understood by readers skilled in the art, repeating units of formula (a1), (b1), or (b3) having two valencies (i.e., open bonds to adjacent units) lead to the propagation of the copolymer structure in a linear manner. Therefore, linear copolymers may contain repeating units of formula (a1), as well as one or more types of repeating units of formulas (b1) and (b3), but may not contain repeating units of formula (a2), (b2), or (b4). As will be further understood, the presence of repeating units of formula (a2), (b2), or (b4) having three valencies provides branching points in the copolymer structure. Thus, branched copolymers may contain one or more types of repeating units of formulas (a2), (b2), and (b4), and may further contain one or more types of repeating units of formulas (a1), (b1), and (b3). Such copolymers may contain a plurality of repeating units (a) independently selected from the repeating units of formulas (a1) and (a2) defined above, as well as a plurality of repeating units (b) independently selected from the repeating units of formulas (b1) to (b4) defined above. A copolymer comprising a plurality of repeating units (a) independently selected from the repeating units of formulas (a1) and (a2) defined above, and a plurality of repeating units (b) independently selected from the repeating units of formulas (b1) and (b2) defined above, is preferred.Preferably, such copolymers are branched copolymers comprising one or more types of repeating units selected from repeating units (a2), (b2), and (b4), and optionally further comprising one or more types of repeating units of formulas (a1), (b1), and (b3), in particular a copolymer comprising one or more types of repeating units of formula (a2), as well as formulas (b2) and (b4), and optionally further comprising one or more types of repeating units of formulas (a1), (b1), and (b3). Accordingly, a more preferred copolymer is a branched copolymer comprising repeating units of formula (a2) and repeating units of formula (b2), and optionally further comprising one or more types of repeating units of formulas (a1) and (b1). In the copolymer, the total number of repeating units (a) and repeating units (b) is typically 20 or more, preferably 50 or more, and more preferably 100 or more. Typically, the total number of repeating units (a) and (b) is 10,000 or less, preferably 5,000 or less, and more preferably 1,000 or less. Furthermore, it is preferable for the copolymer that repeating units (a) and (b) account for 80 mol% or more, more preferably 90 mol% or more of the total repeating units in the copolymer. A copolymer in which repeating units (a) selected from (a1) and (a2), and repeating units (b) selected from (b1) and (b2) account for 80 mol% or more, more preferably 90 mol% or more of the total repeating units in the copolymer is even more preferable. It is most preferable that all of the repeating units in the copolymer are repeating units (a) or (b), and in particular all of the repeating units in the copolymer are repeating units (a) selected from (a1) and (a2), or repeating units (b) selected from (b1) and (b2). The weight-average molecular weight of copolymers measured against a linear poly(ethylene oxide) standard, for example by size exclusion chromatography, generally ranges from 1,000 to 500,000 Da, preferably 2,500 to 250,000 Da, and more preferably 5,000 to less than 50,000.The terminal groups of such copolymers typically include one or more types of group (c) independently selected from the groups of formulas (c1) to (c3) below, preferably from the groups of formulas (c1) and (c2) below:

[0198] [ka]

[0199] Preferably, the end groups in the copolymer consist of one or more types of group (c) independently selected from the groups of formulas (c1) to (c3) below, preferably from the groups of formulas (c1) and (c2). As will be understood by those skilled in the art, the number of end groups depends on the structure of the copolymer. Linear copolymers have only two ends, while branched, and especially dendritic copolymers, contain a greater number of end groups. As will be further understood, one or more nitrogen atoms of the end groups (c) contained in the copolymer can also be protonated to provide a cationic copolymer. In the copolymer, the molar ratio of the total repeating units (a) to the total repeating units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, preferably in the range of 0.8 / 1.0 to 1.0 / 0.8. This molar ratio can be determined, for example, by NMR. Therefore, the ratio is usually determined for multiple polymers of the copolymer and will be understood to typically represent the overall ratio of the total repeating units (a) to the total repeating units (b) in the multiple polymers. As shown above, one or more nitrogen atoms of the copolymer may be protonated to result in a copolymer in cationic form, typically oligocation or polycation form. Primary, secondary, or tertiary amino groups in repeating units (a) or (b) or in terminal groups (c) may act as proton acceptors in aqueous solutions, particularly water and physiological fluids. Therefore, such copolymers typically have an overall positive charge in aqueous solutions with a pH below 7.5. As referred to herein, an aqueous solution is a solution in which the solvent contains 50% (vol. / vol.) or more, preferably 80 or 90% or more, most preferably 100% water. Furthermore, when the composition comes into contact with physiological fluids having a pH below 7.5, such as blood and lung fluid, they typically contain repeating units (a) and (b) in which the nitrogen atom is protonated. The pK value of the copolymer used in the composition can be determined by acid-base titration using an automated pK titrator. The net charge at a given pH value can then be calculated, for example, from the Henderson-Hasselbalch formula.Any charge can be shared across some of the basic centers and does not necessarily belong to a single point. Typically, in a solution at physiological pH, the copolymer used in the composition contains repeating units with protonated amino groups and repeating units with non-protonated amino groups. However, as will be understood by the skilled reader, the copolymer can also be provided in a dry salt form containing the cationic form of the copolymer. Further, as will be understood, the counterions (anions) to the positive charge of the protonated amino groups in the composition containing the copolymer and nucleic acid, especially mRNA, are typically provided by the anionic moieties contained in the nucleic acid. When the positively charged groups are present in excess compared to the anionic moieties in the nucleic acid, the positive charge is balanced by other anions, especially Cl or HCO3 - etc., which are typically encountered in physiological fluids. Along the above lines, a preferred copolymer is 80 mol% or more of all repeating units, more preferably all repeating units are a plurality of repeating units (a) independently selected from the repeating units of the following formulas (a1) and (a2)

[0200]

Chemical formula

[0201] as well as a plurality of repeating units (b) independently selected from the repeating units of the following formulas (b1) and (b2):

[0202]

Chemical formula

[0203] is a random copolymer formed by, and the molar ratio of the total of the repeating units (a) to the total of the repeating units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, more preferably in the range of 0.8 / 1.0 to 1.0 / 0.8; the end groups of the copolymer are formed by a group (c) independently selected from the groups of the formulas (c1) and (c2): One or more nitrogen atoms of repeating units (a) and / or (b) contained in the copolymer, and / or of terminal groups (c), may be protonated to provide a cationic copolymer. The copolymer is more preferably a branched copolymer containing units (a2) and (b2) together with units (a1) and / or (b1) as an option. The preparation of the copolymer is specified in EP4 223 306 A2, which is incorporated herein by reference as a whole. In principle, lipidoids, in particular compared to oligomers, and more particularly compared to polymers, are preferred nanoparticles to be employed.

[0204] At least one disease-associated antigen may be an antigenic protein encoded by RNA (e.g., modified or unmodified mRNA), the RNA may be encapsulated within lipid nanoparticles (LNPs) for delivery to phDCs according to the present invention. The encapsulated RNA (e.g., mRNA) is combined with the phDC. The uptake of RNA (e.g., mRNA, optionally modified mRNA) by the phDC may be monitored or assessed by means known in the art. These means include, but are not limited to, cascade reactions or detection of expressed antigenic proteins encoded by fluorescent LNPs, luminescent LNPs, radioactive LNPs, or RNA encapsulated within LNPs. In one embodiment, the transfection efficacy of a phDC, e.g., human phDC, is assessed by co-incubating a phDC generated from an isolated PBMC with mRNA encoding GPI-anchored nanoluciferase (NLuc) (LNP[NLuc]) and observing the luminescence signal. In some embodiments, the spatial-temporal distribution of the LNP is monitored.

[0205] III. Pharmaceutical Compositions or Therapeutic Compositions The present invention also provides a pharmaceutical composition or therapeutic composition comprising a phDC and at least one RNA (e.g., mRNA) containing a coding sequence encoding at least one antigen protein. The terms pharmaceutical composition and therapeutic composition are used synonymously herein. The at least one disease-related antigen may be in any form described under the First Embodiment and in the Detailed Description. In one embodiment, the pharmaceutical composition of the present invention may contain at least 1, at least 10, at least 100, at least 1000, or at least 1 × 10 4 each, at least 1 × 10 5 , or at least 1 × 10 6 It contains phDCs. The number of phDCs can be estimated, for example, from the volume of apheresed whole blood passed through a flow chamber (e.g., plate, bag, or hybrid) or directly through a flow chamber (e.g., plate, bag, or hybrid). At least one mRNA may be provided as RNA encapsulated in nanoparticles (e.g., LNPs), particularly mRNA, and the pharmaceutical composition contains at least about 0.1 μg, 0.5 μg, 5 μg, 10 μg, 100 μg, 500 μg, or 1000 μg of encapsulated RNA, particularly mRNA. The therapeutic composition provides a pharmaceutically effective amount of antigen-specific phDCs. In some embodiments, the therapeutic composition according to the present invention contains at least 1, at least 10, at least 100, at least 1000, or at least 1 × 10 4 each, at least 1 × 10 5 pieces, or at least 1 × 10 6It contains antigen-specific phDCs. From a dose-saving perspective, the dose of nanoparticles containing RNA, particularly mRNA (e.g., LNPs), can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the dose of nanoparticles (e.g., LNPs) used in other RNA therapeutics that do not contain phDCs. Typical doses for mRNA therapeutics can be 0.1 to 100 μg, e.g., 1 μg, 5 μg, 10 μg, 30 μg, or 50 μg. In one particular embodiment, the phDC of the present invention is combined with up to 4.5 μg of nanoparticles (e.g., LNPs) containing RNA, particularly mRNA.

[0206] The pharmaceutical or therapeutic compositions of the present invention optionally comprise pharmaceutically acceptable carriers and / or diluents. Additionally, the pharmaceutical or therapeutic compositions may comprise adjuvants and / or immunomodulators to enhance the activity of the pharmaceutical or therapeutic composition and the response of the target. Such adjuvants and / or immunomodulators are well understood by those skilled in the art and readily described in available publications. The pharmaceutical compositions of the present invention may comprise one or more T cell activators. As intended herein and depending on the type of composition produced, the production of antigen-specific phDCs may be extended, if necessary, by culturing cells in a bioreactor or fermenter, or other such vessel or device suitable for the growth of large quantities of cells. Other steps in composition preparation may be individualized to meet the requirements of a particular therapeutic agent. Such additional steps will be well understood by those skilled in the art.

[0207] In all embodiments of the present invention, the pharmaceutical or therapeutic composition is used to treat diseases such as cancer and / or infectious diseases. The pharmaceutical or therapeutic composition of the present invention may induce antigen-specific T cell and / or high-titer antibody responses, thereby eliciting an immune response that is directed towards or reactive to a disease associated with the expression of each antigen (e.g., cancer or infectious disease). In some embodiments, the induced or elicited immune response may be cellular, humoral, or both cellular and humoral immune responses. In some embodiments, the induced or elicited cellular immune response may include the induction or secretion of interferon-gamma (IFN-γ) and / or tumor necrosis factor alpha (TNF-α). In certain embodiments, the pharmaceutical or therapeutic composition acts by one or more of the following: (i) inducing humoral immunity via a B cell response to produce antibodies; (ii) increasing cytotoxic T lymphocytes (CTLs) such as CD8+ to attack and kill disease-causing particles expressing their respective antigens; (iii) increasing the helper T cell response; (iv) increasing the inflammatory response via IFN-γ and / or TNF-α; (v) increasing the natural killer cell response; (vi) increasing the central memory T cell and stem-like T cell subsets. In one embodiment, the pharmaceutical or therapeutic composition acts by all of the above. In one embodiment, the pharmaceutical or therapeutic composition acts by at least (ii), (v), and (vi). In one embodiment, the pharmaceutical or therapeutic composition acts by at least (vi).

[0208] In other embodiments, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote disease development. In the case of cancer, the induced or elicited immune response may reduce or inhibit one or more immunosuppressive factors that promote the growth of antigen-expressing tumors or cancers, including, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells, cytokines such as PD-L1, FasL, IL-10, and TFG-β, tumor-associated macrophages, tumor-associated fibroblasts, soluble factors produced by immunosuppressive cells, CTLA-4, PD-1, MDSC, MCP-1, and immune checkpoint molecules. The pharmaceutical composition may be combined with other pharmaceutical products or compounds. The pharmaceutical composition of the present invention may be used to treat diseases such as cancer or infectious diseases in individuals. The pharmaceutical composition of the present invention may be particularly useful for subjects having an impaired immune system.

[0209] In some embodiments, subjects treated with the pharmaceutical or therapeutic compositions of the present invention experience fewer post-administration side effects compared to subjects treated with dendritic cell-free therapeutics, particularly RNA therapeutics (control subjects). In one embodiment, subjects exhibit less fatigue, pain, headache, and / or fever than control subjects. Comparisons may be made against a control group of subjects treated with dendritic cell-free therapeutics, particularly RNA therapeutics.

[0210] Depending on the target, the pharmaceutical composition may be administered by various routes, including subcutaneous, intravenous, intramuscular, intra-articular, intra-sacral, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, percutaneous, intradermal, intrapulmonary, intraperitoneal, intracardiac, intraarterial, or sublingual. Preferred routes of administration are intradermal or intramuscular. The composition may also be administered by inhalation. In one example, the composition of the present invention is administered intradermally, subcutaneously, or intramuscularly to the limbs, arms, and legs of the subject being treated. In another example, the composition of the present invention is administered intravenously. The pharmaceutical composition of the present invention may be administered in one or more doses. In one embodiment, the pharmaceutical composition of the present invention is administered in one dose. In one embodiment, the pharmaceutical composition of the present invention is administered in two, three, four, or five doses. In one embodiment, the pharmaceutical composition of the present invention is administered in two doses. In one embodiment, the pharmaceutical composition of the present invention is administered in five doses.

[0211] The present invention provides a product, such as a kit, that provides phDC in one container and at least one disease-related antigen (e.g., at least one mRNA encoding at least one antigen protein, optionally encapsulated within nanoparticles) in the other container. The container may be a pre-treated glass or plastic vial or ampoule. The product may include instructions for use.

[0212] IV. Definition Where the term “includes” is used in this description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered a preferred embodiment of the term “includes.” Hereafter, where a group is defined as including at least a certain number of embodiments, this should also be understood as disclosing a group preferably consisting only of these embodiments.

[0213] For the purposes of this invention, the term “obtained” is considered a preferred embodiment of the term “obtainable.” Hereafter, where it is stated, for example, that phDC is obtainable by a specific method, this should also be understood as disclosing phDC obtained by this method.

[0214] When referring to a singular noun, if an indefinite or definite article, such as "a," "an," or "the," is used, this includes the plural form of that noun unless otherwise specifically stated. The term "or" means the term "and / or" and is used interchangeably unless otherwise clearly indicated in the context. In the context of this invention, the terms "about" or "approximately" represent an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the given numerical value.

[0215] Furthermore, terms such as “First,” “Second,” “Third,” or “(a),” “(b),” “(c),” “(d),” or “(i),” “(ii),” “(iii),” “(iv),” and similar terms in this description and claims are used to distinguish similar elements and do not necessarily indicate order or chronology. It should be understood that such terms are interchangeable under appropriate circumstances, and that embodiments of the invention described herein may operate in any order other than those described or illustrated herein.

[0216] Where terms such as “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” or “(i),” “(ii),” “(iii),” “(iv)” relate to a method, use, or assay step, unless otherwise indicated, there is no consistency in time or time interval between steps; that is, unless otherwise indicated in this application as described above or below, steps may be performed simultaneously, or there may be time intervals of a few seconds, a few minutes, a few hours, a few days, a few weeks, a few months, or even several years between such steps.

[0217] The term "approximately" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 15, 30, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length of reference. When the term "approximately" is used with a range of numbers, it modifies that range by widening the boundaries above and below the stated numerical value. In the context of this invention, the term "approximately" represents an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the property in question. Generally, the term "approximately" is intended to modify a numerical value by ±10% above and below the stated value.

[0218] Technical terms are used in their general sense. When a specific meaning is assigned to a particular term, the definition of that term will then be given within the context in which it is used.

[0219] nucleic acids and proteins As used herein, the terms “polynucleotide” or “nucleic acid” are intended to include DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. Nucleic acids may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA or synthetic RNA. In one embodiment of all aspects of the present invention, mRNA encoding an antigen protein is expressed in phDCs obtained from donor or subject monocytes to be treated. The supported phDCs present the immunogenic antigen to the subject's immune system.

[0220] The nucleic acids described herein may be recombinant and / or isolated molecules. In this disclosure, the term “RNA” refers to nucleic acid molecules comprising ribonucleotide residues. In preferred embodiments, 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 the β-D-ribofuranosyl group. RNA includes, but is not limited to, isolated RNA such as double-stranded RNA, single-stranded RNA, partially purified RNA, substantially pure RNA, mRNA, circular RNA, synthetic RNA, recombinantly produced RNA, synthetic RNA, auto-amplified RNA, and modified RNA (e.g., modified mRNA) that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The above RNA species, e.g., “RNA,” “mRNA,” “auto-amplified RNA,” “modified mRNA,” or “synthetic RNA,” always include their fragments, which are still functional. “Fragment” may refer to a portion of a nucleotide sequence, i.e., a sequence shortened at the 5' or 3' end. An RNA sequence fragment contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the residues derived from the RNA sequence. Preferably, an RNA sequence fragment contains at least 18 consecutive residues derived from the RNA sequence, particularly at least 24, at least 36, at least 45, at least 60, at least 90, at least 150, or at least 300 residues.

[0221] Modification may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends of RNA. It is also intended herein that the nucleotides within the RNA (e.g., mRNA) may be chemically synthesized nucleotides, naturally occurring modified nucleotides, or non-standard nucleotides such as deoxynucleotides. For the purposes of this disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0222] In certain embodiments of the present invention, RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As is established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a coding region, and a 3' untranslated region (3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemosynthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to nucleic acid containing deoxyribonucleotides. In one embodiment, RNA is in vitro transcription RNA (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.

[0223] DNA templates for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0224] In one embodiment, the RNA, in particular mRNA, described herein may have modified nucleosides. In some embodiments, the RNA, in particular mRNA, contains modified nucleosides instead of at least one (e.g., any) uridine.

[0225] "Pseudouridine" is an example of a modified nucleoside, which is an isomer of uridine in which uracil is attached to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond. Another exemplary modified nucleoside is N1-methyl-pseudridine. Another exemplary modified nucleoside is 5-methyl-uridine. In some embodiments, one or more uridines in the RNA (e.g., mRNA) described herein are replaced by modified nucleosides. In one embodiment, the RNA contains other modified nucleosides, or further modified nucleosides, such as modified cytidine. For example, in one embodiment, in the RNA, cytidine is partially or completely, preferably completely replaced with 5-methyl-cytidine. In one embodiment, the RNA contains 5-methylcytidine and one or more selected from pseudouridine, N1-methyl-pseudridine, and 5-methyl-uridine. In one embodiment, the RNA comprises 5-methylcytidine and N1-methylpseudridine. In some embodiments, the RNA comprises 5-methylcytidine instead of each cytidine and N1-methylpseudridine instead of each uridine. In some embodiments, the RNA (e.g., mRNA) may contain more than one type of modified nucleoside.

[0226] In some embodiments, the modified nucleoside is modified uridine. Exemplary nucleic acid bases and nucleosides having modified uracil include pseudouridine (Ψ), pyridine-4-onyribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5 - Methoxyuridine (mo5U), Uridine 5-oxyacetic acid (cmo5U), Uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-aminomethyl-2- Thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 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-pseuduridine, 5-taurinomethyl-uridine (τ m5U), 1-taurinomethyl-pseuduridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseuduridine, 5-methyl-uridine (m5U, i.e., having the nucleic acid base deoxythymine), 1-methyl-pseuduridine (m1Ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseuduridine (m1s4Ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m3Ψ), 2-thio-1-methyl-pseuduridine,1-Methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine (D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseuduridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseuduridine, 4-methoxy-2-thio-pseuduridine, N1-methylpseuduridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseuduridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5S2U), α-thio-uridine, 2 '-O-methyluridine (Um), 5,2'-O-dimethyluridine (m5Um), 2'-O-methylpseudridine (Ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine It contains din (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thiouridine, deoxythymidine, 2'-F-ala-uridine, 2'-F-uridine, 2'-OH-ala-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine.

[0227] In some embodiments, the modified nucleoside is a modified cytidine. Exemplary nucleic acid bases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methylcytidine (m3C), N4-acetylcytidine (ac4C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 5-methylcytidine (m5C), 5-halocytidine (e.g., 5-iodocytidine), and 5-hydrocytidine. Xymethylcytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolocytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine (s2C), 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine Zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thiocytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl It contains thyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-af-cytidine, 2'-F-cytidine, and 2'-OH-af-cytidine.

[0228] In some embodiments, the modified nucleoside is modified adenosine.Exemplary nucleic acid bases and nucleosides having modified adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- Methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl α-Adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenine It contains denosine, 2'-O-methyladenosine (Am), N6,2'-O-dimethyladenosine (m6Am), N6,N6,2'-O-trimethyladenosine (n62Am), 1,2'-O-dimethyladenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methylpurine, 1-thioadenosine, 8-azidoadenosine, 2'-F-aa-adenosine, 2'-F-adenosine, 2'-OH-ala-adenosine, and N6-(19-amino-pentaoxa-nonadecyl)-adenosine.

[0229] In some embodiments, the modified nucleoside is a modified guanidine. Exemplary nucleic acid bases and nucleosides having modified guanines include inosine (I), 1-methyl-inosine (m1l), uiosine (imG), methyluiosine (mimG), 4-demethyluiosine (imG-14), isouiosine (imG2), wibutosine (yW), peroxywibutosine (02yW), hydroxywibutosine (OhyW), low-modified hydroxywibutosine (OhyW*), 7-deaza-guanosine, keuosine (Q), epoxykeuosine (oQ), and galactosyl-keuosine. Syn (galQ), Mannosyl-keuosin (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQ1), Alkaeosin (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine ( m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl - Contains guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1lm), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ala-guanosine, and 2'-F-guanosine.

[0230] In some embodiments, the mRNA contains a 5'-cap. In one embodiment, the mRNA does not have an uncapped 5'-triphosphate. In one embodiment, the mRNA may be modified by a 5'-cap analog. The term "5'-cap" refers to a structure found at the 5' end of an mRNA molecule, generally consisting of a guanosine nucleotide attached to the mRNA via a 5'-to-5' triphosphate linkage. In one embodiment, this guanosine is methylated at position 7. Providing a 5'-cap or 5'-cap analog to mRNA may be achieved by in vitro transcription, the 5'-cap may be co-transcribed within the mRNA chain, or it may be attached to the mRNA post-transcriptionally using a capping enzyme.

[0231] The terms “untranslated region” or “UTR” refer to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region within an RNA molecule such as an mRNA molecule. Untranslated regions (UTRs) can be located at the 5' (upstream) (5'-UTR) and / or 3' (downstream) (3'-UTR) of the open reading frame. The 5'-UTR, if present, is located at the 5' end, upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. The 3'-UTR, if present, is located at the 3' end, downstream of the stop codon of the protein-coding region, although the term “3'-UTR” preferably does not include a poly(A) sequence. Thus, the 3'-UTR is upstream of a poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0232] As used herein, the terms “poly(A) sequence” or “poly-A tail” refer to an uninterrupted or interrupted sequence of adenylate residues typically located at the 3' end of an RNA molecule (e.g., an mRNA molecule). Poly(A) sequences are known to those skilled in the art and may follow the 3'-UTR in the RNA (e.g., mRNA) described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, uninterrupted poly(A) sequences are typical. RNA (e.g., mRNA) according to the present invention may have a poly(A) sequence attached to the free 3' end of the RNA (e.g., mRNA) after transcription by a template-independent RNA polymerase, or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase. A poly(A) sequence of approximately 120 A nucleotides has beneficial effects on RNA and protein levels in transfected eukaryotic cells, the protein being translated from an open reading frame located upstream (5') of the poly(A) sequence (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017). The poly(A) sequence can be of any length. In some embodiments, the poly(A) sequence is essentially composed of, or consists of, at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 800, up to 400, up to 300, up to 200, or up to 150 A nucleotides. In one embodiment, the poly(A) sequence is essentially composed of, or consists of, 300 nucleotides. In this context, "essentially consisting of" means that most of the nucleotides in a poly(A) sequence, 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% based on the number of nucleotides in the poly(A) sequence, are A nucleotides, but the remaining nucleotides are allowed to be other nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid).In this context, "consists of" means that all nucleotides in the poly(A) sequence, i.e., 100% based on the number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate. In some embodiments, the poly(A) sequence is attached during RNA transcription, for example, during the preparation of in vitro transcription RNA (e.g., mRNA), based on a DNA template containing repeating dT nucleotides (deoxythymidylate) on a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is referred to as the poly(A) cassette.

[0233] The term "codon-optimized" refers to modifications of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, without preferably altering the amino acid sequence encoded by the nucleic acid molecule. In the context of the present invention, the coding region is preferably codon-optimized for optimal expression in the target to be treated with the RNA (e.g., mRNA) molecule described herein. Codon optimization is based on the finding that translation efficiency is also determined by the different frequencies of tRNA appearances within the cell. Therefore, the sequence of RNA (e.g., mRNA) can be modified to insert codons where frequently present tRNA is available in place of "rare codons".

[0234] In some embodiments of the present invention, the guanosine / cytosine (G / C) content of the coding region of the RNA (e.g., mRNA) described herein is increased compared to the G / C content of the corresponding coding sequence of wild-type RNA, and the amino acid sequence encoded by the RNA (e.g., mRNA) is preferably unmodified compared to the amino acid sequence encoded by wild-type RNA. This modification of the RNA (e.g., mRNA) sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of that RNA (e.g., mRNA). Sequences with increased G / C content are more stable than sequences with increased A (adenosine) / U (uracil) content. Taking into account the fact that several codons code for the same amino acid (so-called degeneracy of the gene code), the most preferred codon for stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA (e.g., mRNA) sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by substituting these codons with other codons that encode the same amino acids but do not contain A and / or U, or contain lower amounts of A and / or U nucleotides.

[0235] In the context of this invention, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (e.g., mRNA). The RNA (e.g., mRNA) can then be translated into peptides or proteins.

[0236] According to the present invention, the term "transcription" includes "in vitro transcription," and the term "in vitro transcription" relates to the process by which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is used for the production of the transcript. These cloning vectors are generally called transcription vectors and are encompassed by the term "vector" according to the present invention.

[0237] With respect to RNA (e.g., mRNA), the terms “expression” or “translation” refer to the process in cellular ribosomes in which a chain of mRNA directs the assembly of amino acid sequences to produce a peptide or protein. In one embodiment, after the combination of RNA (e.g., mRNA) as described herein, formulated as RNA (e.g., mRNA) lipid particles, and a phDC, at least a portion of the RNA (e.g., mRNA) is delivered to the phDC. In one embodiment, the RNA (e.g., mRNA) is translated by the phDC to produce the peptide or protein it encodes. RNA (e.g., mRNA) particles, such as the RNA (e.g., mRNA) lipid particles described herein, may be used to deliver RNA to a phDC.

[0238] "Code" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide such as a gene, cDNA, RNA, or mRNA, which has either a predetermined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a predetermined amino acid sequence, and the biological properties that result from it, acting as a template for the synthesis of other polymers and macromolecules in biological processes. Therefore, when RNA (e.g., mRNA) translation occurs in a cell, the RNA (e.g., mRNA) sequence can code for a protein (e.g., an antigen).

[0239] As used herein, terms such as “reduce,” “effectively reduce,” “decrease,” “inhibit,” or “impair” preferably relate to an overall reduction or the ability to cause an overall reduction of at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or higher percentages of a level. These terms include complete or substantially complete inhibition, i.e., a reduction to zero or substantially zero. Terms such as “increase,” “boost,” or “exceed” preferably relate to an increase or boost of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher percentages.

[0240] According to this disclosure, the term “peptide” includes oligopeptides and polypeptides and refers to substances containing about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and up to about 50, about 100, or about 150 consecutive amino acids linked to one another via peptide bonds. The terms “protein” or “polypeptide” refer to larger peptides, in particular peptides having at least about 150 amino acids, but the terms “peptide,” “protein,” and “polypeptide” are used herein as synonyms unless otherwise described.

[0241] The term "fragment" can refer to antigens such as disease-related antigens, amino acid sequences, antigenic proteins, RNA (including the RNA species listed above), and especially parts of mRNA.

[0242] When a fragment relates to an amino acid sequence (e.g., an antigen protein), the term refers to a sequence representing a portion of the amino acid sequence, i.e., a shortened amino acid sequence (e.g., of an antigen protein) at the N-terminus and / or C-terminus. A C-terminus shortened fragment (N-terminal fragment) can be obtained by translation of a truncated open reading frame, for example, lacking the 3' end of the open reading frame. An N-terminus shortened fragment (C-terminal fragment) can be obtained by translation of a truncated open reading frame, for example, lacking the 5' end of the open reading frame, insofar as the truncated open reading frame contains a start codon that plays a role in initiating translation. An amino acid sequence fragment contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues derived from the amino acid sequence. Preferably, an amino acid sequence fragment contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids derived from the amino acid sequence.

[0243] In this specification, “variant” means an amino acid sequence that differs from the parent amino acid sequence due to at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or a modified version of the wild-type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, for example, 1 to about 20 amino acid modifications, 1 to about 10, or 1 to about 5 amino acid modifications compared to the parent.

[0244] In this specification, “wild-type,” “WT,” or “natural” means an amino acid sequence found in nature, including allelic mutations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0245] A “variant” of an amino acid sequence (peptide, protein, 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 variants, splice variants, post-translational modification variants, conformations, isoforms, allele variants, species variants, and species homologs, especially those occurring in nature. The term “variant” specifically includes fragments of an amino acid sequence. Preferably, the degree of similarity (or identity) between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence will be 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%. Alignment for determining sequence similarity, preferably sequence identity, may be performed using tools known in the art, preferably using the best sequence alignment, for example, using Align with standard settings, preferably EM-BOSS::needle, matrix:Blosum62, and similar. "Sequence similarity" refers to the percentage of amino acids that are identical or represent conserved 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. The identity percentage is obtained by determining the number of identical positions corresponding to the sequences being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0246] In one embodiment, the fragment or “variant” is preferably a “functional fragment” or “functional variant.” The term “functional fragment” or “functional variant” is used with respect to any fragment or variant that exhibits one or more functional properties identical or similar to those of the unfragmented active substance from which it is derived (e.g., an unfragmented amino acid sequence, an unfragmented RNA sequence, an unfragmented mRNA sequence, etc.), i.e., it is functionally equivalent. With respect to an antigen or antigenic protein, one particular function is one or more immunogenic (synonymous herein with “antigenicity”) 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” refers in particular to a variant molecule or sequence that includes a sequence in which one or more amino acids or nucleotides are altered compared to the parent molecule or sequence, and which is still capable of performing one or more functions of the parent molecule or sequence, such as inducing an immune response (i.e., being antigenic). In one embodiment, modifications 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 a different embodiment, the function of the functional fragment or functional variant may be reduced but still significantly present; for example, the immunogenicity of the functional fragment or variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent substance, molecule, or sequence. However, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0247] Composition and administration In all embodiments of the present invention, the therapeutic compositions of the present invention induce an immune response in cells, tissues, or subjects (e.g., humans) to at least one disease-associated antigen (e.g., an antigenic protein). The therapeutic compositions of the present invention are administered to treat a disease or disorder, or to reduce the severity of the symptoms of a disease or disorder. In the context of the present invention, “treating,” “treating,” or “treatment” of a disease or disorder occurs after the onset of a pathological event (e.g., the onset or manifestation of a disease or disorder). The term “treatment” or “therapeutic treatment” includes treatment of a subject or cells to alter the current course of a subject (e.g., a mammal such as a human) or cells. In the context of the present invention, treatment refers to a subject or cells that have been previously diagnosed or determined to have a disease or disorder (as opposed to inhibition). Treatment includes, but is not limited to, the administration of the therapeutic compositions of the present invention and is performed after the onset of a pathological event or contact with an infectious agent. If the therapeutic composition of the present invention is administered more than once, the next administration is typically performed at least 2, 3, 4, 5, 7, 14, 21, 28, 35, 42, 49, or 56 days, or more, after the previous administration.

[0248] In the context of the present invention, prophylactic administration (or vaccination) is distinguished from therapeutic treatment with respect to the timing of administration of the active substance or composition to the subject. Vaccination occurs before the onset of a pathological event, while therapeutic treatment or treatment occurs afterward, for example, after the subject has been previously diagnosed with the disease.

[0249] As used herein, “to induce / stimulate an immune response” means that an immune response to a particular antigen may not have existed before induction / stimulation, or that there was a baseline level of immune response to a particular antigen before induction, and that it was enhanced after induction. Therefore, “to induce / stimulate an immune response” includes “to enhance an immune response.”

[0250] immunity As used herein, the terms “antigen” or “antigenic protein” refer to a compound, composition, or substance, including compositions injected into or absorbed by an animal, that can stimulate antibody production or a T-cell response in an animal. Antigens or antigenic proteins react with specific humoral or cellular immunity products, including those induced by heterologous immunogens. The term “antigen” is used interchangeably with the term “immunogen.” The terms “antigen” or “antigenic protein” include all relevant antigenic epitopes. The terms “antigen,” “antigen molecule / protein” or “immunogen” include their fragments that can still act as antigens. “Antigenic proteins” include a large number of amino acids, ranging from small peptides to large proteins, e.g., 4 to 2000 or more amino acids, 4 to 1800 amino acids, 4 to 1600 amino acids, or 4 to 1400 amino acids. In one embodiment, the antigen protein encoded by RNA (e.g., mRNA) contains 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 1000, at least 1200, at least 2000, or at least 3000 amino acids. In one embodiment, the antigen protein encoded by RNA (e.g., mRNA) contains at least 50 amino acids. In one embodiment, the antigen protein encoded by RNA (e.g., mRNA) contains at least 500 amino acids. In one embodiment, the antigen protein encoded by RNA (e.g., mRNA) contains at least 1000 amino acids. In one embodiment, the antigen protein contains 100 to 1500 amino acids. In one embodiment, the antigen protein contains 200 to 1300 amino acids. In one embodiment, the antigen protein contains 400 to 1300 amino acids.

[0251] An "epitope" refers to the site of an antigen to which B and / or T cells respond. As used herein, the term "immunogenicity" refers to the ability of a substance, cell, or part thereof, such as an antigen or antigenic protein, to elicit an immune response in the body of a human or animal.

[0252] The terms "cell-mediated immunity," "cellular immunity," "cellular immune response," or similar terms are intended to include cellular responses directed at cells characterized by the expression of antigens or antigenic proteins, particularly those characterized by the presentation of antigens by class I or class II MHCs. Cellular responses relate to immune effector cells, specifically cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (also called CD4+ T cells) play a central role by modulating the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill affected cells, such as virus-infected cells, and prevent the production of more affected cells.

[0253] Humoral immunity, or humoral immune response, is a form of immunity mediated by macromolecules found in the extracellular fluid, such as secreted antibodies, complement proteins, and certain antimicrobial peptides. It is in contrast to cell-mediated immunity. The form involving antibodies is often called antibody-mediated immunity. Humoral immunity refers to antibody production and its associated support processes, including Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation, and memory cell generation.

[0254] In this specification, "dendritic cells," also referred to as "DCs," are antigen-presenting immune cells that process antigenic material and present it to other cells of the immune system, most notably T cells. DCs perform the function of capturing and processing antigens. When DCs take up antigens by endocytosis, they process the antigens into smaller fragments, generally peptides, which are presented on the DC surface, and these peptides are then presented to antigen-specific T cells, for example, via MHC molecules. After antigen uptake, DCs migrate to lymph nodes. During maturation, DCs can express costimulatory signals, spurred by various signals, including signaling via Toll-like receptors (TLRs), that induce, activate, and proliferate associated effector T cells (Teffs), thereby initiating a T-cell-mediated immune response to the antigen. Alternatively, DCs may present antigens to antigen-specific T cells without providing costimulatory signals (or while providing co-inhibitory signals), resulting in improper activation of Teffs. Such presentations may, for example, cause the death or anergy of antigen-recognizing T cells, or induce the generation and / or increase of regulatory T cells (Tregs). The term “dendritic cells” includes differentiated, immature, and mature dendritic cells. These cells may be characterized by the expression of certain cell surface markers (e.g., CD11c, MHC class II, and at least low levels of CD80 and CD86, CD11b, CD304 (BDCA4)). In some embodiments, DCs express CD8, CD103, CD1d, etc. Other DCs may be identified by the absence of lineage markers such as CD3, CD14, CD19, CD56, etc. In addition, dendritic cells may be functionally characterized by their ability to stimulate alloresponse and mixed lymphocyte reactions (MLRs). In one example, a phDC combined with a SARS-CoV-2-derived spike protein antigen (antigen protein) or the mRNA encoding it would process and present the spike protein-related antigen; that is, the phDC would be antigen-specific to the spike protein.

[0255] The terms "AB serum" or "human AB serum" refer to cell culture reagents well known in the art for certain human cell types that provide growth factors, vitamins, nutrients, as well as trace elements and transport factors. Human AB serum is collected from healthy volunteer male donors of the AB serotype.

[0256] The term "FBS" refers to a growth supplement widely used in cell culture media. It typically contains a high content of embryonic growth-promoting factors.

[0257] disease The terms “disease” or “disorder” refer to any disease or disorder involving an antigen or antigenic protein, such as a disease characterized by the presence of an antigen or antigenic protein. A disease may be, for example, an infectious disease or a neoplastic disease (cancer). As mentioned above, the antigen may be a disease-associated antigen such as a viral antigen or a tumor antigen. In one embodiment, an antigen-related disease is preferably a disease involving cells that express the antigen on the cell surface. In the context of the present invention, the disease is caused by disease-causing particles. The concentration of disease-causing particles in a subject may be determined by any suitable means known to those skilled in the art. For example, viral load may be determined by quantitative PCR and expressed as copy number per tissue ng. Where appropriate, expression markers may also be used to quantify disease-causing particles.

[0258] The term “hyperproliferative disorders” encompasses tumors and cancers. The term “tumor” refers to a disease in which some cells of the body grow uncontrollably and spread to other parts of the body. Tumors can be cancerous or non-cancerous (benign). In the context of this invention, the terms tumor and cancer are used interchangeably. Cancerous tumors can spread to or invade neighboring tissues and travel to distant locations within the body to form new tumors (metastasis). Cancerous tumors may also be called malignant tumors. Many cancers form solid tumors, but blood cancers such as leukemia do not. Benign tumors do not spread to or invade neighboring tissues. Blood cancers include leukemia, lymphoma, myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), multiple myeloma, and all their subtypes.

[0259] The term "infectious disease" refers to any disease (e.g., the common cold) caused by a microbial factor that can be transmitted from individual to individual or from organism to organism. Infectious diseases are well known in the art and include, for example, viral diseases, bacterial diseases, fungal diseases, or parasitic diseases, which are caused by viruses, bacteria, fungi, and parasites, respectively. In this regard, infectious diseases may include, for example, hepatitis, sexually transmitted infections (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), COVID-19, avian influenza, and influenza.

[0260] "Novel Coronavirus Disease" or COVID-19 is a contagious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The SARS-CoV-2 outbreak, which causes atypical pneumonia, raged in China from mid-December 2019 and developed into a public health emergency of international concern. SARS-CoV-2 (MN908947.3) belongs to the beta-coronavirus lineage B. It has at least 70% sequence similarity to SARS-CoV. In particular, COVID-19 refers to the disease as defined in the current International Classification of Diseases (ICD-11, World Health Organization, version: 09 / 2020). More specifically, COVID-19 is used to describe the disease as diagnosed clinically, epidemiologically, or otherwise, regardless of whether clinical testing is definitive, indefinitive, or unavailable. Generally, coronaviruses possess four structural proteins: the envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins play crucial roles in viral association, while the N protein is necessary for viral RNA synthesis. The critical glycoprotein S is involved in viral binding and entry into target cells. The S protein is synthesized as a single-chain inactive precursor, which is cleaved by a furin-like host protease within the producing cell into two non-covalently linked subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) that recognizes host cell receptors. The S2 subunit contains a fusion peptide, two heptad repeats, and a transmembrane domain, all of which are required to mediate the fusion of the virus and host cell membranes through a major conformational rearrangement. The S1 and S2 subunits trimerize to form a large pre-fusion spike. The SARS-CoV-2 S precursor protein can be proteolytically cleaved into S1(685aa) and S2(588aa) subunits. The S1 subunit consists of a receptor-binding domain (RBD), which mediates viral entry into susceptible cells via the host angiotensin-converting enzyme 2 (ACE2) receptor.Therapeutic treatment of COVID-19 may include treating, improving, or curing at least one of the following conditions in a subject with COVID-19: pulmonary fibrosis, interstitial pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), alveolar injury, renal injury, vascular disease, cardiac injury, acute myocardial injury, chronic cardiovascular injury, thrombosis, and venous thromboembolism. In specific embodiments, pulmonary fibrosis, interstitial pneumonia, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), renal injury such as proteinuria and acute kidney injury, and vascular disease are induced by COVID-19. According to the ICTV, SARS-CoV-2 is used to represent all variants of viruses belonging to the Riboviria realm, Orthornavirae kingdom, Pisuviricota phylum, Pisoniviricetes class, Nidovirales order, Coronaviridae family, Betacoronavirus genus, Sarbecovirus subgenus, Severe Acute Respiratory Syndrome-related Coronavirus species, and the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) lineage.

[0261] The term "HIV" refers to the human immunodeficiency virus. HIV is a member of the genus Lentivirus, which is part of the family Retroviridae. Two types of HIV are characterized: HIV-1 and HIV-2. HIV-1 is the first virus discovered and was named both lymphadenopathy-associated virus (LAV) and human T-lymphotropic virus 3 (HTLV-III). HIV-1 is more virulent and infectious than HIV-2 and is responsible for the majority of HIV infections worldwide. HIV infects humans and, over time, causes acquired immunodeficiency syndrome (AIDS). AIDS manifests as a progressive failure of the immune system, leading to the proliferation of deadly opportunistic infections and cancers.

[0262] As used herein, the terms “animal” or “mammal” encompass all mammals, including humans. Preferably, the mammal of the present invention is a human subject. In one embodiment, the subject is a human.

[0263] The terms “individual” and “subject” are used interchangeably herein. They refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that is susceptible to, but may or may not have, a disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms “individual” and “subject” do not imply a specific age and therefore encompass adults, the elderly, children, and newborns. In some embodiments, the term “subject” includes a human being who is at least 50 years old, at least 55 years old, at least 60 years old, at least 65 years old, at least 70 years old, or older. In some embodiments, the term “subject” includes a human being who is at least 65 years old, such as 65–80 years old, 65–75 years old, or 65–70 years old. In some embodiments of the present invention, “individual” or “subject” is a “patient.”

[0264] The term "patient" refers to an individual or subject for treatment. Herein, the present invention is described with reference to some specific examples, however, these are for illustrative purposes only and should not be taken as limiting.

[0265] The present invention further relates to the following embodiments. 1. A therapeutic composition comprising a pharmaceutically effective amount of physiological DC (phDC) and at least one mRNA containing a coding sequence encoding at least one antigen protein.

[0266] 2. The therapeutic composition according to 1, further comprising a pharmaceutically acceptable carrier or diluent. 3. The therapeutic composition according to claim 1 or 2, wherein the at least one mRNA is contained in lipid nanoparticles.

[0267] 4. The therapeutic composition according to any one of 1 to 3, wherein the at least one mRNA comprises at least one modified nucleoside.

[0268] 5. The therapeutic composition according to 4, wherein at least one modified nucleoside is modified uridine, and all uridine residues are optionally replaced with modified uridine.

[0269] 6. The therapeutic composition according to 5, wherein the modified uridine is N1-methyl-pseudouridine.

[0270] 7. The therapeutic composition according to any one of 1 to 6, wherein at least one mRNA is partially modified with N1-methyl-pseuduridine.

[0271] 8. The therapeutic composition according to any one of 1 to 6, wherein at least one mRNA is completely modified with N1-methyl-pseuduridine.

[0272] 9. A therapeutic composition according to any one of 1 to 8, wherein the at least one mRNA comprises a 5'UTR, a 3'UTR, a 5' cap, and / or a poly(A) tail.

[0273] 10. The therapeutic composition according to any one of 1 to 9, wherein the at least one mRNA comprises an optimized sequence; optionally, the 5'UTR, 3'UTR sequences, and / or coding sequences for the antigen protein are optimized.

[0274] 11. The therapeutic composition according to any one of 1 to 10, wherein the lipid nanoparticles comprise a cationic lipid, a polyethylene glycol (PEG)-modified lipid, a cholesterol-based lipid, and / or a non-cationic lipid.

[0275] 12. The therapeutic composition according to 11, wherein the cationic lipid is present in a molar ratio of 30% to 40%, the PEG-modified lipid is present in a molar ratio of 1.5% to 4.0%, the cholesterol-based lipid is present in a molar ratio of 40% to 52%, and the non-cationic lipid is present in a molar ratio of 11% to 21%, and all molar ratios are relative to the total lipid content of LNP.

[0276] 13. The therapeutic composition according to 11 or 12, wherein the cationic lipid is a cKK-E12 lipid.

[0277] 14. A therapeutic composition according to any one of 1 to 13, wherein at least one antigen protein is associated with a disease.

[0278] 15. The therapeutic composition according to 14, wherein the antigen protein associated with the disease is an infectious disease-associated antigen protein.

[0279] 16. The therapeutic composition according to 15, wherein the infectious disease-related antigen protein is a viral antigen protein, a bacterial antigen protein, a fungal antigen protein, a prion antigen protein, or a parasitic antigen protein.

[0280] 17. The therapeutic composition according to 16, wherein the viral antigen protein is a coronavirus antigen protein or a human immunodeficiency virus (HIV) antigen protein.

[0281] 18. The therapeutic composition according to 17, wherein the coronavirus antigen protein is derived from a spike protein, an envelope protein, a nucleocapsid protein, a membrane protein, and / or an Orf1ab polyprotein.

[0282] 19. The therapeutic composition according to 17 or 18, wherein the coronavirus antigen protein is derived from a spike protein.

[0283] 20. The therapeutic composition according to any one of 17 to 19, wherein the coronavirus antigen protein is a beta-coronavirus antigen protein.

[0284] 21. The therapeutic composition according to 20, wherein the beta-coronavirus antigen protein is the SARS-CoV-2 antigen protein.

[0285] 22. The therapeutic composition according to any one of 1 to 21, wherein the at least one mRNA comprises a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0286] 23. The therapeutic composition according to any one of 1 to 22, wherein the at least one mRNA comprises a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to Sequence ID No. 1.

[0287] 24. The therapeutic composition according to any one of 1 to 21, wherein the at least one mRNA comprises a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to SEQ ID NO: 19.

[0288] 25. The therapeutic composition according to 16, wherein the bacterial antigen protein is an antigen protein of the Borrelia species or Mycobacteria species.

[0289] 26. The therapeutic composition according to 16, wherein the fungal antigen protein is the antigen protein of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans.

[0290] 27. The therapeutic composition according to 16, wherein the parasitic antigen protein is the antigen protein of Plasmodium malariae.

[0291] 28. The therapeutic composition according to 14, wherein the antigen protein associated with the disease is a tumor-associated antigen protein.

[0292] 29. The therapeutic composition according to 28, wherein the tumor-associated antigen protein is a blood cancer antigen protein.

[0293] 30. The therapeutic composition according to 29, wherein the blood cancer antigen protein is a leukemia antigen protein, a lymphoma antigen protein, or a myeloma antigen protein.

[0294] 31. The therapeutic composition according to 28, wherein the tumor-associated antigen protein is a solid tumor antigen protein.

[0295] 32. The therapeutic composition according to 31, wherein the solid tumor antigen protein is melanoma antigen protein, endometrial cancer antigen protein, kidney cancer antigen protein, brain tumor antigen protein, cervical cancer antigen protein, liver cancer antigen protein, head and neck cancer antigen protein, gastrointestinal cancer antigen protein, lymph node cancer antigen protein, pancreatic cancer antigen protein, otolaryngological (ENT) cancer antigen protein, breast cancer antigen protein, prostate cancer antigen protein, ovarian cancer antigen protein, or lung cancer antigen protein.

[0296] 33. A therapeutic composition according to any one of 1 to 32, wherein phDC can be obtained by subjecting monocytes to a physical force.

[0297] 34. The therapeutic composition according to 33, wherein the physical force is applied by passing the monocytes through a flow chamber.

[0298] 35. The therapeutic composition according to 34, wherein the flow chamber is a plate or a flexible bag, optionally a flexible plastic bag.

[0299] 36. The therapeutic composition according to any one of 33 to 35, wherein the monocytes are of autologous origin.

[0300] 37. A therapeutic composition for use in a method of treating a disease in a subject, wherein the subject has been previously diagnosed with the disease, and the method comprises administering the therapeutic composition to the subject, according to any one of 1 to 36.

[0301] 38. A therapeutic composition for use in a method of treating an infectious disease in a subject, wherein the subject has been previously diagnosed with an infectious disease, and the method comprises administering the therapeutic composition to the subject, according to any one of 15 to 27.

[0302] 39. A therapeutic composition for use in a method of treating a viral disease in a subject, wherein the subject has been previously diagnosed with a viral disease, and the method comprises administering the therapeutic composition to the subject, according to any one of 16 to 24.

[0303] 40. A therapeutic composition for use in a method of treating coronavirus disease, preferably COVID-19, in a subject, wherein the subject has been previously diagnosed with coronavirus disease or COVID-19, and the method comprises administering the therapeutic composition to the subject, according to any one of 17 to 24.

[0304] 41. A therapeutic composition for use in a method of treating a tumor or cancer in a subject, wherein the method comprises administering the therapeutic composition to the subject, according to any one of 28 to 32.

[0305] 42. A therapeutic composition for use in a method for treating a blood cancer in a subject, the therapeutic composition according to 29 or 30, wherein the method comprises administering the therapeutic composition to the subject.

[0306] 43. A therapeutic composition for use in a method for treating a solid tumor in a subject, wherein the method comprises administering the therapeutic composition to the subject, according to 31 or 32.

[0307] 44. A therapeutic composition for use according to any one of 37 to 43, wherein the therapeutic composition is administered intradermally, intravenously, or intramuscularly.

[0308] 45. A therapeutic composition for use according to any one of 37 to 43, wherein the administration of the therapeutic composition is by inhalation.

[0309] 46. ​​A therapeutic composition for use according to any one of 37 to 45, administered once or multiple times.

[0310] 47. A therapeutic composition for use according to any one of 37 to 46, wherein at least two doses are administered to the subject.

[0311] 48. The therapeutic composition for use according to any one of 37 to 47, wherein the at least two doses are administered to the subject with an interval of 2 to 6 weeks between each dose, or optionally 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks between each dose.

[0312] 49. A method for preparing antigen-specific phDCs, comprising combining physiological dendritic cells (phDCs) obtained from a donor with at least one mRNA encoding at least one antigen protein.

[0313] 50. The method according to Embodiment 49, wherein the phDC is produced by subjecting monocytes obtained from the donor to physical force.

[0314] 51. The method according to Embodiment 49 or 50, wherein the physical force is applied by passing the monocytes obtained from the donor through a flow chamber.

[0315] 52. The method according to any one of Embodiments 49 to 51, further comprising the step of incubating the phDC together with the at least one mRNA.

[0316] 53. The method according to Embodiment 52, wherein the incubation step is carried out for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, or 24 hours.

[0317] 54. The method according to any one of embodiments 49 to 53, wherein the monocytes obtained from the donor are derived from an in vitro blood sample or PBMC from the donor.

[0318] 55. The method according to any one of embodiments 49 to 54, wherein the flow chamber is a plate.

[0319] 56. The method according to any one of embodiments 49 to 55, wherein the flow chamber is a bag, optionally a flexible bag or a plastic bag.

[0320] 57. The method according to Embodiment 56, wherein the plastic bag is made from a material including polyolefin, polyethylene, fluoropolymer, polyvinyl chloride, ethylene-vinyl acetate copolymer, ethylene vinyl alcohol, polyvinylidene fluoride, and / or other plastics including materials approved for medical use.

[0321] 58. The method according to any one of embodiments 49 to 57, wherein the at least one mRNA comprises at least one modified nucleoside.

[0322] 59. The method according to Embodiment 58, wherein the at least one modified nucleoside is a substitution of some or all uridine residues with at least one modified uridine, and optionally some or all uridine residues are substituted with N1-methyl-pseudridine.

[0323] 60. The method according to Embodiment 59, wherein some or all of the modified uridine residues comprise one or more uniquely modified uridine residues.

[0324] 61. The method according to Embodiment 60, wherein the one or more uniquely modified uridine residues include N1-methyl-pseudouridine.

[0325] 62. The method according to any one of embodiments 49 to 61, wherein at least one mRNA is partially modified with N1-methyl-pseuduridine.

[0326] 63. The method according to any one of embodiments 49 to 61, wherein at least one mRNA is fully modified with N1-methyl-pseuduridine.

[0327] 64. The method according to any one of embodiments 49 to 63, wherein the at least one mRNA comprises a 5'UTR, a 3'UTR, a 5' cap, and / or a poly(A) tail.

[0328] 65. The method according to embodiment 64, wherein the 5' cap is a cap 1 structure or an m7GpppG cap.

[0329] 66. The method according to any one of embodiments 49 to 65, wherein the mRNA includes a coding sequence encoding an antigen protein.

[0330] 67. The method according to any one of embodiments 49 to 66, wherein the at least one mRNA comprises an optimized sequence; preferably, the 5'UTR, 3'UTR sequences, and / or coding sequences are optimized for the antigen protein.

[0331] 68. The method according to any one of embodiments 49 to 67, wherein the at least one mRNA encoding at least one antigen protein is provided within a lipid nanoparticle.

[0332] 69. The method according to Embodiment 68, wherein the lipid nanoparticles include cationic lipids, PEG-modified lipids, cholesterol, and / or non-cationic lipids.

[0333] 70. The method according to any one of Embodiments 49 to 69, wherein the antigen protein is associated with a disease and is optionally an infectious disease-associated antigen protein or a tumor-associated antigen protein.

[0334] 71. The method according to Embodiment 70, wherein the infectious disease-related antigen protein is a viral antigen protein, a bacterial antigen protein, a fungal antigen protein, a prion antigen protein, or a parasitic antigen protein.

[0335] 72. The method according to Embodiment 71, wherein the viral antigen protein is coronavirus or HIV antigen protein.

[0336] 73. The method according to Embodiment 72, wherein the coronavirus antigen protein is SARS-CoV antigen protein, optionally SARS-CoV-2 antigen protein.

[0337] 74. The method according to Embodiment 73, wherein the SARS-CoV or SARS-CoV-2 antigen protein is derived from a spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein.

[0338] 75. The method according to Embodiment 71, wherein the bacterial antigen protein is an antigen protein of the Borrelia species or the Mycobacteria species.

[0339] 76. The method according to Embodiment 71, wherein the fungal antigen protein is the antigen protein of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans.

[0340] 77. The method according to Embodiment 71, wherein the parasitic antigen protein is the antigen protein of Plasmodium malariae.

[0341] 78. The method according to Embodiment 70, wherein the tumor-associated antigen protein is leukemia antigen protein, melanoma antigen protein, lymphoma antigen protein, endometrial cancer antigen protein, kidney cancer antigen protein, brain tumor antigen protein, cervical cancer antigen protein, liver cancer antigen protein, head and neck cancer antigen protein, gastrointestinal cancer antigen protein, lymph node cancer antigen protein, pancreatic cancer antigen protein, otolaryngological (ENT) cancer antigen protein, breast cancer antigen protein, prostate cancer antigen protein, ovarian cancer antigen protein, or lung cancer antigen protein.

[0342] 79. The method according to any one of Embodiments 49 to 74, wherein the mRNA comprises a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to a sequence or portion of a sequence selected from SEQ ID NO: 1 (spike protein transcript), SEQ ID NO: 2 (envelope protein transcript), SEQ ID NO: 3 (nucleocapsid protein transcript), SEQ ID NO: 4 (membrane protein transcript), and / or SEQ ID NO: 5 (Orf1ab polyprotein transcript).

[0343] 80. An antigen-specific phDC obtainable by the method described in any one of Embodiments 49 to 79.

[0344] 81. A therapeutic composition comprising a pharmaceutically effective amount of antigen-specific phDC and at least one mRNA encoding at least one antigen protein.

[0345] 82. The therapeutic composition according to Embodiment 81, further comprising a pharmaceutically acceptable carrier or diluent.

[0346] 83. The therapeutic composition according to Embodiment 81 or 82, wherein the mRNA is contained in lipid nanoparticles.

[0347] 84. The therapeutic composition according to any one of embodiments 81 to 83, wherein the at least one mRNA comprises at least one modified nucleoside.

[0348] 85. The therapeutic composition according to Embodiment 84, wherein the at least one modified nucleoside is a substitution of some or all uridine residues.

[0349] 86. The therapeutic composition according to Embodiment 85, wherein some or all of the modified uridine residues comprise one or more uniquely modified uridine residues.

[0350] 87. The therapeutic composition according to Embodiment 85 or 86, wherein the one or more modified uridine residues comprise N1-methyl-pseudouridine.

[0351] 88. The therapeutic composition according to any one of embodiments 81 to 87, wherein at least one mRNA is partially modified with N1-methyl-pseuduridine.

[0352] 89. The therapeutic composition according to any one of embodiments 81 to 87, wherein at least one mRNA is completely modified with N1-methyl-pseuduridine.

[0353] 90. The therapeutic composition according to any one of embodiments 81 to 89, wherein the at least one mRNA comprises a 5'UTR, a 3'UTR, a 5' cap, and / or a poly(A) tail.

[0354] 91. The therapeutic composition according to Embodiment 90, wherein the 5' cap is a cap 1 structure or an m7GpppG cap.

[0355] 92. The therapeutic composition according to any one of Embodiments 81 to 91, wherein the mRNA comprises a coding sequence encoding at least one antigen protein.

[0356] 93. The therapeutic composition according to any one of embodiments 81 to 92, wherein the at least one mRNA comprises an optimized sequence; optionally, the 5'UTR, 3'UTR sequences, and / or coding sequences for the antigen protein are optimized.

[0357] 94. The therapeutic composition according to any one of Embodiments 83 to 93, wherein the lipid nanoparticles include a cationic lipid, a PEG-modified lipid, cholesterol, and / or a non-cationic lipid.

[0358] 95. The therapeutic composition according to any one of Embodiments 81 to 94, wherein the antigen protein is associated with a disease and is optionally an infectious disease-associated antigen protein or a tumor-associated antigen protein.

[0359] 96. The therapeutic composition according to Embodiment 95, wherein the infectious disease-related antigen protein is a viral antigen protein, a bacterial antigen protein, a fungal antigen protein, a prion antigen protein, or a parasitic antigen protein.

[0360] 97. The therapeutic composition according to Embodiment 96, wherein the viral antigen protein is a coronavirus or HIV antigen protein, preferably a SARS-CoV-2 antigen protein.

[0361] 98. The therapeutic composition according to Embodiment 97, wherein the SARS-CoV-2 antigen protein is derived from a spike protein, an envelope protein, a nucleocapsid protein, a membrane protein, and / or an Orf1ab polyprotein.

[0362] 99. The therapeutic composition according to Embodiment 96, wherein the bacterial antigen protein is an antigen protein of the Borrelia species or the Mycobacteria species.

[0363] 100. The therapeutic composition according to Embodiment 96, wherein the fungal antigen protein is the antigen protein of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans.

[0364] 101. The therapeutic composition according to Embodiment 96, wherein the parasitic antigen protein is the antigen protein of Plasmodium malariae.

[0365] 102. The therapeutic composition according to Embodiment 96, wherein the tumor-associated antigen protein is leukemia antigen protein, melanoma antigen protein, lymphoma antigen protein, endometrial cancer antigen protein, kidney cancer antigen protein, brain tumor antigen protein, cervical cancer antigen protein, liver cancer antigen protein, head and neck cancer antigen protein, gastrointestinal cancer antigen protein, lymph node cancer antigen protein, pancreatic cancer antigen protein, otolaryngological (ENT) cancer antigen protein, breast cancer antigen protein, prostate cancer antigen protein, ovarian cancer antigen protein, or lung cancer antigen protein.

[0366] 103. The therapeutic composition according to any one of Embodiments 81 to 102, wherein the at least one mRNA comprises a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, or 99%, or 100%, identical to a sequence or a portion of a sequence selected from the group comprising SEQ ID NO: 1 (spike protein transcript), SEQ ID NO: 2 (envelope protein transcript), SEQ ID NO: 3 (nucleocapsid protein transcript), SEQ ID NO: 4 (membrane protein transcript), and / or SEQ ID NO: 5 (Orf1ab polyprotein transcript).

[0367] 104. The therapeutic composition according to any one of embodiments 81 to 103, wherein the phDCs are produced by subjecting monocytes obtained from a donor to physical force before combining them with the at least one mRNA.

[0368] 105. The therapeutic composition according to Embodiment 104, wherein the physical force is applied by passing the monocytes obtained from the donor through a flow chamber.

[0369] 106. The therapeutic composition according to Embodiment 104 or 105, wherein the monocytes obtained from the donor are derived from an in vitro blood sample or PBMC from the donor.

[0370] 107. The therapeutic composition according to any one of embodiments 104 to 106, wherein the flow chamber is a plate.

[0371] 108. The therapeutic composition according to any one of embodiments 104 to 106, wherein the flow chamber is a bag, optionally a flexible bag or a plastic bag.

[0372] 109. The therapeutic composition according to any one of Embodiments 104 to 108, comprising the further step of incubating the at least one mRNA together with the phDC.

[0373] 110. The therapeutic composition according to Embodiment 109, wherein the incubation step is carried out for at least 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, or 24 hours.

[0374] 111. An antigen-specific phDC or therapeutic composition for use in a method of treating a disease in a subject, wherein the subject has been previously diagnosed with a disease, and the method comprises administering the antigen-specific phDC or therapeutic composition to the subject, as described in Embodiment 80 or any one of Embodiments 81 to 110.

[0375] 112. The antigen-specific phDC or therapeutic composition for use according to Embodiment 111 is administered subcutaneously, intravenously, intramuscularly, intraarticularly, intrasacrally, intrasternally, intrathecally, intrahepatically, intralesionally, intracranially, percutaneously, intradermally, intrapulmonaryly, intraperitoneally, intracardiacly, intranasally, or sublingually; preferably intradermally, intravenously, or intramuscularly.

[0376] 113. The disease is caused by disease-causing particles, and optionally the disease-causing particles are viruses, bacteria, fungi, parasites, and / or tumor cells, an antigen-specific phDC or therapeutic composition for use according to Embodiment 111 or 112.

[0377] 114. The antigen-specific phDC or therapeutic composition for use according to Embodiment 113, wherein the concentration of the disease-causing particles is lower at the time of infection in the subject compared to the concentration of the disease-causing particles before administration of the therapeutic composition.

[0378] 115. The antigen-specific phDC or therapeutic composition for use according to Embodiment 113, wherein the concentration of the disease-causing particles is lower in a subject compared to the concentration of the disease-causing particles in a second subject that has been previously diagnosed with the same disease, vaccinated for the same disease, and treated with the same disease-causing particles with a different therapeutic treatment.

[0379] 116. The different therapeutic agents are antigen-specific phDCs or therapeutic compositions for use as described in Embodiment 115, wherein the therapeutic agents do not contain dendritic cells.

[0380] 117. The different therapeutic treatment is an RNA vaccine, an antigen-specific phDC or therapeutic composition for use as described in Embodiment 115 or 116.

[0381] 118. The antigen-specific phDC or therapeutic composition for use according to Embodiment 115 or 116, wherein the concentration of disease-causing particles is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% lower in the subject compared to the concentration of disease-causing particles in the second subject.

[0382] 119. The antigen-specific phDC or therapeutic composition for use according to Embodiment 113, wherein the concentration of disease-causing particles is lower systemically in the subject at the time of infection.

[0383] 120. An antigen-specific phDC or therapeutic composition for use according to Embodiment 113, wherein the concentration of disease-causing particles is lower locally, and optionally the concentration of disease-causing particles is lower in the brain.

[0384] 121. An antigen-specific phDC or therapeutic composition for use according to any one of Embodiments 111 to 120, wherein the subject exhibits an increased proportion of central memory T cells and / or stem-like T cells that are specific to at least one antigen protein, compared to the proportion of central memory T cells and / or stem-like T cells in a second subject previously diagnosed with the same disease and treated with the same disease-causing particles with a different therapeutic agent.

[0385] 122. The subject exhibits a reduction in the proportion of exhausted effector T cells compared to the proportion of exhausted effector T cells in a second subject previously diagnosed with the same disease and treated with the same disease-causing particles with a different therapeutic agent, according to any one of Embodiments 111 to 121.

[0386] 123. The subject is an antigen-specific phDC or therapeutic composition for use according to any one of embodiments 111 to 122, for a person previously diagnosed with an infectious disease.

[0387] 124. The subject has been previously diagnosed with a viral infectious disease; preferably, the subject has been diagnosed with COVID-19, an antigen-specific phDC or therapeutic composition for use according to any one of Embodiments 111 to 123.

[0388] 125. The subject is an antigen-specific phDC or therapeutic composition for use according to any one of embodiments 111 to 123, for a person previously diagnosed with having a tumor or cancer.

[0389] 126. The cancer can be classified as stage I, II, III, or IV according to the Tumor Nodule Metastasis (TNM) Anatomical / Prognostic Classification System of the Joint Commission on Cancer (USA) Cancer Staging System, and the antigen-specific phDC or therapeutic composition for use as described in Embodiment 125.

[0390] 127. The subject is an elderly person, an infant, has a chronic medical condition, has recently undergone cancer treatment, has a lung disease, or has immunodeficiency, and is an antigen-specific phDC or therapeutic composition for use according to any one of Embodiments 111 to 126.

[0391] 128. The antigen-specific phDC or therapeutic composition for use according to any one of Embodiments 111 to 127, wherein administration of the antigen-specific phDC described in Embodiment 80 or the therapeutic composition described in any one of Embodiments 81 to 110 additionally provides a vaccine effect in the subject.

[0392] 129. Use of any one of the therapeutic compositions according to Embodiments 113 to 125 for vaccination.

[0393] 130. A kit comprising physiological dendritic cells (phDCs) and at least one mRNA, wherein the at least one mRNA encodes at least one antigen protein.

[0394] 131. The kit according to Embodiment 127, wherein the phDC is obtained by passing monocytes acquired from a donor through a flow chamber.

[0395] 132. The kit according to embodiment 127 or 128, wherein the at least one mRNA encodes at least one viral antigen protein.

[0396] 133. The kit according to Embodiment 127 or 128, wherein the at least one mRNA encodes at least one tumor-associated antigen protein.

[0397] 134. The kit according to Embodiment 129, wherein the viral antigen protein is a coronavirus antigen protein, preferably a SARS-CoV-2 antigen protein.

[0398] 135. The kit according to Embodiment 131, wherein the SARS-CoV-2 antigen protein is derived from a spike protein, an envelope protein, a nucleocapsid protein, a membrane protein, and / or an Orf1ab polyprotein.

[0399] 136. The kit according to any one of embodiments 127 to 132, wherein at least one mRNA is formulated within lipid nanoparticles.

[0400] 137. A method for treating a disease in a subject, the method comprising administering to the subject an antigen-specific phDC described in Embodiment 80 or a therapeutic composition described in any one of Embodiments 81 to 110, wherein the subject has been previously diagnosed with the disease.

[0401] 138. The method according to Embodiment 137, wherein the therapeutic composition is administered subcutaneously, intravenously, intramuscularly, intraarticularly, intrasacrally, intrasacrally, intrasternally, intrathecally, intrahepatically, intralesionally, intracranially, percutaneously, intradermally, intrapulmonaryly, intraperitoneally, intracardiacly, intraarterially, or sublingually; preferably intradermally, intravenously, or intramuscularly.

[0402] 139. The method according to Embodiment 137 or 138, wherein the disease is caused by disease-causing particles, and optionally the disease-causing particles are viruses, bacteria, fungi, parasites, prions, and / or tumor cells.

[0403] 140. The method according to Embodiment 138, wherein the concentration of disease-causing particles in the subject is lower than the concentration of disease-causing particles before administration of the therapeutic composition.

[0404] 141. The method according to Embodiment 138, wherein the concentration of disease-causing particles is lower in a subject compared to the concentration of disease-causing particles in a second subject that has been previously diagnosed with the same disease and treated with the same disease-causing particles with a different therapeutic treatment.

[0405] 142. The method according to Embodiment 141, wherein the different therapeutic procedure does not involve dendritic cells. 143. The method according to Embodiment 141 or 142, wherein the different therapeutic treatment is RNA therapeutic treatment.

[0406] 144. The method according to any one of Embodiments 141 to 143, wherein the concentration of disease-causing particles is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% lower in the subject compared to the concentration of disease-causing particles in the second subject.

[0407] 145. The method according to Embodiment 139, wherein the concentration of disease-causing particles is lower systemically in the subject at the time of infection.

[0408] 146. The method according to Embodiment 139, wherein the concentration of disease-causing particles is lower locally, and optionally the concentration of disease-causing particles is lower in the brain.

[0409] 147. The method according to any one of Embodiments 137 to 146, wherein the subject shows an increase in the proportion of central memory T cells and / or stem-like T cells that are specific to at least one antigen protein, compared to the proportion of central memory T cells and / or stem-like T cells in a second subject that was previously diagnosed with the same disease and treated with the same disease-causing particles with a different therapeutic agent.

[0410] 148. The method according to any one of Embodiments 137 to 147, wherein the subject shows a reduction in the proportion of exhausted effector T cells compared to the proportion of exhausted effector T cells in a second subject previously diagnosed with the same disease and treated with the same disease-causing particles with a different therapeutic agent.

[0411] 149. The method according to any one of embodiments 137 to 148, relating to a subject who has been previously diagnosed with an infectious disease.

[0412] 150. The method according to any one of Embodiments 137 to 148, wherein the subject has been previously diagnosed with a viral infectious disease; preferably, the subject has been diagnosed with COVID-19.

[0413] 151. The method according to any one of embodiments 137 to 139, relating to a subject who has been previously diagnosed with a tumor or cancer.

[0414] 152. The method according to Embodiment 151, wherein the cancer can be classified as stage I, II, III, or IV according to the Tumor Nodule Metastasis (TNM) Anatomical / Prognostic Classification System of the Joint Commission on Cancer (USA) Cancer Staging System.

[0415] 153. The method according to any one of Embodiments 137 to 152, wherein the subject is elderly, an infant, has a chronic medical condition, has recently undergone cancer treatment, has a lung disease, or has immunodeficiency.

[0416] 154. The method according to any one of Embodiments 137 to 153, wherein administration of the antigen-specific phDC described in Embodiment 80 or the therapeutic composition described in any one of Embodiments 81 to 110 additionally provides a vaccine effect in the subject. [Examples]

[0417] Example 1. Preparation of phDC transduced with LNP particles Isolation of murine peripheral blood mononuclear cells (PBMCs) Peripheral blood (100–200 uL / mouse) is collected from experimental mice and control mice (and any additional mice for blood collection if necessary) as needed for the experiment; for example, on days 1 and 7 for prophylactic vaccination and booster studies, or twice a week for the duration of treatment for therapeutic tumor treatment studies. Whole blood is collected in 1:100 5,000 U / mL heparin (McKesson Packaging Services). Platelet-containing peripheral blood mononuclear cells (PBMCs) are isolated from peripheral whole blood by Lympholyte M gradient separation (Cedarlane Labs). Autologous serum is collected from a separate cohort of syngeneic donor mice and stored for subsequent steps (overnight culture).

[0418] Transimnization Chamber A small ECP device suitable for use in animal models, called a transimmunization (TI) chamber, was designed and fabricated by Transimmune AG in collaboration with the Fraunhofer Institute for Biomedical Engineering, Saarland, Germany, for Dr. Edelson's laboratory. The sterile polystyrene TI chamber has external dimensions of 25 x 75 mm, along with a flow path of 18 x 66 mm and a flow path height of 290 ± 15 μm.

[0419] PBMC TI Treatment Protocol Isolated murine platelet-containing platelet-containing microcentrifuges (PBMCs) are resuspended in fetal bovine serum (FBS). The cells are then incubated in a TI chamber at 37°C for 1 hour. This step allows for the precipitation of platelet-activating plasma proteins within the chamber and platelet adhesion to the coated chamber surface, as confirmed by light microscopy. The cells are then passed through the TI chamber at a rate of 0.09 mL / min using a syringe pump. After plate passage, the cells are collected and the TI chamber is washed with 100% FBS at 0.49 mL / min, while physically perturbing the plate surface by lightly tapping or swatting, to help detach any adherent cells from the chamber. The collected cells are washed and cultured overnight under standard conditions in RPMI without phenol red (Gibco), supplemented with 15% autologous mouse serum and 1% penicillin / streptomycin / L-glutamine (Invitrogen).

[0420] phDC mRNA-containing LNP transfection A desired amount of LNP containing mRNA for the antigen of interest (such as SARS-CoV-2 spike protein or sample tumor antigen) (cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16 (Santangelo laboratory, Emory University)) is added directly to the phDC culture overnight during culture setup. In our experience, the amount of LNP can vary from 20ug to 10ng depending on the LNP and antigen used.

[0421] Reintroduction of phDC into laboratory animals The following day, cells transfected with LNP were collected by scraping, washed, resuspended in sterile PBS (Gibco), and administered intravenously at a rate of 100 μL / animal via the posterior orbital plexus.

[0422] Example 2. Internalization of viral antigen-containing LNPs into phDCs method PhDCs are prepared from the blood of healthy C57BL / 6 donor mice according to the standard protocol described in Example 1. A desired amount of LNP containing mRNA for the viral antigen of interest (SARS-CoV-2 spike protein mRNA), such as 20 ug / mL of cKK-E12-based LNP containing SARS-CoV-2 spike protein (LNP prepared and provided by Santangelo Laboratory, Emory University, mRNA sequence corresponding to SEQ ID NO: 19), is added to plate-passing PBMCs under culture. The plate-passing PBMCs are incubated overnight with the LNP to allow LNP uptake and antigen expression.

[0423] After an overnight incubation, a suitable fluorescently labeled detection antibody (anti-human SARS-CoV-2 spike protein antibody, generously provided by Santangelo Laboratory, Emory University), as well as any necessary antibodies to identify cells of interest (e.g., CD11b against murine phDCs in plate-passing PBMCs) were applied.+ Ly6G - Antigen expression is monitored using the following: CD11b Biolegend clone M1 / 70; Ly6G Biolegend clone 1A8).

[0424] Antibody binding to phDC can be detected by either flow cytometry (analysis using a Cytoflex cytometer and FlowJo v10 software) or confocal microscopy.

[0425] result Murid phDCs, among all other immune cell subsets contained in PBMCs, specifically internalize cKK-E12 LNPs and efficiently express spike proteins encoded by mRNA contained in such LNPs. FACS analysis showed that CD11b was superior in cultures without LNP transfection compared to cultures without LNP transfection. + PhDC specifically shows positivity for the spike protein (Figure 1A, B). This was confirmed by confocal microscopy analysis, and CD11b + Ly6G - phDCs specifically exhibit spike protein expression on the cell surface and in the cytoplasm (projected Z-stack images, Figure 1C, D; Z-planar slice image, Figure 1E).

[0426] Example 3. Therapeutic phDC administration in an EG7-OVA lymphoma mouse model using tumor antigen mRNA-containing LNP (ovalbumin (OVA) antigen). The experimental design is schematically shown in Figure 2A.

[0427] For tumor induction, 3 × 10 6 Individual EG7-OVA tumor cells are subcutaneously injected into the right flank of recipient wild-type C57BL / 6J mice at a dose of 100 mL. Therapeutic treatment is initiated on day 2 post-tumor transplantation. For each treatment, phDC is prepared from the blood of each experimental group (200 μL of blood collected per animal) according to the standard protocol described in Example 1. Blood from groups not treated with phDC is discarded.

[0428] To plate-passed PBMCs under culture, 10 ng (0.5 ug / kg LNP, equivalent dose to the COVID-19 Pfizer mRNA LNP vaccine in the current human vaccine setting) of cKK-E12-based LNP (SEQ ID NO: 20, also used in all subsequent experiments related to OVA mRNA-containing LNPs) containing mRNA for the tumor antigen of interest, here, ovalbumin (OVA), a model antigen introduced into EL4 lymphoma cells to produce an EG7-OVA tumor model, is added. All mRNAs and LNPs are prepared and provided by the Santangelo laboratory, Emory University.

[0429] After overnight incubation, cells co-cultured with LNP containing OVA mRNA are collected and intravenously injected into the posterior orbital plexus of "phDC" treated experimental animals in 100 μL volume of sterile PBS. Simultaneously, an equivalent dose of LNP containing OVA mRNA in 10–50 μL volume of sterile PBS is injected into the thigh of each animal in the "IM" treated experimental group. Animals in the "untreated" ("no Tx") group receive no therapy.

[0430] The treatment is repeated twice a week until the experiment is completed, determined by the maximum tumor growth rate permitted by the animal breeding facility. Mice are typically collected for phDC therapy on Mondays and Thursdays, and phDC or im treatment is performed on Tuesdays and Fridays. Typically, 5-6 therapeutic immunization treatments can be performed every other week.

[0431] Tumor volume is monitored by bi-weekly measurements of the vertical tumor diameter and height using a measuring instrument, and the tumor volume is calculated as (tumor length × width × height) / 2.

[0432] For further analysis, splenocytes were collected from all treatment and control groups at the end of the experiment (30 days after tumor inoculation) to characterize the resulting anti-tumor immune response. Elispot analysis: CD8+ splen T cells from treated mice (negative selection using Miltenyi CD8 T cell isolation kit) were analyzed at a rate of 1*10 per well in the presence or absence of 10 ug / mL SIINFEKL peptide. 5 Individual cells are immediately subjected to an 18-hour IFN-g Elispot assay. Antigen (SIINFEKL)-specific CD8 T cell evaluation: CD8 (BioRad clone KT1.5) and specific H2d-SIINFEKL dextramer staining (Immudex), followed by flow cytometry (Cytoflex). Tem (effector) / Tcm (central memory) type phenotypic analysis: Staining for CD44 (Biolegend clone IM7) and CD62L (Biolegend clone MEL-14) expression on antigen-specific T cells, followed by flow cytometry (Cytoflex). Stem-like T cell analysis: Staining for IL7Ra (Biolegend clone A7R34) and SCA-1 (Biolegend clone E13-161.7) expression on antigen-specific T cells, followed by flow cytometry (Cytoflex). Evaluation of T cell exhaustion markers: PD-1 (Biolegend clone 29F.1A12) expression on antigen-specific T cells, followed by flow cytometry (Cytoflex).

[0433] result Compared to the untreated control group ("no Tx"), therapeutic phDC treatment with a clinically relevant dose of 10 ng OVA mRNA-containing LNP (0.5 ug / kg LNP, equivalent to the dose of the COVID-19 Pfizer mRNA LNP vaccine in the current human vaccine setting) successfully controlled EG7-OVA tumor growth, while direct intramuscular administration of the same amount of OVA mRNA-containing LNP ("IM" group) did not significantly alter the kinetics of tumor growth (Figure 2B).

[0434] To better understand the therapeutic effects of the phDC versus IM treatment method, splenocytes were collected and further analyzed at the end of the experiment (30 days after tumor inoculation) (Figure 3A-C, schematic diagram of the experiment). The overall responsiveness to the OVA immunodominance peptide SIINFEKL, measured by IFNg Elispot analysis of splenocytes from various groups, showed a partial response in the control untreated group, as expected for mice with OVA-expressing tumors; a stronger response in the phDC-treated group; and, interestingly, the strongest response in the IM-treated group, despite this response being clinically unproductive (Figure 2B) (Figure 3D-E).

[0435] Therefore, it became clear that it is not the overall quantity of the model tumor antigen OVA-specific T cell response that controls tumor growth. Thus, to explain the strong therapeutic effect of phDC treatment, it had to be the quality of the T cell response generated. The quality of the T cell response at day 30 in splenocytes of treated mice with tumors was further investigated. This analysis demonstrated that antigen-specific T cells in phDC-treated mice exhibit a phenotype characterized by the presence of central memory T cells and stem-like T cell subsets associated with productive anti-cancer immune responses in human and mouse studies (Figure 4A). In contrast, T cells from the IM-treated group showed a phenotype of strong effector T cell dominance (Figure 4A), which is consistent with the strong response observed in the IFNg Elispot assay (Figures 3D-E).

[0436] This effector-like response generated by IM treatment appears clinically unproductive in tumor settings, likely due to the potentially exhausted state of such effector T cells, as evidenced by their high levels of PD1 expression compared to antigen-specific T cells derived from phDC-treated animals (Figure 4B).

[0437] Example 4. Analysis of spontaneous immune response in prophylactic phDC vaccination using LNPs containing ovalbumin (OVA) sample antigen mRNA. method For each treatment, phDC was prepared from the blood of healthy C57BL / 6 mice from each experimental group (200 μL of blood collected per animal) according to the standard protocol described in Example 1. Blood from groups that were not treated with phDC was discarded.

[0438] To plate-passed PBMCs under culture, 6ug of cKK-E12-based LNPs containing mRNA for the tumor antigen of interest, in this case the model antigen ovalbumin (OVA), are added. All LNPs are prepared and provided by the Santangelo laboratory, Emory University.

[0439] Plate-passed PBMCs are incubated overnight with LNP to enable LNP uptake and antigen expression. The following day, PBMCs are collected and prepared as described in Example 1 and administered intravenously at 100 μL / animal via the posterior orbital plexus to the "phDC[ova]" vaccinated group. In parallel, an equivalent dose of LNP containing OVA mRNA is injected intramuscularly ("IM[ova]") into the thigh of each animal in the "untreated" group in 10–50 μL volume of sterile PBS. Animals in the "untreated" group receive no therapy.

[0440] Mice were given blood samples on experimental days 8 and 1, and prophylactically vaccinated with either phDC or intramuscular LNP on days 7 and 0. Whole undifferentiated splenocytes were collected on days 0, 5, and 13 post-vaccination for analysis of the spontaneous immune response using IFNg Elispot. Elispot analysis: Whole undifferentiated splenocytes from all experimental groups were measured at a rate of 1*10⁶ per well in the absence of any additional stimulation. 5 Individual cells were immediately subjected to an 18-hour IFN-g Elispot assay. To further identify the cells involved in IFNg production, some day 13 samples were further fractionated into CD8 T cells (negative selection using the Miltenyi CD8 T cell isolation kit) or NK cells (positive selection using the Miltenyi NK1.1 cell isolation kit).

[0441] result Interestingly, animals prophylactically vaccinated and boosted with phDC, rather than by intramuscular LNP injection, exhibit spontaneous IFNg production in the absence of any additional stimulation (Figure 5A-C). This spontaneous response is detectable for at least 13 days after vaccination (Figure 5C).

[0442] Further analysis of the source of this spontaneous IFNg signaling, through isolation of either CD8 T cells or NK cells, identified NK cells as a subset of IFNg-secreting cells. This suggests that phDC vaccination not only initiates antigen-specific T and B cell responses (Figures 4 and 5), but also broadly involves the innate immune system, including NK cells.

[0443] Example 5. Prophylactic phDC vaccination in an EG7-OVA lymphoma mouse model using tumor antigen mRNA-containing LNP (ovalbumin (OVA) antigen). The experimental design is schematically shown in Figure 6A.

[0444] For each vaccination procedure, phDC was prepared from the blood of each experimental group (200 μL of blood collected per animal) according to the standard protocol described in Example 1. Blood from groups not treated with phDC was discarded.

[0445] For the initial vaccination (-14 days), 1 ug of cKK-E12-based LNP containing mRNA for the tumor antigen of interest, in this case ovalbumin (OVA), a model antigen introduced into EL4 lymphoma cells to produce an EG7-OVA tumor model, is added to plate-passed PBMCs under culture. All LNPs are prepared and provided by the Santangelo laboratory, Emory University. After overnight incubation, cells co-cultured with the LNP containing OVA mRNA are collected and intravenously injected into the posterior orbital plexus of "phDC"-treated experimental animals in 100 uL of sterile PBS. Simultaneously, 50 ug of soluble OVA protein in 100 uL of sterile PBS is injected into the posterior orbital plexus of "soluble OVA"-treated experimental animals.

[0446] Following the initial vaccination, a booster vaccine is administered one week later (-7 days later), using the same format as described above.

[0447] Regarding the tumor burden on day 0, 1 × 10 6 EG7-OVA tumor cells were subcutaneously injected in 100 mL doses into the right flank of mice from all treatment groups. Tumor volume was monitored by bi-weekly measurements of the vertical tumor diameter and height using a measuring instrument, and the tumor volume was calculated as (tumor length × width × height) / 2.

[0448] result Prophylactic phDC vaccination using OVA mRNA-containing LNPs successfully inhibited the growth of EG7-OVA tumors, while vaccination with soluble OVA ("soluble OVA" group) did not protect against EG7-OVA tumor burden (Figure 6B).

[0449] Experiment 6. Detection of OVA protein in OVA mRNA LNP-transfected phDCs. method PhDCs are prepared from the blood of healthy C57BL / 6 donor mice according to the standard protocol described in Experiment 1. A desired amount of LNP containing mRNA for the antigen of interest, such as 1 ug / mL of cKK-E12-based LNP containing OVA or SARS-CoV-2 spike protein mRNA (LNP prepared and provided by Santangelo Laboratory, Emory University), is added to plate-passing PBMCs under culture. The plate-passing PBMCs are incubated overnight with the LNP to allow LNP uptake and antigen expression.

[0450] After overnight incubation, antigen expression is monitored by intracellular staining of cells with a fluorescently labeled anti-OVA antibody (Rockland Immunochemicals) and any necessary antibodies to identify cells of interest (e.g., CD11b+ against murine phDCs in plate-passing PBMCs; CD11b Biolegend clone M1 / 70).

[0451] Antibody binding to phDC can be detected by flow cytometry (analysis using a Cytoflex cytometer and FlowJo v10 software).

[0452] result Muridae phDCs specifically express the OVA protein encoded by the mRNA contained in cKK-E12 LNP, among all other immune cell subsets contained in PBMCs. FACS analysis specifically shows OVA protein positivity in CD11b+ phDCs compared to cultures using mock (spike protein) LNP transfection (Figure 7).

[0453] Experiment 7. Detection of SIINFEKL-bound MHC I (H-2Kb) on soluble OVA, OVA-expressing tumor cells, or mouse phDCs carrying OVA mRNA LNP. method PhDCs are prepared from the blood of healthy C57BL / 6 donor mice according to the standard protocol described in Experiment 1. Antigen sources such as soluble OVA protein (10 ug / mL; 50 ug / mL); EG7-OVA tumor cells treated with 8-MOP / UVA (200 ng / mL 8-MOP, UVADEX, Therakos; 2 or 4 J / cm2 UVA); or LNPs containing OVA protein mRNA (1 ug / mL or 5 ug / mL cKK-E12-based LNPs) (LNPs prepared and provided by Santangelo laboratory, Emory University) are added to plate-passing PBMCs under culture. Plate-passing PBMCs are incubated overnight with their respective antigen sources to allow for antigen uptake and / or expression, as well as processing.

[0454] After an overnight incubation, the antigenic OVA peptide SIINFEKL presentation on phDC H-2Kb MHCI molecules is detected by staining the cells with a fluorescently labeled 25.D1 TCR-like antibody (Biolegend, clone 25-D1.16), as well as any necessary antibodies to identify cells of interest (e.g., CD11b+, Ly6G-; CD11b Biolegend clone M1 / 70; Ly6G Biolegend clone 1A8) against murine phDCs in plate-passing PBMCs.

[0455] Antibody binding to phDC can be detected by flow cytometry (analysis using a Cytoflex cytometer and FlowJo v10 software).

[0456] result Murid phDCs, among all other immune cell subsets contained in PBMCs, specifically process and present the antigenic OVA SIINFEKL peptide in their MHCI molecules. Antigen peptide presentation is significantly higher when the OVA protein is expressed via mRNA LNPs, in contrast to delivery via soluble OVA protein or dystrophic tumor cells expressing the OVA protein (Figure 8).

[0457] Experiment 8. Time kinetics of surface SIINFEKL-MHC I complex expression (25. D1 ab staining) in phDCs transduced with OVA mRNA LNP. method PhDC is prepared from the blood of healthy C57BL / 6 donor mice according to the standard protocol described in Experiment 1. A desired amount of LNP containing mRNA for the antigen of interest, such as 1 ug / mL cKK-E12-based LNP containing OVA protein mRNA (LNP prepared and provided by Santangelo Laboratory, Emory University), is added to plate-passed PBMCs under culture. The plate-passed PBMCs are incubated with the LNP for 2, 4, 6, 8, 12, or 20 hours, then washed and fixed for staining.

[0458] After incubation for 2–20 hours, antigenic OVA peptide SIINFEKL expression on phDC H-2Kb MHCI molecules is detected by staining the cells with a fluorescently labeled 25.D1 TCR-like antibody (Biolegend, clone 25-D1.16) and any necessary antibodies to identify cells of interest (e.g., CD11b+;CD11b Biolegend clone M1 / 70 against murine phDCs in plate-passing PBMCs).

[0459] Antibody binding to phDCs can be detected by flow cytometry, and the percentage of antibody-positive cells / mean fluorescence intensity (MFI) of positive cells can be quantified (analysis using a Cytoflex cytometer and FlowJo v10 software).

[0460] result The percentage of phDCs exhibiting antigenic OVA SIINFEKL peptide bound to MHCI molecules on their surface, as well as the amount of SIINFEKL / MHCI per cell, increased with phDC incubation time with OVA mRNA LNP (Figure 9). 25.D1 expression levels are shown at various time points in CD11b+ cells (FACS plot, upper panel). The percentage of 25.D1-positive cells and 25.D1 MFI levels in the CD11b+ subset are shown in bar graphs (lower panel).

[0461] Experiment 9. Detection of SIINFEKL-bound MHC I (H-2Kb) on mouse phDC or BMDC transduced with OVA mRNA LNP (with reduced LNP concentration). method PhDCs were prepared from the blood of healthy C57BL / 6 donor mice according to the standard protocol described in Experiment 1. BMDCs were cultured from the bone marrow of healthy C57BL / 6 donor mice according to the standard protocol. Briefly, bone marrow cells were seeded in cell culture medium containing GM-CSF (20 ng / ml); after 5 days, non-adherent cells were washed, re-seeded in fresh GM-CSF supplement medium, and incubated for at least 48 hours; then, non-adherent BMDC cells were removed, washed, and immediately used in the experiment.

[0462] To the plate-transfer PBMCs or BMDCs under culture, add a desired amount of LNP containing mRNA for the antigen of interest, such as 0.1 ug / mL cKK-E12-based LNP containing OVA protein mRNA (LNP prepared and provided by Santangelo Laboratory, Emory University). Incubate the plate-transfer PBMCs or cultured BMDCs with the LNP overnight to allow LNP uptake and antigen expression.

[0463] After incubation, antigenic OVA peptide SIINFEKL expression on phDC H-2Kb MHCI molecules is detected by staining cells with a fluorescently labeled 25.D1 TCR-like antibody (Biolegend, clone 25-D1.16) and any necessary antibodies to identify cells of interest (e.g., CD11b+ against murine phDCs in plate-passing PBMCs; CD11b Biolegend clone M1 / 70; CD11c against murine BMDCs, Biolegend clone N418).

[0464] Antibody binding to phDC or BMDC can be detected by flow cytometry (analysis using a Cytoflex cytometer and FlowJo v10 software).

[0465] result The percentage of phDCs exhibiting antigenic OVA SIINFEKL peptide bound to MHCI molecules on their surface was significantly higher than the percentage of BMDCs exhibiting the same peptide, suggesting that phDCs are superior in transfection with OVA mRNA LNP, antigen expression and processing from LNP mRNA antigen sources, or all of the above (Figure 10).

[0466] Experiment 10. Surface staining of SIINFEKL peptide mRNA versus OVA protein mRNA in transduced phDC 25. D1 staining method PhDCs are prepared from the blood of healthy C57BL / 6 donor mice according to the standard protocol described in Experiment 1. A desired amount of LNP containing mRNA for the antigen of interest is added to plate-passing PBMCs under culture. This includes 1 ug / mL of cKK-E12-based LNP containing OVA protein mRNA, or 0.05, 0.1 ug, 0.5 ug, 1 ug, or 5 ug of LNP containing immunogenic OVA SIINFEKL peptide mRNA (LNPs prepared and provided by Santangelo Laboratory, Emory University). The plate-passing PBMCs are incubated overnight with the LNPs to allow LNP uptake and antigen expression.

[0467] After an overnight incubation, antigenic OVA peptide SIINFEKL expression on phDC H-2Kb MHCI molecules is detected by staining the cells with a fluorescently labeled 25.D1 TCR-like antibody (Biolegend, clone 25-D1.16) and any necessary antibodies to identify cells of interest (e.g., CD11b+;CD11b Biolegend clone M1 / 70 against murine phDCs in plate-passing PBMCs).

[0468] Antibody binding to phDC or BMDC can be detected by flow cytometry (analysis using a Cytoflex cytometer and FlowJo v10 software).

[0469] result The percentage of phDCs exhibiting the antigenic OVA SIINFEKL peptide bound to the MHCI molecule on their surface was higher for phDCs transduced with SIINFEKL peptide mRNA LNP than for phDCs transduced with OVA protein mRNA LNP at the same LNP concentration (1 ug / mL) (Figure 11). Transduction of phDCs with SIINFEKL peptide mRNA LNP is efficient even at much lower LNP concentrations, with surface antigen / MHCI complexes being exhibited using as little as 0.05 ug / mL of LNP. In summary, these data suggest that loading only immunogenic peptides, rather than the whole protein, as antigens onto phDCs can be highly efficient.

[0470] Experiment 11. OT1 Growth Assay: PhDCs pulsed with a titration of LNP or soluble OVA protein are cultured together with OT1 T cells. method PhDCs are prepared from the blood of healthy C57BL / 6 donor mice using the standard protocol described in Experiment 1. Antigen sources such as soluble OVA protein (50 ug / mL; 200 ug / mL) or LNPs containing OVA protein mRNA (cKK-E12-based LNPs at 1, 0.1, 0.01, or 0.001 ug / mL) (LNPs prepared and provided by Santangelo laboratory, Emory University) are added to plate-passing PBMCs under culture. The plate-passing PBMCs are incubated overnight with their respective antigen sources to allow for antigen uptake and / or expression, as well as processing.

[0471] After overnight incubation, antigen-carrying cells were collected and 1*10 5 Individual cells / mL CFSE-labeled OVA-specific OT1 CD8 T cells (isolated from the spleen of C57Bl / 6-Tg(TcraTcrb)1100Mjb / J mice, Jackson Laboratory, recognizing OVA peptide residues 257-264 in an H-2Kb background) were subjected to 2*10 cubic feet in a 96-well plate under standard conditions.5 cells / mL (note that phDCs are not purified; cell numbers are for total PBMCs, 3 - 10% of which are CD11+ phDCs; therefore, the number of phDC cells in PBMCs is at most about 2*10 4 cells / mL) for 3 days. At the end of the co - culture, cells are stained with anti - CD8 antibody to identify antigen - reactive T cells (Biolegend, clone 53 - 6.7).

[0472] Antigen - specific CD8 T cell proliferation, shown by CFSE dilution in OT1 CD8+ T cells, can be assayed by flow cytometry (analysis using a Cytoflex cytometer and FlowJo v10 software).

[0473] Results The percentage of proliferated OVA - reactive OT1 CD8 T cells when cultured with OVA protein mRNA LNP - transfected phDCs is very high even at the lowest LNP concentration (0.001 μg / mL, Figure 12). This demonstrates that LNP - transfected phDCs are extremely potent in stimulating antigen - specific CD8 T cell responses. Furthermore, phDC transfection with the lowest LNP concentration (0.001 μg / mL) is as potent as phDC loading with the highest soluble OVA protein concentration (200 ng / mL) in stimulating CD8 T cell responses, suggesting that mRNA LNP is an extremely good antigen source.

[0474] Experiment 12. OT1 proliferation assay: Culture OT1 T cells with titrated phDCs (1 μg / mL LNP) Methods phDCs are prepared from the blood of healthy C57BL / 6 donor mice by the standard protocol described in Experiment 1. Antigen sources such as LNP containing OVA protein mRNA (1 μg / mL cKK-E12-based LNP, LNP prepared and provided by the Santangelo laboratory, Emory University) are added to the plate-passed PBMCs under culture. The plate-passed PBMCs are incubated overnight with each antigen source to enable antigen uptake and / or expression, as well as processing.

[0475] After overnight incubation, the cells are harvested and, in 96-well plates under standard conditions, with 1 × 10 5 cells / mL CFSE-labeled OVA-specific OT1 CD8 T cells (isolated from the spleen of C57Bl / 6-Tg(TcraTcrb)1100Mjb / J mice, which recognize OVA peptide residues 257 - 264 in the background of H-2Kb, Jackson Laboratory), at 2 × 10 5 cells, 1 × 10 5 cells, 5 × 10 4 cells, 2.5 × 10 4 cells, or 1.2 × 10 4 cells / mL (note that phDCs are not purified; the cell numbers are for total PBMCs, of which 3 - 10% are CD11+ phDCs; therefore, the number of phDC cells in PBMCs is at most about 2 × 10 4 cells, 1 × 10 4 cells, 5 × 10 3 cells, 2.5 × 10 3 cells, or 1.2 × 10 3 cells / mL) and co-cultured for 3 days. At the end of the co-culture, the cells are stained with anti-CD8 antibody to identify antigen-reactive T cells (Biolegend, clone 53-6.7).

[0476] CD8 T cell proliferation, indicated by CFSE dilution in CD8+ T cells, can be assayed by flow cytometry (analysis using a Cytoflex cytometer and FlowJo v10 software).

[0477] result When cultured with titrated OVA protein mRNA LNP-transduced phDCs, the percentage of proliferated OVA-reactive OT1 CD8 T cells remained remarkably high, with as few as 1,200 phDCs per mL (120 cells per well in a 96-well plate) retaining the ability to stimulate proliferation in up to 47% of OT1 CD8 T cells (Figure 13). This further demonstrates that mRNA LNP-transduced phDCs are an extremely potent and efficient stimulator of antigen-specific CD8 T cell responses.

[0478] Experiment 13. Therapeutic phDC administration in an EG7-OVA lymphoma mouse model using tumor antigen mRNA-containing LNP (ovalbumin (OVA) antigen). method The experimental protocol is as described for Experiment 3, with the only difference being that the control mice received the same amount (10 ng / mouse) of mock mRNA LNP (expressing SARS-CoV-2 spike protein unrelated in the EG7-OVA tumor model setting) via intramuscular injection every other week, instead of remaining untreated.

[0479] Therefore, the experimental group is as follows: - Mock (SARS-CoV-2 spike protein) mRNA LNP intramuscular vaccination - OVA mRNA LNP intramuscular vaccination - OVA mRNA LNP phDC posterior orbital vaccination The treatment was administered on days 4, 8, 11, 15, 18, and 25 after EG7-OVA tumor transplantation (Figure 14a).

[0480] result Compared to the control mock treatment group ("mock mRNA IM"), therapeutic phDC administration using a clinically relevant dose of 10 ng OVA mRNA-containing LNP (0.5 ug / kg LNP, equivalent to the dose of the COVID-19 Pfizer mRNA LNP vaccine in the current human vaccine setting) ("OVA mRNA phDC") successfully controlled EG7-OVA tumor growth, while direct intramuscular administration of the same amount of OVA mRNA-containing LNP ("OVA mRNA IM (without phDC)") did not significantly alter the kinetics of tumor growth (Figure 14b).

[0481] Experiment 14. SIINFEKL tetramer analysis and adoptive T cell transfer method For further analysis, splenocytes were collected from all treatment and control groups at the end of Experiment 13 described above (collected at the end of the in vivo tumor monitoring period (32 days after tumor inoculation)) to characterize the resulting anti-tumor immune response.

[0482] Antigen (SIINFEKL)-specific CD8 T cell evaluation: CD8 (BioRad, clone KT1.5) and specific H2d-SIINFEKL dextramer (Immudex) staining of splenocytes isolated above, followed by flow cytometry and analysis (Cytoflex).

[0483] Adoptive T cell transfer: CD3+ T cells (Miltenyi T cell isolation kit) isolated from splenocytes of the three experimental groups in Experiment 13 were transferred to antigen-naive C57BL / 6 mice newly inoculated with EG7-OVA tumors (tumor inoculation as described in Experiment 3) in a 1.5*10 ratio. 7 T cells were intravenously transferred to mice, and tumor development was monitored over a 19-day period.

[0484] result Both therapeutic OVA protein mRNA LNP transduction phDC vaccination ("OVA phDC") and intramuscular OVA protein mRNA LNP vaccination ("OVA IM") successfully induced OVA antigen-specific T cells, as measured by the percentage of H-2Kb SIINFEKL tetramer-positive CD8 T cells (Figure 15a). Mock-treated animals ("Mock IM") also showed some tetramer positivity, reflecting the presence of background innate T immunity in mice with EG7-OVA tumors.

[0485] However, despite the presence of tumor-reactive T cells in all groups, only T cells isolated from the spleen of mice treated with OVA protein mRNA LNP transducible phDCs ("OVA phDCs") conferred protective immunity against EG7-OVA tumors in antigen-naive, untreated mice (Figure 15b). This indicates that only phDC therapy provides true anti-tumor immunity.

[0486] Experiment 15. Detection of the spike protein ELISpot in SARS-CoV-2-reactive T cells. Transduction of human phDCs with spike protein mRNA-containing LNPs Plate-passed PBMCs containing newly synthesized phDCs derived from healthy human donors, produced according to the method described in Experiment 1, were placed in human IFNγ ELISpot plates in sets of three per well, 5*10 per well. 5Individual cells are seeded. Each experimental well is supplemented with 200 μL of culture medium consisting of RPMI without phenol red (Gibco) and 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen), containing 62.5–250 ng / well of cKK-E12-based LNP (cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16) containing spike protein mRNA (LNP prepared and provided by Santangelo Laboratory, Emory University, RNA sequence corresponding to SEQ ID NO: 19). Positive control wells are treated with a duplicate pool of spike class I & II peptides (Miltenyi Biotec, PepTivatorSARS-CoV-2 Prot-S Complete). Negative control wells are treated with 62.5–250 ng / well of cKK-E12-based LNP containing unrelated (e.g., nanoluciferase) protein mRNA.

[0487] Elispot assay readout After overnight incubation, the wells are washed, and IFNγ spots are detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrate according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFUs) for each experimental condition are quantified using the ELISpot plate reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). By convention, an ELISpot signal is considered positive if it exceeds 50 SFUs / million cells and / or if the experimental group signal is at least twice as high as the control (mock) group signal.

[0488] CD4 and CD8 T cell depletion To test the contribution of T cell subsets to cytokine production, CD4 or CD8 T cells can be selectively depleted from PBMCs after plate passaging, but before overnight incubation with LNPs in Elispot wells. T cells are depleted using a standard depletion kit (Miltenyi; CellSep).

[0489] result Figure 16A: An example of phDC ELISpot using PBMCs isolated from a single human donor 4 weeks after SARS-CoV-2 infection. Compared to negative controls, IFNγ release was significantly increased in the presence of phDC[spike]. This demonstrates the detection of human spike-specific T cells via mRNA-transduced phDC in Covid convalescent donors.

[0490] Figure 16B: An example of phDC ELISpot dose response using single-human donor PBMCs 4 weeks after SARS-CoV-2 infection. When LNP [spike] antigen was added at 62.5 ng / well, IFNγ release increased to levels exceeding 150 SFU / million. T cell activation further improved at higher doses, but showed evidence of a response plateau at 250 ng / well. This demonstrates a detectable phDC LNP spike dose response even at low levels of 62.5 ng / well.

[0491] Figure 17A: Eighteen previously vaccinated human donors were screened for SARS-CoV-2 spike antigen in phDC ELISpot. Donors were divided into two cohorts based on whether they had previously been infected with SARS-CoV-2 (black) or not (blue). Statistical analysis was performed using the unpaired two-sided Mann-Whitney U test. This demonstrates that human phDCs transduced with LNP [spike] can distinguish between T-cell responses associated with innate immunity and responses associated solely with vaccination.

[0492] Figure 17B: Eleven previously vaccinated and convalescent human donors were screened for SARS-CoV-2 spike antigen using phDC ELISpot. The dotted line represents the standard threshold cutoff (50 SFU / million cells) for a positive response. This demonstrates that 1) response intensity generally correlates negatively with convalescence duration; and 2) the phDC-induced IFNγ response is persistent and detectable up to one year after Covid infection.

[0493] Figure 17C: Either CD8 or CD4 T cells were depleted from plate-passed PBMCs of vaccinated and convalescent donors before incorporation into a standard 18-hour phDC[spike]ELISpot IFNγ assay. CD8 T cell depletion eliminated most of the phDC[spike] response, while CD4 depletion had minimal effect. This demonstrates that the convalescent phDC[spike]IFNγ response is primarily driven by CD8+ T cells.

[0494] Figure 17D: An example of phDC ELISpot using PBMCs isolated from a single human donor, before and 6 weeks after SARS-CoV-2 infection. Post-infection, IFNγ release increased beyond the positive threshold of 50 SFU / million cells. This demonstrates an increase in phDC-induced IFNγ T cell response after spontaneous Covid infection, thus showing the feasibility and practicality of long-term immune monitoring.

Claims

1. A therapeutic composition comprising a pharmaceutically effective amount of physiological DCs (phDCs) and at least one mRNA containing a coding sequence encoding at least one antigen protein.

2. The therapeutic composition according to claim 1, wherein at least one of the mRNAs is contained in lipid nanoparticles.

3. The therapeutic composition according to claim 1 or 2, wherein at least one mRNA is modified with N1-methyl-pseudridine instead of uridine.

4. The therapeutic composition according to any one of claims 1 to 3, wherein the lipid nanoparticles comprise cKK-E12 lipid, SM-102 lipid, or MC3 lipid.

5. The therapeutic composition according to any one of claims 1 to 4, wherein the aforementioned phDC can be obtained by passing monocytes obtained from a donor through a flow chamber and subjecting the monocytes to shear force.

6. The therapeutic composition according to any one of claims 1 to 5, wherein the at least one antigen protein is an infectious disease-related antigen protein, optionally a viral antigen protein, a bacterial antigen protein, a fungal antigen protein, a prion antigen protein, or a parasitic antigen protein.

7. The therapeutic composition according to claim 6, wherein the viral antigen protein is a coronavirus antigen protein or a human immunodeficiency virus (HIV) antigen protein.

8. The therapeutic composition according to any one of claims 1 to 7, wherein the at least one antigen protein is exogenous with respect to the phDC.

9. The therapeutic composition according to any one of claims 1 to 7, wherein the at least one mRNA comprises a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

1.

10. The therapeutic composition according to any one of claims 1 to 7, wherein the at least one mRNA comprises a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

19.

11. The therapeutic composition according to any one of claims 1 to 9, wherein the at least one mRNA is heterologous mRNA or exogenous mRNA relative to the phDC.

12. The therapeutic composition according to any one of claims 1 to 10, wherein the mRNA is at least partially contained in the phDC.

13. The therapeutic composition according to claim 11, wherein substantially all of the mRNA is contained in the phDC.

14. The therapeutic composition according to claim 12, comprising unintegrated mRNA.

15. The therapeutic composition according to any one of claims 1 to 10, wherein the mRNA is not at least partially incorporated into the phDC.

16. The therapeutic composition according to claim 14, wherein a proportion exceeding 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the mRNA is not incorporated into the phDC.

17. The therapeutic composition according to any one of claims 1 to 16, wherein the at least one antigen protein is a tumor-associated antigen protein.

18. The therapeutic composition according to claim 17, wherein the tumor-associated antigen protein is a blood cancer antigen protein.

19. A therapeutic composition for use in a method of treating an infectious disease, preferably a viral disease, in a subject, wherein the method comprises administering the therapeutic composition to the subject, according to any one of claims 6 to 16.

20. A therapeutic composition for use in a method of treating coronavirus disease, preferably COVID-19, in a subject, wherein the method comprises administering the therapeutic composition to the subject, according to any one of claims 7 to 16.

21. A therapeutic composition for use in a method of treating a tumor or cancer in a subject, wherein the method comprises administering the therapeutic composition to the subject, according to claim 17 or 18.

22. A therapeutic composition for use in a method for treating blood cancer in a subject, wherein the method comprises administering the therapeutic composition to the subject, according to claim 18.

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