Methods for analyzing immune responses and antigen profiling

Abstract

This invention relates to a method for testing a subject's immune response to at least one antigen. The method of this invention can also screen for immunogenic antigens, select subjects for specific protocols based on the tested immune response, and identify or isolate T cells that specifically recognize antigens in the subject.

Description

Invention Field

[0001] This invention relates to methods for testing immune responses. Methods for determining whether a subject is suitable for a particular protocol and methods for identifying or screening antigens are also provided. Furthermore, this invention relates to methods for identifying and / or isolating T cells. Background of the Invention

[0003] Even considering the emergence and promise of cutting-edge technologies, including combinatorial chemistry, high-throughput screening, genomics, and proteomics, the frequency with which new drugs and vaccines enter the market has not increased significantly. The development and biological testing of human therapeutics (such as vaccines or cancer drugs) have traditionally relied on various in vitro assays and small animal models (e.g., mouse and rabbit models) and non-human primate models. A major challenge lies in the translation from testing systems to human immunology. Successful transfer between traditional testing systems and human biology requires a complex understanding of disease pathogenesis and immune responses at various levels.

[0004] The mammalian immune system uses two universal mechanisms to protect the body from environmental pathogens. When pathogen-derived molecules are encountered, an immune response is activated to ensure protection against the original pathogen. The first immune system mechanism is the nonspecific (or innate) inflammatory response. The innate immune system acts by recognizing specific molecules present on the pathogen but not within the body itself. The second immune system mechanism is the specific or acquired (or adaptive) immune response. The innate response is essentially the same for every injury or infection; conversely, the acquired response is tailored to the specific pathogen or type of cancer in question.

[0005] The acquired immune system has evolved specific immunoglobulin (antibody) responses against many different molecules or antigens derived from pathogens. In addition, numerous T-cell receptors (TCRs) are sampled based on their ability to bind processed peptides derived from antigens to proteins of the major histocompatibility complex (MHC) classes I and II on the surface of antigen-presenting cells (APCs) such as dendritic cells (DCs). Acquired immunity is mediated by specialized immune cells known as B and T lymphocytes (or simply B and T cells).

[0006] In particular, dendritic cells (DCs) are considered crucial for initiating and controlling cellular immune responses via T cells, including CD4+ and CD8+ T cells. A rapid and physiological process has recently been developed to generate DCs in vitro by applying physical forces (including interactions with surface-bound platelets) to monocytes (Durazzo et al., 2014 (see, for example, “Induction of Monocyte-to-Dendritic Cell Maturation by Extracorporeal Photochemotherapy: Initiation via Direct Platelet Signaling”) and Ventura et al., 2018 (“Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity”). Since monocytes do not require cytokine induction to differentiate into DCs, it is hypothesized that DCs obtained by exposing monocytes to physical forces (and optionally platelet interactions) more closely resemble the characteristics of naturally occurring dendritic cells. Therefore, DCs obtained through the latter process are referred to in the literature and herein as physiological dendritic cells (phDCs).

[0007] Advances in the design, creation, and testing of more complex therapeutics, particularly mRNA therapeutics, have stalled for various reasons. First, only a limited number of therapeutics can be tested in humans. Second, predicting which immunodominant epitopes are optimal for inducing effective CD4+ and CD8+ T cell responses remains challenging. Therefore, an in vitro assay system that utilizes phDCs (which present specific antigens to T cells) for rapid generation and analysis of subsequent immune responses would be of great importance. Such an assay system could have various uses, such as assessing a patient's individual immune response (and potentially, for example, altering the course of therapy), screening for the most effective antigens that can be included in drug formulations, or identifying or isolating T cells activated upon contact with antigen-specific phDCs. Summary of the Invention

[0008] One object of the present invention is to provide a method for testing the immune response of a subject. Another object of the present invention is to provide a method for identifying immunogenic antigens. Another object of the present invention is to provide a method for screening antigens. Another object of the present invention is to provide a method for determining whether a subject is suitable for a particular protocol, such as a clinical trial or a specific therapy. Finally, an object of the present invention is to provide a method for identifying and / or isolating T cells capable of recognizing antigens from a subject.

[0009] These and other objectives, which will become apparent from the following description, are addressed by the subject matter of the independent claims. Some preferred embodiments of the invention form the subject matter of the dependent claims. Other embodiments of the invention may be obtained from the following description. The invention, as illustratively described below, can be suitably practiced without the presence of any one or more elements or limitations not specifically disclosed herein. The invention is described below with reference to specific embodiments and certain drawings, but is not limited thereto; rather, it is limited only by the claims.

[0010] This invention is based on the inventors' insight that physiological dendritic cells (phDCs) can be used as an effective diagnostic tool for assessing in vitro immune responses. Different types of immune cells (e.g., CD4+ and CD8+ T cells) rely on specialized antigen-presenting cells (such as dendritic cells) for antigen presentation. To date, it has been difficult to obtain sufficient quantities of functional dendritic cells from patients, let alone equip these cells with specific antigens through methods such as mRNA transfection. On the other hand, the inventors have found that phDCs can be rapidly generated from monocytes and, moreover, can be efficiently transfected with defined antigens compared to dendritic cells obtained through different methods. At the heart of this invention is the ability of such modified phDCs to stimulate immune cells such as CD4+ and CD8+ T cells in vitro. Therefore, this invention overcomes the limitation of predicting which epitopes or antigens are optimal for inducing effective CD4+ and CD8+ T cell responses. It is assumed that in vitro assays using antigen-presenting phDCs will closely reflect in vivo immune responses. Therefore, this invention enables researchers or clinicians to predict which antigens or drugs (e.g., prophylactic or therapeutic vaccines) will prove effective in vivo. Other conceivable applications include phenotyping or monitoring immune responses in subjects, guiding patient treatment, adjusting treatment interventions, determining patient prognosis or assessing the likelihood of a patient responding to a particular therapy, or selecting subjects for clinical trials. Furthermore, this invention allows for screening the potency of different antigens in evoking effective immune responses. Therefore, this invention enables the immediate incorporation of identified antigens into existing antigen delivery systems to produce vaccine formulations with a high probability of generating protective cell-mediated immunity. Finally, T cells activated by antigen-presenting phDCs can be isolated and used for therapeutic purposes, such as adoptive T cell transfer.

[0011] First aspect: Methods for testing immune responses

[0012] In a first aspect, the present invention relates to a method for testing an immune response in a subject to at least one antigen in vitro, comprising:

[0013] - Provides a mixture containing antigen-specific phDCs and cells capable of secreting IFNγ;

[0014] - Identify the activation of cells capable of secreting IFNγ.

[0015] Antigen-specific phDCs and cells capable of secreting IFNγ can be provided together or separately and then combined to provide a mixture containing antigen-specific phDCs and cells capable of secreting IFNγ.

[0016] Testing immune response

[0017] In one embodiment, activation of cells capable of secreting IFNγ indicates an immune response. In another embodiment, if cells capable of secreting IFNγ are activated, the subject is determined to have an immune response.

[0018] Activation of IFNγ-secreting cells can be determined by measuring the secretion of cytokines such as IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IFNγ, and / or TNF-α. In one embodiment, activation of IFNγ-secreting cells is determined by measuring IFNγ secretion. Activation of IFNγ-secreting cells is measured after IFNγ-secreting cells have been stimulated with antigen-specific phDCs. In one embodiment, the secretion of one or more of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IFNγ, and / or TNF-α by IFNγ-secreting cells indicates an immune response. In one embodiment, the secretion of IFNγ by IFNγ-secreting cells indicates an immune response. In one embodiment, activation of IFNγ-secreting cells (e.g., measured by cytokine secretion, such as IFNγ secretion) is correlated with the strength of the immune response; for example, stronger cytokine secretion indicates a stronger immune response.

[0019] In some embodiments, cytokine secretion levels are measured in response units per volume of blood. In some embodiments, cytokine secretion levels are measured in response units (e.g., spot-forming units) per cell number (e.g., PBMC number or number of cells capable of secreting IFNγ). In some embodiments, cytokine secretion levels are measured in the amount of cytokine per volume (e.g., pg of IFNγ per ml).

[0020] Activation of cells capable of secreting IFNγ can be measured by comparison with appropriate controls. It should be recognized that negative controls (i.e., samples run without the addition of antigen) and positive controls (i.e., samples run using antigens known to trigger the secretion of IFNγ-secreting factors in cells) will be run under conditions that are otherwise replicated to validate the results of this method.

[0021] An immune response is indicated if cells capable of secreting IFNγ are more activated than cells capable of secreting IFNγ in a negative control sample (e.g., without added antigen or with a mimic antigen). An immune response is not indicated if cells capable of secreting IFNγ are equally or less activated than cells capable of secreting IFNγ in a negative control sample (e.g., without added antigen or with a mimic antigen). In one embodiment, an immune response is indicated if the cytokine secretion (e.g., IFNγ) level of cells capable of secreting IFNγ is greater than 5%, greater than 10%, greater than 15%, greater than 20%, or greater than 25% compared to the cytokine secretion (e.g., IFNγ) level of cells capable of secreting IFNγ in a negative control. In another embodiment, an immune response is indicated if the cytokine secretion (e.g., IFNγ) level of cells capable of secreting IFNγ is greater than 50 SFU / million cells compared to the cytokine secretion (e.g., IFNγ) level of cells capable of secreting IFNγ in a negative control. In another embodiment, if the cytokine secretion (e.g., IFNγ) level of cells capable of secreting IFNγ is greater than 2 times (e.g., 3 times, 4 times, 5 times, or 10 times) compared to the cytokine secretion (e.g., IFNγ) level of cells capable of secreting IFNγ in a negative control, it indicates the presence of an immune response.

[0022] Activation of IFNγ-secreting cells can be determined by any suitable method. In one embodiment, activation of IFNγ-secreting cells is determined by ELIspot, FluoroSpot, FACS, lymphocyte proliferation assay, calcium response measurement, and / or ELISA. In one embodiment, activation of IFNγ-secreting cells is determined by ELIspot.

[0023] Cells capable of secreting IFNγ

[0024] In one embodiment, cells capable of secreting IFNγ include CD8+ T cells, natural killer cells, γδ T cells, natural killer T cells, CD4+ T cells, group 1 innate lymphoid cells, IFN-producing killer dendritic cells, memory CD8+ T cells, and memory CD4+ T cells. In one embodiment, cells capable of secreting IFNγ include T cells (e.g., CD4+ T cells, CD8+ T cells, memory CD4+ T cells, and memory CD8+ T cells). In one embodiment, cells capable of secreting IFNγ include CD8+ T cells or CD4+ T cells. In one embodiment, cells capable of secreting IFNγ include CD8+ T cells. In one embodiment, cells capable of secreting IFNγ include CD4+ T cells. In one embodiment, activated cells capable of secreting IFNγ are specific to at least one antigen.

[0025] In one embodiment, the cells capable of secreting IFNγ do not contain CD4+ T cells, i.e., they are depleted of CD4+ T cells. In one embodiment, the cells capable of secreting IFNγ do not contain CD8+ T cells, i.e., they are depleted of CD8+ T cells. The depletion of CD4+ T cells and / or CD8+ T cells can be performed using standard methods known in the art, such as using a depletion kit (e.g., from Miltenyi or CellSep). In one embodiment, the cells capable of secreting IFNγ are T cells, optionally selected from CD8+-enriched T cells and CD4+-enriched T cells. In one embodiment, the cells capable of secreting IFNγ are rich in CD8+ T cells. In one embodiment, the cells capable of secreting IFNγ are rich in CD4+ T cells. In a preferred embodiment, the cells capable of secreting IFNγ are rich in CD8+ T cells.

[0026] In some implementations, further characterization of cells capable of secreting IFNγ may be useful, especially if they are activated by antigen-specific phDCs. Further characterization may include analyzing one or more of the biomarkers CD4, CD8, and gdTCR (for T cell subset grouping). Further characterization may include analyzing one or more T cell differentiation biomarkers such as CD25, CD27, CD44, CD45RA, CD62L, CXCR3, CXCR5, CCR6, and CCR7. Further characterization may include analyzing one or more cytokine and degranulation biomarkers such as TNFα, IL-17, granzyme, and CD107a. Further characterization may include analyzing one or more T cell activation and exhaustion biomarkers such as CD69, OX40, CD40L, CD200, PD-1, 4.1BB, LAG3, and TIM3. This can be accomplished using, for example, flow cytometry that allows for the analysis of multiple biomarkers or a combination of flow cytometry / mass spectrometry (e.g., CyTOF).

[0027] Cells capable of secreting IFNγ (such as T cells) can also be characterized by single-cell RNA sequencing, which allows for the analysis of gene expression characteristics in these cells.

[0028] Obtain antigen-specific phDC

[0029] Antigen-specific phDCs can be obtained alone (separately) or in a mixture with cells capable of secreting IFNγ.

[0030] In one implementation, antigen-specific phDCs can be obtained through the following steps:

[0031] - To subject monocytes to physical forces;

[0032] - Add at least one antigen.

[0033] This will produce antigen-specific phDCs. An incubation step can be added at any suitable point, such as after the addition of at least one antigen.

[0034] In one implementation, antigen-specific phDCs can be obtained through the following steps:

[0035] - Combine monocytes with at least one antigen;

[0036] - subject a mixture of monocytes and at least one antigen to physical forces.

[0037] This will produce antigen-specific phDCs. An incubation step can be added at any suitable point, for example, after subjecting the mixture of monocytes and at least one antigen to physical forces.

[0038] In these embodiments, the resulting antigen-specific phDCs are combined with cells capable of secreting IFNγ to obtain a mixture of antigen-specific phDCs and cells capable of secreting IFNγ in the first aspect.

[0039] In one embodiment, a mixture of antigen-specific phDCs and cells capable of secreting IFNγ can be obtained through the following steps:

[0040] - Expose the sample containing monocytes and cells capable of secreting IFNγ to physical forces;

[0041] - Add at least one antigen.

[0042] In one embodiment, a mixture of antigen-specific phDCs and cells capable of secreting IFNγ can be obtained through the following steps:

[0043] - Combine a sample containing monocytes and cells capable of secreting IFNγ with at least one antigen;

[0044] - subject a mixture of a sample containing monocytes and cells capable of secreting IFNγ with at least one antigen to physical force.

[0045] The incubation step can be performed at any suitable time and can last for, for example, at least 0.5 h, at least 1 h, at least 2 h, at least 3 h, at least 6 h, or at least 12 h. For example, the incubation step can be performed before physical force is applied (e.g., before subjecting the monocytes to physical force, etc.). 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 for the incubation step. In one embodiment, autologous human plasma is added for the incubation step.

[0046] In another example, a mixture of antigen-specific phDCs and cells capable of secreting IFNγ can be incubated for at least 0.5 h, at least 1 h, at least 2 h, at least 3 h, at least 6 h, or at least 12 h. Incubation can be performed under standard conditions for culturing human cells, such as at 37°C and 5% CO2 in a standard medium such as RPMI-1640. In one embodiment, the standard medium is supplemented 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 standard medium is supplemented with autologous human plasma.

[0047] In one embodiment, the monocytes are autologous. Autologous monocytes can be obtained from the subject. In one embodiment, the monocytes are allogeneic. Allogeneic monocytes can be obtained from a donor, optionally from a blood sample, graft, or tissue sample. In one embodiment, the IFNγ-secreting cells are allogeneic. Allogeneic IFNγ-secreting cells can be obtained from a cell line (e.g., DMF5) or a donor, optionally from a blood sample, graft, or tissue sample. In one embodiment, the IFNγ-secreting cells are derived from a cell line (e.g., DMF5), and the antigen-specific phDCs are autologous.

[0048] In one embodiment, the monocytes or IFNγ-secreting cells are autologous (generating autoantigen-specific phDCs or autologous IFNγ-secreting cells). The graft can be a cell graft, optionally a hematopoietic cell graft or a bone marrow graft.

[0049] In one implementation, the sample containing monocytes and cells capable of secreting IFNγ does not contain CD4+ T cells, i.e., CD4+ T cells are depleted.

[0050] A sample containing monocytes and IFNγ-secreting cells but not CD4+ T cells is subjected to physical force to obtain antigen-specific phDCs and IFNγ-secreting cells. In one embodiment, the CD4+ T cell depletion step is performed before the sample containing monocytes and IFNγ-secreting cells is subjected to physical force. Thus, in one embodiment, the sample containing monocytes and IFNγ-secreting cells but not CD4+ T cells produces a mixture containing antigen-specific phDCs and IFNγ-secreting cells, wherein the mixture does not contain CD4+ T cells.

[0051] In one embodiment, the sample containing monocytes and IFNγ-secreting cells does not contain CD8+ T cells, i.e., CD8+ T cells are depleted. Depletion can be performed before or after subjecting the sample containing monocytes and IFNγ-secreting cells to physical force. In one embodiment, depletion is performed after subjecting the sample containing monocytes and IFNγ-secreting cells to physical force.

[0052] Monocytes can be obtained by any suitable means, for example, by obtaining a blood sample or fraction thereof from a subject or donor. The blood sample or fraction thereof may be, for example, a buffycoat containing leukocytes and platelets. Alternatively, the blood sample or fraction thereof may be isolated peripheral blood mononuclear cells (PMBCs). Cells capable of secreting IFNγ can be obtained by any suitable means, for example, by obtaining a blood or tissue sample or fraction thereof from a subject or donor. The blood sample or fraction thereof may be, for example, a buffycoat containing leukocytes. Alternatively, the blood sample or fraction thereof may be isolated peripheral blood mononuclear cells (PMBCs). Tissue samples may be lymphoid or non-lymphoid tissue. Lymphoid tissue may in particular include the spleen and lymph nodes. Non-lymphoid tissue includes the lungs, intestines, and skin.

[0053] Generate phDC

[0054] Typically, the phDCs of this invention are generated at least from monocytes by applying physical forces to the monocytes. In the context of this invention, monocytes are present in various mixtures or samples. If mentioned...

[0055] - "Exposing monocytes to physical forces";

[0056] - "To subject a mixture of a monocyte and at least one antigen to physical force";

[0057] - "Exposing a sample obtained from the subject, containing monocytes and cells capable of secreting IFNγ, to physical forces"; or

[0058] - "Exposing a mixture of a sample containing monocytes and cells capable of secreting IFNγ and at least one antigen to physical force";

[0059] This means that monocytes or a mixture or sample move through a flow chamber, move within a bag, or move through a combination of a flow chamber and a bag. This movement applies a physical force to the monocytes, inducing their differentiation into phDCs. In a preferred embodiment, the physical force is a shear force. The flow chamber can be a plate or a device flow chamber, such as a large ECP device, like a clinical ECP device, or a miniaturized ECP device, such as a transimmunization plate as described in WO2017 / 005700A1. Combinations of flow chambers and bags (e.g., hybrid bags and flow chambers) can also be used to apply physical forces (e.g., shear forces). Methods for preparing phDCs and the term itself have previously been described, for example, in Hanlon et al., 2020 (“Rapid Production of Physiologic Dendritic Cells (phDC) for Immunotherapy”). In one embodiment, phDCs are obtained by a cytokine-independent method.

[0060] In one embodiment, the antigen-specific phDC of the present invention can be obtained by a method comprising combining activated phDC with exogenous mRNA encoding at least one cytokine. In one embodiment, the method includes incubating phDC with an LNP containing exogenous mRNA encoding at least one cytokine for a period of time sufficient for the phDC to take up the LNP. In one embodiment, incubating the phDC with the LNP means adding the LNP to the phDC. In one embodiment, the period of time sufficient for the phDC to take up the LNP is at least 0.1 h.

[0061] The statement that "the phDC of the present invention is generated from at least monocytes" implies that the presence of, for example, platelets may enhance the process. Platelets may be derived from a blood sample of the subject or a fraction thereof, or provided separately. Alternatively, plasma components may be present, which may be derived from a blood sample of the subject or a fraction thereof, or provided separately.

[0062] The flow chamber, bag, plate, or combination of flow chamber and bag 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 mixed 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 mixed flow chamber is coated with autologous human plasma.

[0063] At least one antigen

[0064] In one embodiment, at least one antigen comprises a nucleic acid, peptide, protein, cell extract, or apoptotic cell. In one embodiment, the nucleic acid comprises DNA or RNA encoding at least one antigen. In a preferred embodiment, at least one antigen is (in the form of) at least one RNA, and optionally at least one antigen is (in the form of) at least one mRNA. After a monocyte or phDC is combined with an antigen, the monocyte or phDC is able to take up, process, and present the antigen on its surface. This process is also referred to as “loading”. Such loaded phDCs can elicit an effective immune response. Loaded phDCs are referred to herein as antigen-specific phDCs. At least one antigen may be loaded as the antigen itself (e.g., protein, peptide, epitope, cell, cell extract, apoptotic cell, cell or tissue lysate, virus, viral particle, etc., including immunogenic fragments of each of the foregoing) or may be loaded as a nucleic acid (e.g., DNA or RNA) encoding the antigen.

[0065] In one embodiment, at least one antigen may be included in a drug such as a Covid-19 vaccine. Therefore, in some embodiments, at least one antigen includes a vaccine antigen or a therapeutic antigen. In one embodiment, at least one antigen includes a preventative vaccine antigen or a therapeutic vaccine antigen. A preventative vaccine is a vaccine administered before the onset of illness. A therapeutic vaccine is a vaccine administered after the onset of illness. Therapeutic vaccines work by activating a patient's immune system to fight infection or cancer. In one embodiment, at least one antigen is included in a preventative mRNA vaccine (such as an mRNA coronavirus disease 2019 (Covid-19) vaccine) or a therapeutic mRNA vaccine (such as a therapeutic mRNA vaccine against cancer).

[0066] In one embodiment, at least one antigen comprises a protein or peptide, or mRNA encoding a protein or peptide. In one embodiment, at least one antigen corresponds to at least one RNA, optionally at least one mRNA (encoding at least one antigen). In one embodiment, at least one antigen corresponds to at least one DNA (encoding at least one antigen). In one embodiment, at least one antigen is a tumor neoantigen or cancer neoantigen (i.e., a protein or peptide) or mRNA encoding them. In a preferred embodiment of the invention, at least one antigen is present in the form of at least one mRNA encoding said at least one antigen (i.e., encoding an antigenic peptide or protein).

[0067] The antigens that can be used in the methods of the present invention are described below. It should be understood that the following antigens may exist in the different forms described above, particularly in the form of mRNA encoding the antigen.

[0068] In one implementation, at least one antigen is a disease-related antigen, such as an infectious disease-related antigen or a tumor-related antigen.

[0069] In one embodiment, the infectious disease-associated antigen is a viral antigen, bacterial antigen, fungal antigen, or parasitic antigen. In one embodiment, the infectious disease-associated antigen is a bacterial antigen. In one embodiment, the bacterial antigen is derived from a species of *Borrelia* or *Mycobacteria*. In another embodiment, the infectious disease-associated 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-associated antigen is a parasitic antigen. In one embodiment, the parasitic antigen is derived from *Plasmodium malariae*.

[0070] In one embodiment, the infectious disease-related antigen is a viral antigen. In one embodiment, the viral antigen is a coronavirus antigen or an HIV antigen. In one embodiment, the viral antigen is a coronavirus antigen. In one embodiment, the viral antigen is a beta-coronavirus antigen. In another embodiment, the viral antigen is a SARS-CoV-2 antigen. In one embodiment, the coronavirus antigen or beta-coronavirus antigen is an antigen of the spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or a fragment of each of the foregoing. In one embodiment, the SARS-CoV-2 antigen is an antigen of the spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or a fragment of each of the foregoing. In one embodiment, the SARS-CoV-2 antigen is an antigen of the spike protein or a fragment thereof. 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.

[0071] In one embodiment, at least one antigen is a tumor-associated antigen. In one embodiment, the tumor-associated antigen is a leukemia antigen, melanoma antigen, lymphoma antigen, endometrial cancer antigen, kidney cancer antigen, brain cancer antigen, cervical cancer antigen, liver cancer antigen, head and neck cancer antigen, gastrointestinal cancer antigen, lymph node cancer antigen, pancreatic cancer antigen, ear, nose, and throat (ENT) cancer antigen, breast cancer antigen, prostate cancer antigen, ovarian cancer antigen, or lung cancer antigen. In one embodiment, at least one antigen is a blood cancer-associated antigen. The blood cancer antigen can be a leukemia antigen, lymphoma antigen, or myeloma antigen.

[0072] In one embodiment, at least one antigen is a tumor-associated peptide or protein, and said tumor-associated peptide or protein is a tumor-specific peptide or protein, preferably said tumor-specific peptide or protein comprises at least one tumor-specific neoantigen. In one embodiment, at least one tumor-specific neoantigen is selected from the group consisting of Aatf, Cpne1, Dpagt1, Wbp7, Nle1, Irgq, Zbtb40, Cry1, p15E, Gtf2i, Med12, N4bp2l2, and Syde1 or combinations thereof. In a particular embodiment, at least one tumor-specific neoantigen is a patient-specific tumor-specific neoantigen. In a preferred embodiment, the patient is a human subject. In one embodiment, at least one tumor-specific neoantigen is a patient-specific antigen. In one embodiment, the tumor-specific neoantigen comprises one or more epitopes from HPVE6 / E7, Merkel cell polyoma LTA epitopes, tumor-associated antigens (TAAs) (preferably patient-specific TAAs), lineage-restricted differentiation antigens (such as melanoma MART-1 or TRP2), NY-ESO-1, p53, or Ras.

[0073] Regarding a specific implementation where the at least one antigen is present in the form of a nucleic acid (e.g., mRNA).

[0074] In one embodiment, at least one antigen is provided in the form of mRNA encoding said at least one antigen. In this embodiment, the method is used to test a subject's immune response to at least one antigen, said antigen being provided in the form of mRNA to monocytes or phDCs. Using genetically encoded antigens, especially mRNA-encoded antigens, can facilitate the identification of antigens or neoantigens because DNA or RNA (e.g., mRNA) sequences can be rapidly designed and readily incorporated into the methods of the present invention.

[0075] If at least one antigen is provided in the form of mRNA encoding said at least one antigen, then monocytes or phDCs will take up the mRNA (which may be contained, for example, in nanoparticles such as LNPs), translate the mRNA into a peptide or protein, process it, and present the corresponding peptide antigen on their surface. In other words, at least one antigen can be loaded into monocytes or phDCs by transfection with at least one mRNA encoding said at least one antigen.

[0076] Therefore, in a preferred embodiment, antigen-specific phDCs can be obtained through the following steps:

[0077] - To subject monocytes to physical forces;

[0078] - Add at least one mRNA that contains a coding sequence encoding at least one antigen.

[0079] In one embodiment, platelets and / or plasma components are present when physical force is applied, preferably platelets.

[0080] In another preferred embodiment, antigen-specific phDCs can be obtained through the following steps:

[0081] - Combining a monocyte with at least one mRNA, said mRNA containing a coding sequence encoding at least one antigen;

[0082] - subject a mixture of monocytes and at least one mRNA to physical forces.

[0083] In one embodiment, platelets and / or plasma components are present when physical force is applied, preferably platelets.

[0084] In another preferred embodiment, the mixture of antigen-specific phDCs and cells capable of secreting IFNγ can be obtained through the following steps:

[0085] - Expose the sample containing monocytes and cells capable of secreting IFNγ to physical forces;

[0086] - Add at least one mRNA that contains a coding sequence encoding at least one antigen.

[0087] In one embodiment, platelets and / or plasma components are present when physical force is applied, preferably platelets.

[0088] In another preferred embodiment, the mixture of antigen-specific phDCs and cells capable of secreting IFNγ can be obtained through the following steps:

[0089] - A sample containing monocytes and cells capable of secreting IFNγ is combined with at least one mRNA, said mRNA containing a coding sequence encoding at least one antigen;

[0090] - subject a mixture of a sample containing monocytes and cells capable of secreting IFNγ and at least one antigen to physical force.

[0091] In one embodiment, platelets and / or plasma components are present when physical force is applied, preferably platelets. In one embodiment, at least one mRNA containing a coding sequence encodes at least one infectious disease-associated antigen. In one embodiment, at least one mRNA containing a coding sequence encodes at least one viral antigen, bacterial antigen, fungal antigen, prion antigen, or parasitic antigen. The viral antigen, bacterial antigen, fungal antigen, prion antigen, or parasitic antigen can be any antigen mentioned above or below herein.

[0092] In one embodiment, at least one mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence or a portion thereof selected from the group consisting of: SEQ ID NO: 1 (spike protein transcript), SEQ ID NO: 2 (enveloping 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). In one embodiment, at least one mRNA comprises at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to a sequence or a portion thereof of SEQ ID NO: 1. In one embodiment, the antigen encoded by at least one mRNA comprises 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, at least one mRNA-encoded antigen contains at least 50 amino acids. In one embodiment, at least one mRNA-encoded antigen contains at least 500 amino acids. In one embodiment, at least one mRNA-encoded antigen contains at least 1000 amino acids.

[0093] In one embodiment, the SARS-CoV-2 antigen is a protein or peptide comprising at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical sequences or sequences selected from the group consisting of: SEQ ID NO: 6 (spike amino acid sequence), SEQ ID NO: 7 (enveloping 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 at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical sequences or sequences to SEQ ID NO: 6.

[0094] In one embodiment, at least one antigen is the SARS-CoV-2 hexameric spike protein, having a foldon domain in its extracellular domain immediately above the transmembrane domain (its RNA sequence is shown in bold and underlined in Table 3), or RNA encoding it (e.g., mRNA). The corresponding at least one mRNA may be modified with one or more of N1-methylpseuuridine, a cap1 structure, and a poly-A tail. In one embodiment, at least one mRNA comprises N1-methylpseuuridine (e.g., each uridine is replaced by N1-methylpseuuridine), a cap1 structure, and a poly-A tail. In one embodiment, the codons may be optimized to make at least one mRNA GC-rich, and optimized 5' and 3' UTRs may be used. In one embodiment, at least one mRNA comprises a sequence corresponding to or a portion of 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. In one embodiment, at least one mRNA comprises a sequence or a portion thereof 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 to 400 nucleotides).

[0095] In one embodiment, at least one mRNA containing the coding sequence encodes at least one tumor-associated antigen (TAA). The tumor-associated antigen can be any antigen mentioned above or below. In one embodiment, at least one mRNA encodes the MART1 peptide (a melanoma-associated antigen recognized by T cells-1). In one embodiment, the subject has been diagnosed with a tumor associated with at least one antigen. For example, if at least one antigen corresponds to the MART1 peptide, the subject has been diagnosed with melanoma.

[0096] In one embodiment, the subject has a tumor, and at least one mRNA comprises mRNA derived from said tumor. In another embodiment, the subject has a tumor, and at least one antigen comprises DNA derived from said tumor (e.g., cDNA or genomic DNA). The subject's tumor may correspond to any of the tumors described above, such as leukemia, melanoma, lymphoma, endometrial cancer, kidney cancer, brain cancer, 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. In one embodiment, the tumor is a blood cancer (e.g., leukemia, lymphoma, or myeloma).

[0097] Modified RNA

[0098] In one embodiment, at least one mRNA is a modified mRNA. In one embodiment, at least one mRNA contains at least one chemical modification. If more than one mRNA is used, each mRNA independently contains at least one chemical modification (i.e., the chemical modification from one mRNA to another can be different). 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 its modified form, wherein said modification affects the base moiety within the nucleoside. The modified nucleoside can be a naturally occurring or non-naturally occurring modified nucleoside. Naturally occurring modified nucleosides are preferred.

[0099] It is assumed that replacing A, C, U, and G with naturally occurring or non-naturally occurring modified nucleosides reduces Toll-like receptor (TLR)-mediated immune responses in receptor dendritic cells and / or increases the expression of antigens encoded by mRNA.

[0100] In one implementation, chemical modification is the substitution of one or more nucleosides of mRNA with one or more modifying nucleosides.

[0101] 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).

[0102] Of the four nucleosides A, U, C, and G, it is preferred to replace at least one or more uridine nucleosides in the mRNA with naturally occurring modified uridine nucleosides. It is assumed that the above-mentioned effects on TLR-mediated immune responses and / or the expression levels of antigens encoded by mRNA depend on the degree to which uridine is replaced by naturally occurring modified uridine nucleosides. In a preferred embodiment, all uridine nucleosides in the mRNA encoding the antigen are thus replaced by naturally occurring modified uridine nucleosides. However, the 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 mRNA encoding the antigen are replaced by naturally occurring modified uridine nucleosides. When uridine nucleosides are replaced by their naturally occurring modified forms, pseudouridine is preferred, more preferably N1-methylpseudouridine or N1-ethylpseudouridine. N1-methylpseudouridine is the most preferred. Therefore, the invention considers a particularly preferred embodiment to be an mRNA encoding the antigen in which all uridine is replaced by N1-methylpseudouridine.

[0103] The invention also considers, in addition to uridine nucleosides, the replacement of A, C, and / or G nucleosides with modified forms of A, C, and / or G nucleosides. For such replacements, naturally occurring modified forms of A, C, and / or G are preferred. If such additional replacements are considered, it is preferred that all A, C, and / or G nucleosides be replaced with their modified forms.

[0104] In one embodiment, the 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 mRNA comprises 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 mRNA sequence is optimized. In one embodiment, the sequences of the antigen's 5' UTR, 3' UTR, and / or coding sequence are optimized. In one embodiment, the mRNA sequence has optimized codon usage or optimized G / C content. In one embodiment, codon usage, G / C content, and structural elements are optimized. Optimization of the 3' and / or 5' UTR sequences may additionally include the use of 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, the poly(A) tail is enzymatically added.

[0105] Nanoparticles

[0106] At least one DNA or at least one RNA (e.g., mRNA) may be contained in the nanoparticles. The nanoparticles include polymer nanoparticles, lipid nanoparticles, and lipid complexes. Specifically, the nanoparticles include lipid nanoparticles, lipid complexes, poly(amine-co-ester) particles (PACE), poly-β-amino-ester particles, PACE polyplex particles, lipid complexes, and poly(N,N-cystamine bis(acrylamide)-co-4-amino-1-butanol) (pABOL) particles. In a preferred embodiment, at least one DNA or at least one RNA (e.g., mRNA) is contained in lipid nanoparticles (LNP). The lipid nanoparticles may contain cationic lipids, PEG-modified lipids, cholesterol, DSPE-PEG-maleimide, DSPN-PEG-azide, and / or non-cationic lipids. In one embodiment, the lipid nanoparticles contain cationic lipids, PEG-modified lipids, cholesterol, and / or non-cationic lipids. In another embodiment, the lipid nanoparticles contain cationic lipids, PEG-modified lipids, cholesterol, and non-cationic lipids. Cationic lipids include cKK-E12, cKK-E14, LP01, SM102, Lipid5, MC3 (D-Lin-MC3-DMA), 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. 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 30-40:41-51:1.0-4.0:12-21. In one embodiment, the lipid nanoparticles comprise 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 comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 34-36:45-47:2.2-2.8:15-17. In another embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16.

[0107] In one embodiment, the lipid nanoparticles comprise SM-102, cholesterol, DMG-PEG-2K, and DSPC. In one embodiment, the lipid nanoparticles comprise SM-102, cholesterol, DMG-PEG-2K, and DSPC in a ratio of 45-55:33.5-43.5:0.5-2.5:6-16. In one embodiment, the lipid nanoparticles comprise SM-102, cholesterol, DMG-PEG-2K, and DSPC in a ratio of 48-52:36.5-40.5:1-2:8-12. In one embodiment, the lipid nanoparticles comprise SM-102, cholesterol, DMG-PEG-2K, and DSPC in a ratio of 49-51:37.5-39.5:1.2-1.8:9-11. In one embodiment, the lipid nanoparticles comprise SM-102, cholesterol, DMG-PEG-2K, and DSPC in a ratio of 50:38.5:1.5:10.

[0108] In one embodiment, the lipid nanoparticles comprise MC3, cholesterol, C14-PEG 2000-PE, and DOPE. In one embodiment, the lipid nanoparticles comprise 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 comprise 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 comprise 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.

[0109] Subjects

[0110] It should be understood that in some embodiments, the subject has been vaccinated against a disease, has had the disease, is currently diagnosed with the disease, is at risk of disease recurrence, or is at risk of developing the disease in the future. In these embodiments, at least one antigen may be associated with the corresponding disease to which the subject has been vaccinated, which the subject has had, which the subject is currently diagnosed with, which the subject is at risk of recurrence, or which the subject is at risk of developing the disease in the future. For example, if the subject is diagnosed with melanoma, the preferred antigen included in the method of the present invention is a melanoma-associated antigen, such as MART1.

[0111] In one embodiment, the subject is at high risk of cancer recurrence (e.g., cancer associated with at least one antigen mentioned in the first aspect or specific embodiments). In one embodiment, the subject is receiving immunotherapy (e.g., anticancer vaccination, checkpoint blockade, etc.). In one embodiment, the subject is a mammal, optionally a human.

[0112] Testing more than one subject

[0113] In one embodiment, the method is used to test the immune response of more than one subject (e.g., at least 2, at least 3, at least 6, at least 12, at least 24, at least 36, or at least 48). In this embodiment, statistical methods can be used to classify subjects based on the determined immune response. For example, activation of IFNγ-secreting cells can be determined from a number of subjects (e.g., at least 2, at least 3, at least 6, at least 12, at least 24, at least 36, or at least 48) who differ in at least one characteristic (e.g., whether they have previously been vaccinated; whether they are in the recovery phase; whether they are vaccinated and in the recovery phase or vaccinated and not ill). Based on the activation of IFNγ-secreting cells, a threshold level (e.g., a specific level of IFNγ release) can be determined, which can be used to classify subjects based on at least one characteristic. Characteristics may be, for example, whether a subject has been vaccinated or not; whether a subject has been vaccinated and not ill versus vaccinated and in the recovery phase; whether a subject has previously had the disease. Vaccinated subjects show higher activation of IFNγ-secreting cells than unvaccinated subjects. Vaccinated and diseased subjects showed higher activation of IFNγ-secreting cells than vaccinated and disease-free subjects. Subjects who had previously had the disease showed higher activation of IFNγ-secreting cells than those who had never had the disease. Subjects whose characteristics are unknown can be categorized, for example, as vaccinated / unvaccinated; or vaccinated and disease-free versus vaccinated and in recovery; or diseased versus disease-free, based on the activation threshold level of IFNγ-secreting cells. This disclosure indicates that the method of the present invention can assess whether a subject has been vaccinated against Covid-19 or has been vaccinated and in recovery by detecting (e.g., by ELIspot assay) the activation of IFNγ-secreting cellular responses to the SARS-CoV-2 spike protein antigen provided to phDCs in mRNA form.

[0114] Other purposes and monitoring

[0115] In one embodiment, the method is used to test (e.g., evaluate) a subject's adaptive and / or innate immune response. In one embodiment, the method is used to perform immunophenotyping on a subject.

[0116] In one embodiment, the method is used to evaluate drug efficacy by testing the immune response of a subject. In this embodiment, at least one antigen is included in the drug. The drug may be, for example, an mRNA vaccine such as an mRNA cancer vaccine or any other drug (vaccine / therapeutic agent) mentioned above or below. Activation of cells capable of secreting IFNγ indicates that the test drug will have or is likely to have the desired therapeutic effect in the subject. The absence of activation of cells capable of secreting IFNγ indicates that the test drug will not have or is unlikely to have the desired therapeutic effect in the subject. Therefore, in one embodiment, the present invention relates to a method for guiding therapy. In another embodiment, the method is used to determine the immune function of a subject in response to a drug. In one embodiment, subjects are selected for clinical trials, therapies, or vaccinations based on the measured activation of cells capable of secreting IFNγ. Therapies include, for example, cancer treatments or desensitization therapies.

[0117] In one implementation, the method is performed at multiple time points, such as 2, 3, 4, 5, 6, 7, or more, to analyze the subject's immune response over time. This is particularly useful for monitoring the immune response of subjects receiving a therapy (e.g., a cancer therapy or, for example, a therapy using a prophylactic or therapeutic (mRNA) vaccine) or monitoring the immune response of subjects participating in a (therapeutic or vaccine) trial. Time points can span a day, several days, a week, or even several weeks or years. Time points can also span from before treatment, at the start of treatment, during treatment, to the end of treatment. In principle, the method of the present invention allows for testing the immune response throughout the subject's entire lifespan. This can be important in cases involving long-term protection against cancer treatments or vaccines for infectious diseases of significant importance. Therefore, the method of the present invention contributes to vaccine safety and the effective use of vaccines. Conversely, it may also be meaningful to estimate whether a subject is likely to respond to a therapy before starting a treatment (e.g., a prophylactic or therapeutic (mRNA) vaccine).

[0118] The method of the present invention can also be used to measure the decrease in the titer of IFNγ-secreting cells (e.g., CD8+ or CD4+ T cells, or the sum of both) over time from vaccination or infection, or to measure the corresponding effect of a booster. In one embodiment, a lower level of IFNγ-secreting cells (e.g., CD8+ or CD4+ T cells, or the sum of both) in a subject compared to a suitable control (e.g., a healthy subject) that is responsive to a specific infectious disease-associated antigen (e.g., a spike antigen) indicates that the subject requires vaccination or a booster vaccination against a pathogen associated with that specific infectious disease-associated antigen (e.g., SARS-CoV-2, if at least one of the antigens is a spike antigen).

[0119] The second aspect: Methods for identifying or screening antigens.

[0120] In a second aspect, the present invention relates to a (screening) method for identifying immunogenic antigens in vitro, comprising:

[0121] - Provides a mixture containing antigen-specific phDCs and cells capable of secreting IFNγ;

[0122] - Identify the activation of cells capable of secreting IFNγ.

[0123] All implementation plans in the first aspect are applicable after necessary modifications.

[0124] In one embodiment, activation of cells capable of secreting IFNγ indicates an immunogenic antigen. In one embodiment, activation of cells capable of secreting IFNγ is associated with the immunogenicity of the antigen. Therefore, different antigens can be ranked according to their immunogenicity or potency in evoking an immune response. In one embodiment, the screening method includes testing at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 40, at least 80, or at least 160 different antigens. In one embodiment, the screening method includes testing at least 3 antigens. In one embodiment, the screening method includes testing at least 5 antigens. In one embodiment, the screening method includes testing at least 10 antigens.

[0125] The screening method provided by this invention provides a system that allows for the reliable determination of the protective efficacy of specific antigens, particularly regarding adaptive immune responses. A variety of potential immunogenic antigens can be screened, such as potential drugs or prophylactic or therapeutic (mRNA) vaccines, disease-associated antigens (e.g., tumor-associated antigens, infectious disease-associated antigens, etc.), neoantigens, patient-specific antigens, etc. In one embodiment, the screening method is used to identify immunogenic tumor-associated antigens. In one embodiment, the screening method is used to identify immunogenic patient-specific tumor-associated antigens. In one embodiment, the screening method is used to identify immunogenic neoantigens. In one embodiment, the screening method is used to identify infectious disease-associated antigens.

[0126] Thirdly: Antigens that can be identified through the screening method in the second aspect.

[0127] The present invention also relates to antigens that can be identified by the (screening) method of the second aspect. In this embodiment, the presence of the antigen is associated with the secretion of cytokines by cells capable of secreting IFNγ. In a preferred embodiment, the antigen that can be identified by the (screening) method of the second aspect is a nucleic acid (e.g., mRNA) encoding an antigen, peptide, or protein.

[0128] Antigens that can be identified by the second (screening) method can exist in their complete form or in a partially functional form. This explicitly includes nucleic acids (e.g., mRNA) encoding antigens identified according to the second (screening) method.

[0129] Fourth aspect: Pharmaceutical compositions containing the antigens identified in the third aspect

[0130] The antigens identified in the third aspect can be used in pharmaceutical compositions. The pharmaceutical composition may further comprise pharmaceutically acceptable carriers or diluents. Acceptable carriers or diluents are known in the art. In one embodiment, the pharmaceutical composition additionally comprises phDC. Pharmaceutical compositions comprising the antigens identified in the third aspect can be prophylactic compositions (i.e., vaccines) and / or therapeutic compositions (e.g., therapeutic vaccines). In one embodiment, the pharmaceutical composition induces an immune response against the antigens identified in the third aspect. The immune response can be induced in cells, tissues, or subjects (e.g., humans).

[0131] Fifth aspect: Methods for determining whether a subject is eligible for a specific protocol

[0132] The method used in the first aspect for testing the immune response of a subject can also be used to determine whether a subject is eligible for a specific protocol.

[0133] Therefore, in a third aspect, the present invention relates to a method for determining whether a subject is eligible for a particular protocol, the method comprising:

[0134] - Provides a mixture containing antigen-specific phDCs and cells capable of secreting IFNγ;

[0135] - Identify the activation of cells capable of secreting IFNγ.

[0136] In one embodiment, activation of IFNγ-secreting cells indicates suitability for a particular regimen (e.g., in cancer treatment). In another embodiment, negative or low activation of IFNγ-secreting cells indicates suitability for a particular regimen (e.g., in vaccination). Subjects exhibiting negative or low activation of IFNγ-secreting cells may correspond to subjects at high risk of a disease associated with at least one of the said antigens. Negative or low activation of IFNγ-secreting cells can be determined by comparison with an appropriate control group (e.g., subjects in recovery or subjects known not to be at high risk of a specific disease associated with at least one of the said antigens).

[0137] In one implementation, the subject is a patient. In one implementation, the specific protocol is vaccination, therapy, or clinical trial. Vaccination includes initial and / or booster vaccinations (e.g., second, third, fourth, fifth, etc. vaccinations). In one implementation, the vaccination targets at least one antigen, such as any antigen mentioned in the first aspect or specific embodiments. Therapies include treatments for diseases associated with any antigen mentioned in the first aspect or specific embodiments. For example, a therapy could be cancer treatment, treatment for an infectious disease (e.g., a viral disease, such as SARS-CoV-2), or desensitization therapy.

[0138] In one implementation, the subject is at high risk of an infectious disease (e.g., a viral disease such as SARS-CoV-2). In this implementation, the specific protocol corresponds to a clinical trial involving a SARS-CoV-2 booster vaccination or other vaccination strategies that activate antiviral T-cell responses.

[0139] In one embodiment, the subject is a cancer patient, and the specific regimen is a therapy targeting the cancer. In this embodiment, the method is used to identify cancer patients suitable for a specific therapy targeting the cancer. The cancer may be associated with any antigen mentioned in the first aspect or the specific embodiments. In one embodiment, the specific regimen is a pre-screening of cancer patients at risk of recurrence of said cancer. In this embodiment, activation of cells capable of secreting IFNγ indicates that the subject has an existing reactivity to the antigen. This may further indicate that the subject is suitable for a clinical trial involving antigen-specific phDC vaccination or other treatment strategies involving at least one antigen (e.g., therapeutic vaccination). Available antigens are mentioned in the first aspect and the specific embodiments. It should be understood that the clinical trial, vaccination, or therapy is associated with the selected antigen.

[0140] Sixth aspect: Methods for isolating T cells

[0141] In a sixth aspect, the present invention relates to a method for identifying and / or isolating T cells capable of recognizing antigens from a subject, the method comprising:

[0142] - Provides a mixture containing antigen-specific phDCs and T cells;

[0143] - Determine T cell activation.

[0144] - Identify and / or isolate one or more T cells from the T cell population that recognize antigens presented by antigen-specific phDCs.

[0145] All embodiments of the first aspect are applicable with necessary modifications. In one embodiment, the identified and / or isolated T cells are capable of recognizing one or more antigens from the subject's disease.

[0146] In one embodiment, the subject has a disease, has had a disease, is at risk of disease recurrence, or is at risk of developing a disease in the future. In one embodiment, the subject has a disease. In one embodiment, the T cells are T cells capable of specifically recognizing diseased cells from the subject. Preferably, the T cells are T cells capable of recognizing and killing diseased cells. In one embodiment, the method for identifying and / or isolating T cells capable of recognizing antigens from the subject with the disease is performed in vitro. In a preferred embodiment, the method is used to identify and / or isolate CD8+ T cells.

[0147] Specifically regarding the following methods and steps:

[0148] - Provides a mixture of antigen-specific phDCs and T cells;

[0149] - Determine T cell activation;

[0150] All embodiments of the first aspect are applicable with necessary modifications. The at least one antigen is preferably related to a disease of the subject. Therefore, in one embodiment, the subject suffers from an infectious disease, and the at least one antigen is an infectious disease antigen as described in the first aspect or specific embodiments.

[0151] In one implementation, the subject has a tumor, and at least one antigen is a tumor-associated antigen as described in the first aspect or specific implementation.

[0152] In one implementation, the subject has a tumor, and the identified and / or isolated T cells are capable of recognizing one or more antigens from the subject's tumor.

[0153] In one embodiment, the T cell is a T cell capable of specifically recognizing tumor cells from the subject's tumor. Preferably, the T cell is a T cell capable of recognizing and killing tumor cells from the tumor. Preferably, at least one antigen is a neoantigen, for example, the tumor cell is a tumor cell expressing the neoantigen.

[0154] In one embodiment, the subject is designated as receiving or currently receiving adoptive T-cell therapy. In one embodiment, a T-cell reference tissue culture is established containing duplicate samples of T cells used in the method described in the sixth aspect. This may be useful for isolating T cells from the tissue culture that also demonstrate the ability to recognize antigens in the method described in the sixth aspect. Attached Figure Description

[0155] Figure 1. Detection of human spike-specific T cells by phDC transduced with mRNA in Covid convalescent donors.

[0156] Figure 1B Dose response of human IFN-g Elispot using phDC [spike].

[0157] Figure 2A Human ex vivo phDC assays can differentiate T-cell responses associated with innate immunity. Eighteen previously vaccinated human donors were screened using ex vivo phDC elispot assays against the SARS-CoV-2 spike antigen. Donors were divided into two separate cohorts based on whether they had a prior SARS-CoV-2 infection (black) or were not infected (blue). Elispot responses were reported as spot-forming units (SFU) per million PBMCs. Statistical analysis employed an unpaired two-tailed Mann-Whitney U test.

[0158] Figure 2B Further analysis of a screening cohort of 18 human vaccine donors, with particular focus on convalescent donors (11), is presented in the figure. The correlation between IFNγ response intensity and convalescent duration is shown in the figure. The dashed line in the right figure represents the threshold cutoff for a positive response (50 SFU / million cells). phDC [spike]-induced IFNγ responses are durable and detectable up to one year post-Covid infection. Response intensity is generally negatively correlated with convalescent duration.

[0159] Figure 2C PBMC plates from vaccinated / convalescent donors 1 and 7 had CD8 or CD4 T cells depleted before inclusion in the standard 18-hr phDC [spike] Elispot IFNγ assay. CD8 T cell depletion eliminated most of the phDC [spike] response, while CD4 depletion had the least impact. The convalescent phDC [spike] IFNγ response was primarily driven by CD8+ T cells.

[0160] Figure 2D phDC [spike]-induced IFNγ T cell responses increased after natural Covid infection. *The dashed line indicates the threshold cutoff for a positive response (50 SFU / million cells).

[0161] Figure 3 MART-1-specific IFNγ release can be induced by phDC[MART1]. All three donors produced phDC[MART1]-specific IFNγ in Elispot (A) and DMF5 ELISA (B).

[0162] Figure 4Neoantigen selection and Elispot validation for MC38. This schematic diagram outlines the ELISpot assay procedure used to detect antigen-specific T-cell responses in tumor-bearing mice. Tumor samples underwent whole-exome sequencing (WES) and bulk RNA sequencing to identify potential neoantigens, followed by epitope prediction via a bioinformatic pipeline. Candidate neoantigens were further filtered based on previous literature, resulting in a list of 13 preferred antigens. Lipid nanoparticles (LNPs) encoding these antigens were generated, including single LNPs and LNPs containing a string of antigens. Peripheral blood mononuclear cells (PBMCs) were isolated from tumor-bearing mice, and dendritic cells (phDCs) were generated. The ELISpot assay was then performed by incubating LNPs with phDCs and detecting interferon-γ (INF-g) produced by T cells. Spots corresponding to T-cell activation were quantified to assess the immune response.

[0163] Figure 5 Comparison of antigen-specific T cell responses in PBMC populations with and without CD4+ T cell depletion. PBMCs were isolated from tumor-bearing mice 11 days after MC38 inoculation. ELISPOT T cell responses were assessed using whole PBMCs (A, n=6) or after CD4+ T cell depletion (B, n=5). Counted spots were assessed using a CTL autocounter and plotted as spot-forming units (SFUs) detected under multiple antigen conditions. In the ELISPOT assay, a positive response (gray and black bars) was defined as meeting at least two of the following three classic criteria: 1) greater than 2.5x background (simulant); 2) greater than 50 SFU / 1x10⁻¹. 6 3) After background subtraction, the result is greater than 50 SFU / 1x10 6 .

[0164] Figure 6 Multi-mRNA conjugates capable of inducing phDC-associated T cell stimulation over a wide dose range. (A) Several multi-mRNA constructs were constructed and their stimulatory efficacy was tested in an ELIspot assay. 8x neoantigens were those that had been empirically validated for immunogenicity in the literature, while 4x neoantigens were remaining neoantigens that had only been predicted in the literature but not validated, and p15E was an MC38-associated endogenous retroviral (ERV) antigen. When 8x and 4x conjugates were combined, they were combined at a 1:1 ratio. (B) Single neoantigens (zbtb40 and dpag1) were compared with multiple conjugate formulations or LNP mixtures containing single neoantigens across the entire PBMC at doses ranging from 12.5 ng to 1250 ng. (C) Similarly, CD4-reduced PBMCs were screened against single and conjugate antigens.

[0165] Figure 7 The ELISpot assay was used to detect responses to tumor-associated LCMV antigens in tumor-bearing mice. In the KP-NINJA tumor-bearing mouse model, the ELISpot assay was used to detect T cell responses to the LCMV-derived antigen (GP33). Responses were measured after inoculation with multiple cell doses (2E5 inoculum: 200,000, 5E5 inoculum: 500,000, and 1E6 inoculum: 1,000,000). The assay compared T cell responses to two forms of the antigen: full-length LCMV GP protein and GP33 peptide. CD4 downsampling was performed to enhance the sensitivity of the assay, and CD8 T cell responses were enriched. Detailed Implementation

[0166] 1. Generation of phDC

[0167] The generation of phDCs is achieved by subjecting monocytes (which may be contained in a sample or mixture) to physical forces such as shear forces. Shear forces can be applied by moving the monocytes through a flow chamber, in a bag, or through a combination of flow chambers and bags (hybrid). In one embodiment, platelets and / or plasma components are present when physical forces are applied, preferably with at least additional platelets.

[0168] Optionally, the bag is a flexible bag or a plastic bag. In one embodiment, the bag is a flexible bag. In one embodiment, the bag is a plastic bag. In one embodiment, the material of the flow chamber, bag, or hybrid (e.g., plate, flexible bag, or plastic bag) is plastic. In one embodiment, the material of the flow chamber is a non-plastic material such as glass. If a plastic material is considered for the flow chamber, acrylic resins, polycarbonates, polyetherimides, polysulfones, polyphenylene sulfones, styrene, polyurethanes, polyethylene, Teflon, or any other suitable medical-grade plastics can be used. In a preferred embodiment of the invention, the flow chamber is made of acrylic plastic. If the bag (e.g., a flexible bag) is considered, the material can be plastic, rubber, or silicone. In a preferred embodiment, the material is plastic. Plastic materials include polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymers, ethylene-vinyl alcohol, polyvinylidene fluoride, and / or other plastic-containing materials approved for medical use.

[0169] In some embodiments, the flow chamber comprises or is composed of a plate. The plate can be made of a variety of materials, including but not limited to plastic materials. In one embodiment, the plate is made of plastic. In another embodiment, the plate is made of a non-plastic material such as glass. Non-limiting examples of materials used for the plate include acrylic resins, polycarbonates, polyetherimides, polysulfones, polyphenylene sulfones, styrene, polyurethanes, polyethylene, Teflon, or any other suitable medical-grade plastic. The plate can be rigid or flexible. In some embodiments, the plate may be made of plastic, rubber, or silicone, or composed of plastic, rubber, or silicone. In some embodiments, the plate is elastic, i.e., made of an elastic material. Elastic materials may include cyclic olefin copolymers (COC), polyolefins, polyethylene, fluoropolymers, polyvinyl chloride, ethylene-vinyl acetate copolymers, ethylene vinyl alcohol, polyvinylidene fluoride, polydimethylsiloxane (PDMS), dimethicone, and / or other plastic-containing materials approved for medical use. In one embodiment, the plate is made of PDMS, such as PDMS RTV-615 or PDMS Sylgard 184. In another preferred embodiment of the invention, the plate is made of acrylic plastic.

[0170] In some implementations, a hybrid flow chamber and bag are used to generate phDC. 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, i.e., the bag, plate, or chamber, are as defined herein.

[0171] In one embodiment, the flow chamber, bag, or a combination of flow chamber and bag is made of a material that is adhesive to platelets, plasma components, proteins, etc.

[0172] The flow chamber, bag, or a combination of flow chamber and bag may be coated with platelets. Platelets may be derived from a blood sample of the subject or a fraction thereof or provided separately. Alternatively or additionally, plasma components may be present, which may be derived from a blood sample of the subject or a fraction thereof or provided separately. If provided separately, platelets and / or plasma components may be autologous or heterologous (i.e., already derived from / obtained from the subject or another subject).

[0173] In one embodiment, the cells capable of secreting IFNγ do not contain CD4+ T cells. Preferably, the CD4+ T cell depletion step is performed before the start of the method of the invention. In one embodiment, CD4+ T cell depletion is performed before subjecting a sample containing monocytes and cells capable of secreting IFNγ to the physical forces as described herein.

[0174] Alternatively, in some embodiments, the method of the present invention includes a CD4+ cell reduction step. Therefore, in some embodiments, reduction is performed after or during subjecting a sample containing monocytes and cells capable of secreting IFNγ to the physical forces described herein.

[0175] Therefore, in some embodiments, the method of the present invention includes using cells that do not contain CD4+ T cells but are capable of secreting IFNγ.

[0176] Therefore, in one embodiment, the present invention also relates to a method for testing an immune response in a subject to at least one antigen in vitro, comprising:

[0177] - Provides a mixture containing antigen-specific phDCs and cells capable of secreting IFNγ;

[0178] - Identify the activation of cells capable of secreting IFNγ.

[0179] The cells that can secrete IFNγ do not include CD4+ T cells.

[0180] In one implementation, the reduction of CD4+ T cells increases the sensitivity of the method as described in the first aspect herein.

[0181] In one implementation, when the assay does not provide a positive result, a CD4+ T cell depletion step is used to increase the sensitivity of the assay.

[0182] An LNP containing at least one antigen may be added to a blood sample before, during, or after exposure to shear stress suitable for monocyte activation (non-limiting examples of such shear stress include blood bags, flow chambers, or transimmunoassay chambers). The blood sample may be whole blood, apheresis products, isolated monocytes, or monocyte concentrates. In a preferred embodiment, the blood sample contains concentrated monocytes. In another preferred embodiment, the blood sample is whole blood. Thus, in one embodiment, the LNP is mixed with the blood sample (e.g., concentrated monocytes, apheresis products, or whole blood) before the activation procedure begins, and thus to generate phDC before monocyte processing and activation. In one embodiment, the LNP is added to the blood sample (e.g., concentrated monocytes, apheresis products, or whole blood) before monocyte activation. In another embodiment, the LNP is added to the blood sample (e.g., concentrated monocytes, apheresis products, or whole blood) during monocyte activation. In another implementation, after monocyte activation, LNP is added to a blood sample (e.g., concentrated monocytes, apheresis products, or whole blood) or phDC.

[0183] 2. Determine the activation of cells capable of secreting IFNγ.

[0184] Various methods can be used to identify cells capable of secreting IFNγ, particularly activated T cells. These methods include, but are not limited to, ELISpot, FluoroSpot, flow cytometry, fluorescence activated cell sorting (FACS), lymphocyte proliferation assays, ELISA, and / or assays measuring calcium response, each of which will be described below.

[0185] Enzyme-linked immunosorbent assay (ELISpot) is a focused quantitative measurement of cytokine secretion by a single cell or cell population. ELISpot assays are also a form of immunostaining, as they are classified as a technique that uses antibodies to detect analytes or proteins, such as biological or chemical substances being identified or measured. In a preferred embodiment, activation of cells capable of secreting IFNγ is measured via ELISpot assay.

[0186] The ELISpot works as follows.

[0187] Antibody coating (e.g., attachment, fixation, or coating): Throughout the ELISpot assay, different substances are added to and washed away from the wells. The first substance added to the well may be a cytokine-specific monoclonal antibody (e.g., an IFNγ-specific antibody). These antibodies may coat the well walls for future binding to cytokines. It should be noted that the surface does not have to be a flat surface, such as the surface of the well, but can also be a surface suitable for readout by flow cytometry on beads, which will be described further below.

[0188] Cell incubation: Desired cells (e.g., antigen-specific phDCs and cells capable of secreting IFNγ) are added to wells. Each well may or may not contain a stimulus that activates the secretion of cytokines in the cells (e.g., antigen-specific phDCs or phDCs equipped with or without antigen mimics). During cell incubation, cells are allowed to respond to any present stimuli and secrete cytokines.

[0189] Cytokine capture: As the cells are surrounded by cytokine-specific monoclonal antibodies that coat the pore walls, cytokines secreted by the incubated cells will begin to attach to the antibodies at specific epitopes.

[0190] Detection Antibodies: At this point, the wells can be flushed to remove cells and any other unwanted material. What remains are cytokine-specific monoclonal antibodies and any cytokines that have bound to the antibodies. Biotinylated cytokine-specific detection antibodies can then be added to the wells. These cytokine-specific detection antibodies will bind to any cytokines remaining in the wells, as these cytokines are still attached to the first set of antibodies used.

[0191] Streptavidin-enzyme conjugates: Streptavidin-enzyme conjugates can be added to the wells to bind with detection antibodies. The purpose of adding cytokine-specific detection antibodies to the wells in the pre-biotinylation step is to enable the antibodies to bind to the novel streptavidin-enzyme conjugate. Biotinylation creates a strong affinity between biotin on the cytokine-specific antibody and streptavidin on the conjugate.

[0192] Substrate addition: A substrate (e.g., a chromogenic substrate) can be added to the wells and catalyzed by the enzyme conjugate added in the previous step. This reaction forms an insoluble precipitate, creating spots in the wells. The substrate used in this step can depend on the type of enzyme used in the previous step. If streptavidin-ALP (streptavidin and alkaline phosphatase conjugate) is used, then using BCIP / NBT-plus (a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium chloride) as the substrate will produce more distinct and easier-to-analyze spots. If streptavidin-HRP (streptavidin and horseradish peroxidase conjugate) is used, then using TMB (tetramethylbenzidine) as the substrate will produce better results.

[0193] Analysis: The formed spots can then be read on an automated ELISpot reader or counted under a dissecting microscope and further used to determine cytokine secretion.

[0194] The FluoroSpot assay is a variant of the ELISpot assay. The key difference is that the FluoroSpot assay can analyze the presence of multiple analytes on a single plate. The FluoroSpot assay achieves this by using fluorescence instead of an enzyme-catalyzed reaction for detection. The procedure for the FluoroSpot assay is similar, but there are some differences.

[0195] Antibody coating: Similar to ELISpot, cytokine-specific monoclonal capture antibodies are added to wells in a plate. For FluoroSpot assays, a mixture of different types of capture antibodies is attached to the wells for the detection of multiple types of analytes.

[0196] Cell incubation: Cells or cell populations can be added to wells and incubated with or without stimuli that affect protein secretion.

[0197] Cytokine capture: During the first step, proteins / analytes secreted by incubated cells will bind to capture antibodies that are attached, immobilized, or coated onto the pores.

[0198] Detection antibodies: Similar to ELISpot, once the wells are flushed to remove cells and other substances not needed for identification or measurement, biotinylated detection antibodies (which can be specific to one type of analyte to be quantified) can be added, followed by the addition of tagged detection antibodies for optional second or third types of research analytes.

[0199] Fluorophore-tagged conjugates: FluoroSpot uses fluorophore-tagged anti-tag antibodies and streptavidin-fluorophore conjugates to amplify the detection of multiple analytes, instead of adding streptavidin-enzyme conjugates. A fluorescence enhancer solution can also be added during this step to enhance the signal used later in the fluorescence color in the analysis wells. Unlike ELISpot, this fluorescence allows FluoroSpot to analyze and compare multiple analytes.

[0200] Analysis: Phosphores are analyzed using an automated fluorescence reader with individual filters for the different fluorophores being analyzed. These filters can be selected for specific wavelengths of the fluorophores for accurate measurements.

[0201] The terms flow cytometry and fluorescence-activated cell sorting (FACS) are used interchangeably herein. The principle of flow cytometry is the separation and analysis of cells arranged in a single file as they flow through a detector in a stream. A flow cytometer sorts and counts cells that have been labeled or tagged using fluorescent dyes or other methods. The labels used depend on the lamp or laser used in the analyzer. In addition to cells, molecules such as IFNγ can also be labeled and detected. The lamp or light excites the fluorescent dye, which is then detected by the device. Several labels or fluorophores exist for flow cytometry and are well known in the art. These fluorophores are typically attached to antibodies that recognize target features displayed on or within cells, such as CD8+ or IFNγ.

[0202] Lymphocyte proliferation assays involve measuring the proliferation of lymphocytes (e.g., cells capable of secreting IFNγ) in response to stimuli such as antigen-specific phDCs. This can be achieved, for example, by adding radioactive... 3 The proliferation level was detected using H(trityl)thymidine, the radioactive... 3 H(trityl)thymidine is incorporated into the newly synthesized DNA of dividing cells. The amount of radioactivity incorporated into the DNA is measured using a scintillation counter and is proportional to the number of proliferating cells, which in turn is a function of the number of stimulated lymphocytes. Readings can be, for example, counts per well per minute (cpm).

[0203] ELISA (Enzyme-Linked Immunosorbent Assay) is used to measure the amount of a target (e.g., IFNγ) bound between matched antibody pairs. Target-specific antibodies are pre-coated in the wells of a provided microplate. Samples (e.g., antigen-specific phDCs and cells capable of secreting IFNγ), standards, or controls are then added to these wells and bound to the immobilized (capture) antibody. A substrate solution is added to form a sandwich by adding a second (detector) antibody, which reacts with the enzyme-antibody-target complex to produce a measurable signal. The intensity of this signal is proportional to the concentration of the target present in the original sample.

[0204] Measurements of calcium response can also be used to identify the activation of cells capable of secreting IFNγ, particularly T cells. An early activation event in T cell activation is an increase in intracellular free calcium ion concentration, caused by the release of intracellular stores and subsequent influx of calcium ions from the extracellular space. Calcium signaling begins within the first few seconds or minutes after antigen exposure and can continue for hours. Various methods exist to determine the calcium response of T cells, such as flow cytometry analysis. For example, the fluorescence intensity of T cells preloaded with calcium indicators such as Fluo-3 can be monitored.

[0205] 3. At least one antigen

[0206] At least one antigen may be derived from cells, biopsies, tissue lysates, or any other source containing the antigen. In one embodiment, at least one antigen may be derived from tumor cells or cancer cells. For example, a sample of tumor cells may be taken from a subject with a tumor, such as through a biopsy. In one embodiment, at least one antigen may be derived from cells infected by a pathogen. For example, the sample may be obtained from a subject with an infectious disease. The sample may be further treated with an agent suitable for releasing the antigen, such as a photoactivatable agent in combination with light (especially UV light), such as a combination of 8-MOP and UVA. Apoptotic cells may be obtained, for example, by subjecting cells to a combination of 8-MOP and UVA. In another instance, the pathogen may be cultured in a culture medium. The relevant antigen may be shed from the pathogen into the culture medium and then collected for combination with monocytes or phDCs. Those skilled in the art are aware of the various and further methods known in the art for providing antigens derived from pathogenic particles. Pathogens in the context of this invention include viruses, bacteria, fungi, prions, and parasites.

[0207] The following infectious disease-associated antigens can be used in the methods of the present invention (e.g., in the form of proteins, peptides, or nucleic acids encoding proteins or peptides, particularly mRNA):

[0208] In some embodiments, at least one antigen is an infectious disease-associated antigen. In some embodiments, at least one antigen is a viral antigen. Examples of viruses from which antigens can be derived include: Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 (also known as HTLV-III, LAV, or HTLV-III / LAV or HIV-III; and other isolates such as HIV-LP, HIV-2); Picornavirida (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains causing gastroenteritis); Clonorchiviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronaviruses). Viruses; 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., Hantavirus, Bunyavirus, sandfly virus, and Nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, circovirus, and rotavirus); BiRNAviridae; HepatDNAviridae (hepatitis B virus); Parvoviridae (parvovirus); Papillomavirus (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); and unclassified viruses (e.g., infectious agent of spongiform encephalopathy, infectious agent of hepatitis D (considered a defective satellite virus of hepatitis B virus), infectious agents of non-A, non-B hepatitis (enteral-transmitted type; extra-enteral-transmitted type (i.e., hepatitis C), Norwalk virus and related viruses, and astroviruses).

[0209] Preferably, at least one antigen is a viral antigen, more preferably a retroviral or coronavirus antigen, and most preferably an HIV or SARS-CoV-2 antigen. In even more preferred embodiments, at least one antigen is derived from SARS-CoV-2.

[0210] In some implementations, at least one antigen is a bacterial antigen. Examples of bacteria from which disease-associated antigens can be derived include: Helicobacter pylori, *Treponema* species (e.g., *Borrelia burgdorferi*), Legionella pneumophila, *Mycobacterium* species (e.g., *Mycobacterium tuberculosis*, *Mycobacterium avium*, *Mycobacterium intracellulare*, *Mycobacterium kansasense*, *Mycobacterium Gordonii*), *Staphylococcus aureus*, *Neisseria gonorrhoeae*, *Neisseria meningitidis*, *Listeria monocytogenes*, *Streptococcus pyogenes* (Group A Streptococcus), *Streptococcus agalactiae* (Group B Streptococcus), *Streptococcus* (violetish streptococci), *Enterococcus faecalis*, *Streptococcus bovis*, *Streptococcus* (anaerobic species), *Streptococcus pneumoniae*, *Campylobacter* species, *Enterococcus* species, *Haemophilus influenzae*, *Bacillus anthracis*, *Corynebacterium diphtheriae*, *Corynebacterium* species, *Erysipelothrix rhusiopathiae*, *Clostridium perfringens*, *Clostridium tetani*, *Enterobacter aerogenes*, *Klebsiella pneumoniae*, and *Pasteurella multocida*. Multocida), Bacteroides species, Fusobacterium nucleatum, Streptococcus beadida, Treponema pertenue, Leptospira and Actinomyces ylangis, Burkholderia melioides, Burkholderia melioides, and Pseudomonas aeruginosa.

[0211] Preferably, the bacterial antigen is a species antigen of the genus *Treponema* or *Mycobacterium*.

[0212] In some implementations, at least one antigen is a fungal antigen. Examples of fungi from which at least one antigen can be derived include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

[0213] In some implementations, at least one antigen is a parasitic antigen. Parasitic antigens can be derived from protozoa, worms, or ectoparasites. Examples of parasites from which disease-related antigens can be derived include: *Plasmodium falciparum*, *Plasmodium vivax*, *Plasmodium ovale*, *Plasmodium malariae*, species of *Leishmania*, species of *Trypanosoma* (African and American species, such as *Trypanosoma brevicornu* and *Trypanosoma krusei*), *Cryptosporidium*, species of *Isospora*, *Naegleria fowleri*, species of *Echinopodium*, *Balamuthia mandrillaris*, *Toxoplasma gondii*, or *Pneumocystis carinii*. Further examples of parasites from which disease-associated antigens can be derived include: species of *Echinococcus*, *Hookworm*, *American nematode*, *Angiostrongylus*, *Anisakis*, *Pseudo-Ascaris*, *Ascaris*, *Babesia*, *Balconiosis*, *Barramhondra*, *Baramhi*, *Baylisascaris*, bedbugs, *Schistosoma*, *Bacillus hominis*, *Capillaria*, *Lactobacillus mirabilis*, *Clonorchis*, *Cryptosporidium*, *Cyclopa*, *Cyclopa*, *Dinucleus fragilis*, *Spirometra*, *Dipylidium caninum*, *Filaria*, *Dracunculus*, *Filaria*, and *Endolimax*. *Entamoeba coli* species (e.g., *Entamoeba dispar*, *Entamoeba hartmanni*, *Entamoeba histolytica*, *Entamoeba polekiana*), *Pinus* species (e.g., *Entamoeba pulegii*), *Fasciolopsis* species, *Giardia* species, *Gnathostoma* species, *Heterodon* species, hookworms, *Hymenolepis* species, *Isotrophic amoeba* species (e.g., *Isotrophic amoeba bryophyte*), *Isospora* species, *Leishmania* species, lice (body lice, head lice, or pubic lice, lice infestation, pubic lice infestation), liver flukes (clonorchiasis, posterior clonorchiasis, liver fascioliasis), *Loa* species. Species of *L. loa*, species of *Microsporidia*, mites (scabies), species causing myiasis (e.g., *Green Botrytis cinerea*, *Botrytis hygroscopica*), species of *Naegleria*, species of *Toxocara*, species of *Onchocerca*, species of *Omega clonorchis*, species of *Paragonimus*, *Pneumocystis jirovecii*, species of *Bellis*, species of *Sappinia*, species of *Salmonella*, species of *Strongyloides* (e.g., *Strongyloides stercoralis*), species of *Taenia* (e.g., *Taenia vesicae*), species of *Trichinella*, species of *Trichomonas*, species of *Trichuris*, species of *Trichuris*.

[0214] The following tumor-associated antigens can be used in the methods of the present invention (e.g., in the form of proteins, peptides, or nucleic acids encoding proteins or peptides, particularly mRNA):

[0215] In some embodiments, at least one antigen is a tumor or cancer antigen, such as a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). For the purposes of this disclosure, unless otherwise stated, TAA includes the group consisting of TSA. Tumor or cancer antigens may be present on tumors or cancer cells. Tumor or cancer antigens may be derived from solid tumors or blood cancers.

[0216] Examples of tumors or cancers from which at least one antigen can be derived include leukemia, melanoma, lymphoma, endometrial cancer, kidney cancer, brain cancer, 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.

[0217] In some implementations, at least one antigen is a tumor-associated antigen. Tumor-associated 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-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, and cytokeratin. Leukocytes, desmin, glial fibrillary acidic protein (GFAP), giant cystic disease fluid protein (GCDFP-15), HMB-45 antigen, melan-A protein (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, dimer form of M2 type pyruvate kinase (tumor M2-PK), abnormal ras protein or abnormal p53 protein. In some embodiments, tumor-associated antigens (TAAs) are CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), spermin 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T-cell receptor γ-alternating reading frame protein), Trp-p8, or STEAP1 (prostatic six-transmembrane epithelial antigen 1). In some embodiments, TAAs are cancer / testis (CT) antigens, such as BAGE and 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 some other embodiments, the TAA is a carbohydrate or ganglioside, such as fuc-GMI, GM2 (carcinoembryonic antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, etc.In some other embodiments, TAA is α-actin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, β-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 (Pmel17),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 Kirsten rat sarcoma virus oncogene) or Ral-B.In one embodiment, at least one antigen is a multiepitope construct comprising two or more epitopes of the antigens described above. In one embodiment, at least one antigen comprises two or more pools of mRNAs, each encoding an epitope of one of the antigens described above.

[0218] In one embodiment, at least one antigen is derived from a tumor by isolating tumor DNA and / or RNA and then performing nucleic acid sequencing. In one embodiment, the nucleic acid sequencing is total RNA sequencing. In one embodiment, the nucleic acid sequencing is transcriptome sequencing. Therefore, in one embodiment, candidate antigens are identified by transcriptome sequencing. Sequencing methods such as next-generation sequencing are known to those skilled in the art. In one embodiment, the nucleic acid sequencing is exon sequencing. In one embodiment, at least one antigen is derived from a tumor by isolating tumor DNA and / or RNA and then performing nucleic acid sequencing of exons and total RNA.

[0219] In one embodiment, at least one antigen is a tumor-associated peptide or protein, optionally said tumor-specific peptide or protein, preferably said tumor-specific peptide or protein comprises at least one tumor-specific neoantigen.

[0220] In one embodiment, at least one tumor-specific neoantigen is selected from the group consisting of Aatf, Cpne1, Dpagt1, Wbp7, Nle1, Irgq, Zbtb40, Cry1, p15E, Gtf2i, Med12, N4bp2l2, and Syde1, or combinations thereof. In a particular embodiment, at least one tumor-specific neoantigen comprises sequences from Table 4, or combinations thereof.

[0221] In a preferred embodiment, the tumor-associated or tumor-specific antigen is identified from a human subject. In one embodiment, the tumor-associated or tumor-specific antigen is a human equivalent, i.e., a human homolog, of at least one epitope sequence of any one of SEQ ID NO: 25-42 and 44-55.

[0222] In one embodiment, at least one antigen is a combination of tumor-specific neoantigens contained in lipid nanoparticles. Each tumor-specific neoantigen is linked to the next tumor-specific neoantigen via a linker. This is referred to as a neoantigen string. In one embodiment, the LNP load has at least one mRNA encoding an epitope string of a tumor-specific neoantigen. Thus, in one embodiment, the LNP load has a multi-epitope construct.

[0223] In one embodiment, the multi-epitope construct comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 epitopes of tumor-specific neoantigens, each separated by a linker. Sequences encoding short linker peptides, typically used in fusion proteins and primarily composed of the amino acids glycine (G) and serine (S), can be used as GS linkers (glycine-serine linkers). In a preferred embodiment, the multi-epitope construct comprises at least 4 epitopes of tumor-specific neoantigens.

[0224] In one implementation, the multi-epitope construct contains epitopes of two or more tumor antigens disclosed herein.

[0225] In one embodiment, the multiepitope construct comprises two or more epitopes selected from Aatf, Cpne1, Dpagt1, Wbp7, Nle1, Irgq, Zbtb40, Cry1, p15E, Gtf2i, Med12, N4bp2I2, and Syde1. In another embodiment, the multiepitope construct comprises two or more epitopes selected from Aatf, Cpne1, Dpagt1, Wbp7, Nle1, Irgq, Zbtb40, Cry1, p15E, Gtf2i, Med12, N4bp2I2, and Syde1, each separated by a connector. In yet another embodiment, the multiepitope construct comprises at least two or more epitopes listed in Table 4.

[0226] In a preferred embodiment, the multi-epitope construct comprises epitopes of two or more human tumor-associated or tumor-specific antigens (such as tumor neoantigens). In one embodiment, the multi-epitope construct comprises tumor-associated or tumor-specific antigens, which are patient-specific neoantigens, oncoviral antigens (OVAs, such as HPV E6 / E7 or Merkel cell polyoma LTA), tumor-associated antigens (TAAs) such as lineage-restricted differentiation antigens (such as melanoma MART-1 or TRP2), cancer-testis antigens (such as MAGE or NY-ESO-1), or co-mutant antigens (such as p53 or Ras), or any combination thereof.

[0227] In one implementation, the multi-epitope construct includes at least two or more epitopes selected from the epitopes described in Table 4.

[0228] In one embodiment, the multiepitope construct comprises two or more epitopes selected from Aatf, Cpne1, Dpagt1, Wbp7, Nle1, Irgq, Zbtb40, Cry1, and p15E. In another embodiment, the multiepitope construct comprises two or more epitopes selected from Aatf, Cpne1, Dpagt1, Wbp7, Nle1, Irgq, Zbtb40, Cry1, and p15E, each separated by a connector. In another embodiment, the multiepitope construct comprises two or more epitopes selected from Gtf2i, Med12, N4bp2I2, and Syde1. In yet another embodiment, the multiepitope construct comprises two or more epitopes selected from Gtf2i, Med12, N4bp2I2, and Syde1, each separated by a connector.

[0229] In a preferred embodiment, the multi-epitope construct comprises two or more of DPAGT1, GTF2I, SYDE1, and ZBTB40.

[0230] In a particularly preferred embodiment, the multi-epitope construct comprises DPAG1 and / or ZBTB40.

[0231] In one embodiment, the multiepitope construct comprises at least one nucleic acid sequence encoding the LCMV glycoprotein GP33. In one embodiment, the multiepitope construct comprises at least one sequence encoding HPV E6 / E7. In one embodiment, the multiepitope construct comprises at least one sequence encoding the Merkel cell polyomatous LTA epitope. In one embodiment, the multiepitope construct comprises at least one sequence encoding a tumor-associated antigen (TAA). In one embodiment, the multiepitope construct comprises at least one sequence encoding a lineage-restricted differentiation antigen (such as melanoma MART-1 or TRP2). In one embodiment, the multiepitope construct comprises at least one sequence encoding MART-1. In one embodiment, the multiepitope construct comprises at least one sequence encoding TRP2. In one embodiment, the multiepitope construct comprises at least one sequence encoding a cancer-testis antigen. In one embodiment, the multiepitope construct comprises at least one sequence encoding MAGE. In one embodiment, the multiepitope construct comprises at least one sequence encoding NY-ESO-1. In one embodiment, the multiepitope construct comprises at least one sequence encoding p53. In one embodiment, the multiepitope construct comprises at least one sequence encoding Ras. In one implementation, the multi-epitope construct comprises multiple sequences encoding neotumor antigens, wherein the sequences can be linked by a connector.

[0232] In another embodiment, at least one antigen is a combination of tumor-specific neoantigens encoded by multiple mRNAs (i.e., two or more mRNAs), each mRNA encoding a single tumor neoantigen. This is also referred to as an mRNA pool.

[0233] In some implementations, each mRNA in the mRNA pool is loaded into a separate LNP.

[0234] In another implementation, the LNP is loaded with multiple mRNAs, that is, it is loaded with two or more mRNAs, each encoding a single tumor neoantigen.

[0235] In one implementation, the mRNA pool contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 epitopes of tumor-specific neoantigens, each encoded by at least one separate mRNA.

[0236] In one embodiment, the mRNA pool contains two or more epitopes selected from Aatf, Cpne1, Dpagt1, Wbp7, Nle1, Irgq, Zbtb40, Cry1, p15E, Gtf2i, Med12, N4bp2I2, and Syde1. In another embodiment, the mRNA pool contains two or more epitopes selected from Aatf, Cpne1, Dpagt1, Wbp7, Nle1, Irgq, Zbtb40, Cry1, p15E, Gtf2i, Med12, N4bp2I2, and Syde1, each on at least one individual mRNA. In yet another embodiment, the mRNA pool contains at least two or more epitopes selected from those listed in Table 4.

[0237] In one embodiment, the mRNA pool contains mRNAs encoding multiple epitopes in equimolar ratios. In another embodiment, the mRNA pool contains mRNAs encoding multiple epitopes in different specific ratios. Thus, mRNAs encoding some epitopes may be present in higher amounts than mRNAs encoding other epitopes, resulting in pools of various mRNAs with different amounts or concentrations.

[0238] In one implementation, the LNP is loaded with the mRNA pool described herein.

[0239] In one embodiment, each LNP carries the same mRNA pool. In another embodiment, each LNP carries a portion of the total mRNA pool. Therefore, in some embodiments, each LNP contains a different mRNA pool. In some embodiments, each LNP contains different but overlapping portions of the mRNA pool, each selected from the same mRNA pool.

[0240] In some implementations, each LNP contains the same pool of mRNAs.

[0241] In another implementation, each LNP contains a single mRNA derived from the mRNA pool.

[0242] In one embodiment, at least one antigen comprises at least one nucleic acid sequence encoding the LCMV glycoprotein GP33.

[0243] In one embodiment, at least one antigen comprises a pool of two or more mRNAs, each encoding an epitope of any of the antigens disclosed herein.

[0244] Other tumor-associated antigens are known to those skilled in the art and can be combined with the monocytes or phDCs provided herein to generate corresponding antigen-specific phDCs, or may be present in the compositions disclosed herein.

[0245] Antigens expressed from nucleic acids

[0246] At least one antigen may be provided in the form of a nucleic acid such as DNA or RNA. DNA may be cDNA or genomic DNA. RNA may be single-stranded RNA, mRNA, self-amplifying RNA, circular RNA, and / or synthetic RNA. In a preferred embodiment, the RNA is mRNA. Optionally, the mRNA is self-amplifying 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. Thus, in some embodiments, at least one antigen is encoded by mRNA, which is combined with monocytes or phDCs. mRNAs encoding more than one antigen may also be used in the methods of the present invention, for example, for testing multivalent mRNA vaccines. In one embodiment, multiple mRNAs encoding more than one antigen from the same or different pathogens or tumors may be used in the methods of the present invention, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens. Alternatively or additionally, polycistronic mRNAs capable of being translated into more than one antigen may be designed.

[0247] Encoding area

[0248] In one embodiment, the mRNA encodes one or more antigens of the tumor or infectious disease-associated antigens listed above. In one embodiment, the mRNA encodes one or more antigens of the pathogens listed above. In one embodiment, the mRNA encodes one or more antigens of the viruses, bacteria, fungi, prions, or parasites listed above. In one embodiment, the mRNA encodes one or more antigens of the viruses (optionally coronaviruses) listed above. In one embodiment, the mRNA encodes one or more antigens of the tumors or cancers listed above. In one embodiment, the mRNA encodes one or more antigens of the TAAs listed above. In one embodiment, at least one mRNA encodes an antigen.

[0249] SARS-CoV-2 sequence

[0250] In one embodiment, at least one mRNA encodes one or more antigens derived from SARS-CoV (optionally SARS-CoV-2). Antigens derived from SARS-CoV or SARS-CoV-2 include spike proteins, envelope proteins, nucleocapsid proteins, membrane proteins, and / or Orf1ab polyproteins. Therefore, in one embodiment, the antigen is a fragment of a spike protein, envelope protein, nucleocapsid protein, membrane protein, and / or Orf1ab polyprotein, or each of the foregoing. In one embodiment, the fragment comprises at least 10, at least 50, at least 100, at least 200, at least 400, or at least 800 amino acid residues. In a preferred embodiment, the mRNA encodes one or more antigens derived from the spike protein or nucleocapsid protein. Even more preferably, the mRNA encodes one or more antigens derived from the spike protein. The mRNA may encode an antigen derived from the S1 or S2 subunit of the spike protein. Variants of the SARS-CoV-2 protein described above are also explicitly included in this invention. For example, the first-generation spike protein variant, termed "S-2P" (Pallesen et al., 2017), contains two proline substitutions at positions 986 and 987 (see, for example, Polack et al., 2020; Bos et al., 2020; Corbett et al., 2020; Wrapp et al., 2020). The second-generation spike construct, termed "HexaPro," contains four additional proline residues at positions 817, 892, 899, and 942. HexaPro is expressed at higher levels than the wild-type spike protein or S-2P and exhibits improved stability relative to S-2P under cryogenic storage and multiple freeze-thaw cycles (Edwards et al., 2020).

[0251] In some embodiments, the mRNA comprises a sequence or a portion thereof selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and / or SEQ ID NO: 5.

[0252] In some embodiments, the mRNA comprises a sequence or a portion thereof selected from the group consisting of, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the selected sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5. In one embodiment, the mRNA comprises a sequence or a portion thereof selected from the group consisting of, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the selected sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5, wherein the selected mRNA sequence is modified by one or more (e.g., all) naturally occurring modifying nucleosides, as described in the section “Modification of RNA”. In one embodiment, the naturally occurring modifying nucleoside is 1-methylpseuuridine.

[0253] The above sequences are based on the reference genome from NCBI (accession number NC_045512.2) and are also shown in Table 1 below. The corresponding amino acid sequences are given in Table 2 below.

[0254] In some implementations, the mRNA comprises a sequence or a portion thereof 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).

[0255] Those skilled in the art who read this disclosure will further understand that this disclosure describes various mRNA constructs comprising a sequence encoding a full-length SARS-CoV-2 spike protein (e.g., including embodiments in which such encoded SARS-CoV-2 spike protein may contain at least one or more amino acid substitutions, such as proline substitutions as described herein; and / or embodiments in which the mRNA sequence is optimized, such as for mammalian (e.g., human) subjects; and / or embodiments in which the mRNA contains one or more chemical modifications).

[0256] Table 1

[0257]

[0258] Table 2

[0259]

[0260] Table 3

[0261]

[0262] Tumor-associated antigen sequences

[0263] In one embodiment, at least one mRNA encodes one or more antigens derived from a tumor or cancer. In one embodiment, the mRNA encodes a TAA (tumor-associated antigen) polypeptide, such as the amino acid sequence of a TAA. TAAs 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, calretin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, and cytokeratin. Desmin, glial fibrillary acidic protein (GFAP), giant cystic disease fluid protein (GCDFP-15), HMB-45 antigen, melan-A protein (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, dimer form of M2 type pyruvate kinase (tumor M2-PK), abnormal ras protein or abnormal p53 protein. In some embodiments, the TAA is CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), spermin 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T-cell receptor γ-alternating reading frame protein), Trp-p8, or STEAP1 (prostatic six-transmembrane epithelial antigen 1). In some embodiments, the TAA is a cancer / testis (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 some other embodiments, TAA or TSA is α-actin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, β-catenin, CA 125, CA 15-3 (CA 27.29\BCAA), CA195, 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), prolactin, K Ras (V-Ki-ras2 Kirsten rat sarcoma virus oncogene) or Ral-B.

[0264] The mRNA encoding the amino acid sequence of TAA can be a sequence that has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the mRNA sequence of the selected TAA.

[0265] Antigens derived from tumors

[0266] On the other hand, at least one mRNA may be derived from (e.g., isolated from) a tumor source (e.g., poly ARNA), such as a subject-specific tumor or a similar tumor from a different subject. In one embodiment, at least one mRNA encodes one or more tumor antigens, such as one or more subject-specific tumor antigens or one or more tumor antigens from a different subject with a similar tumor. In one embodiment, the subject has a tumor, and the mRNA is derived from the transcriptome of a subject-specific tumor (e.g., an mRNA library representing the transcriptome of a subject-specific tumor). In another embodiment, the subject has a tumor, and the mRNA is derived from the transcriptome of a tumor from a different subject with a similar tumor (e.g., an mRNA library representing the transcriptome of an allogeneic similar tumor). In some embodiments, tumor-specific mRNA is compared with non-tumor mRNA from the subject to determine tumor-related or tumor-specific nucleotide sequences. Various methods known in the art exist for isolating mRNA. For example, mRNA can be isolated using standard one-step phenol extraction or glass binding methods, and methods using oligo-d(T)-selective mRNA. Isolated mRNAs can be sequenced and compared with mRNAs from healthy tissues to identify tumor-associated or tumor-specific mRNAs, for example, through next-generation sequencing.

[0267] In another embodiment, at least one DNA may be derived from (e.g., isolated from) a tumor source (e.g., cDNA), such as a subject-specific tumor or a similar tumor from a different subject. In one embodiment, at least one DNA encodes one or more tumor antigens, such as one or more subject-specific tumor antigens or one or more tumor antigens from a different subject with a similar tumor. In one embodiment, the subject has a tumor, and the DNA is derived from the exome or genomic DNA of a subject-specific tumor (e.g., a DNA library representing the exome or genomic DNA of a subject-specific tumor). In another embodiment, the subject has a tumor, and the DNA is derived from the exome or genomic DNA of a tumor from a different subject with a similar tumor (e.g., a DNA library representing the exome or genomic DNA of an allogeneic similar tumor). In some embodiments, the tumor-specific DNA is compared with non-tumor DNA from the subject to determine tumor-related or tumor-specific nucleotide sequences.

[0268] Various methods are known in the art for DNA isolation. For example, DNA extraction can be accomplished using specific columns or magnetic beads, phenol-chloroform based methods, or filtration-based methods. Commercially available spin column kits containing silica membranes are widely used in clinical applications. The isolated DNA can be sequenced and compared with DNA from healthy tissue to identify tumor-related or tumor-specific sequences, for example, through next-generation sequencing.

[0269] 4. RNA modification

[0270] If at least one antigen contains RNA (e.g., mRNA) or corresponds to at least one RNA (e.g., mRNA), then the RNA (e.g., mRNA) may be modified. In one embodiment, the RNA (e.g., mRNA) is modified RNA, particularly stable mRNA. In some embodiments, the RNA (e.g., mRNA) may be modified to achieve maximum efficacy, such as improved cell lifespan, transcriptional efficiency, non-immunogenic properties (no Toll receptor induction, etc.), and / or structural mRNA stability.

[0271] In one embodiment, the RNA (e.g., mRNA) independently comprises at least one chemical modification. The chemical modification may be, for example, a modified nucleoside. In one embodiment, the chemical modification includes naturally occurring modified nucleosides. Naturally occurring modified nucleosides include 1-methyladenosine (m1A), N6-methyladenosine (m6A), 2'-O-methyladenosine (Am), 5-methylcytidine (m5C), 2'-O-methylcytidine (Cm), 2-thiocytidine (s2C), N4-acetylcytidine (ac4C), 5-formylcytidine (f5C), 2'-O-methylguanosine (Gm), inosine (I), pseudouridine (Ψ), 5-methyluridine (m5U), 2'-O-methyluridine (Um), 1-methylpseudouridine (m1Ψ), 2-thiouridine (s2U), 4-thiouridine (s4U), 5-methoxyuridine (mo5U), and 3-methyluridine (m3U). In one embodiment, the RNA (e.g., mRNA) comprises a modified nucleoside replacing at least one uridine. In one embodiment, the RNA comprises a modified nucleoside replacing each uridine. In one embodiment, the modified nucleoside is independently selected from pseudouridine, N1-methyl-pseudouridine, 5-methyl-uridine, and N1-ethyl-pseudouridine. In one embodiment, the modified nucleoside is N1-methyl-pseudouridine or N1-ethyl-pseudouridine. In one embodiment, the modified nucleoside is N1-methyl-pseudouridine. For example, N1-methyl-pseudouridine has been found to be superior in translational ability to several other nucleoside modifications and combinations thereof. 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.

[0272] RNA, particularly mRNA, can be optimized as an alternative to or complement to chemical modifications. RNA sequence optimization includes, in particular, codon optimization, G / C content optimization, and structural element optimization (e.g., 5' cap, 5' UTR, 3' UTR, and poly(A)-tail).

[0273] In some embodiments, the amino acid sequence of at least one antigen is encoded by a coding sequence with codon-optimized and / or increased G / C content compared to the wild-type coding sequence. This also includes embodiments in which one or more regions of the coding sequence are codon-optimized and / or have increased G / C content compared to the corresponding sequence region of the wild-type coding sequence. In one embodiment, the codon optimization and / or increase in G / C content preferably does not alter the sequence of the encoded amino acid sequence. In some embodiments, the G / C content of the coding region of said RNA is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G / C content of the coding region of wild-type RNA.

[0274] In some embodiments, the RNA (e.g., mRNA) may contain one or more optimized structural elements. These structural elements include a 5' cap, a 5' UTR, a 3' UTR, and a poly(A) tail. Thus, in one embodiment, the RNA (e.g., mRNA) contains 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 some embodiments, the RNA contains a 5'-UTR and / or a 3'-UTR. In one embodiment, the RNA (e.g., mRNA) contains a cap. In some embodiments, the RNA (e.g., mRNA) contains a 3'-poly(A) sequence. In one embodiment, the cap is a Cap1 structure or an m7GpppG cap. In one embodiment, the sequence of the 5' UTR and / or the 3' UTR is optimized. In some embodiments, the mRNA contains a 5' or 3' UTR derived from a gene with a sequence different from that encoding at least one antigen, i.e., the UTR is a heterologous UTR. In some implementations, the 5' and / or 3' UTR sequence may be derived from stable mRNAs (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycling enzymes) to increase mRNA stability. For example, as a 5'-UTR sequence, a 5'-UTR sequence of human α-globin mRNA may be used, optionally with an optimized "Kozak sequence" to improve translation efficiency. Alternatively, a 5' UTR sequence of human cytochrome mRNA may be used, such as human cytochrome b-245α mRNA or cytochrome p4502E1 mRNA. As a 3'-UTR sequence, a combination of two sequence elements (FI elements) derived from a split N-terminal enhancer (AES) mRNA (referred to as F) and a mitochondrial-encoded 12S ribosomal RNA (referred to as I) may be used, positioned between the coding sequence and the poly(A)-tail to ensure higher maximum protein levels and extended mRNA persistence. Alternatively, the 3'-UTR may be the 3'-UTR of two re-iterated human β-globin mRNAs.

[0275] In one embodiment, the poly-A sequence contains at least 100 nucleotides. In another embodiment, the poly-A sequence contains at least 150 nucleotides. In yet another embodiment, the poly-A sequence contains at least 250 nucleotides. Additionally, a poly(A)-tail of 110 nucleotides in length can be used, consisting of a 30-adenosine residue segment followed by a 10-nucleotide linker sequence (random nucleotides) and an additional 70 adenosine residues. This poly(A)-tail sequence enhances RNA stability and improves translation efficiency. In one embodiment, the poly(A)-tail is 300 to 800 nucleotides in length.

[0276] Furthermore, the secretory signal peptide (sec) can be fused to the antigen coding region, preferably in a manner that translates sec into an N-terminal tag. A sequence encoding a short linker peptide, typically composed of the amino acids glycine (G) and serine (S), as is commonly used in fusion proteins, can be used as a GS linker (glycine-serine linker). In other embodiments, the RNA (e.g., mRNA) may have one or more AU-rich sequences removed. These AU-rich sequences, sometimes referred to as AURES, are unstable sequences present in the 3' UTR. AURES can be removed from the RNA (e.g., mRNA). Alternatively, AURES can be retained in the RNA (e.g., mRNA).

[0277] 5. Nanoparticles

[0278] At least one antigen (e.g., at least one mRNA encoding at least one antigen) may be contained (e.g., encapsulated) in nanoparticles, such as polymer nanoparticles, lipid nanoparticles (LNPs), or lipid complexes. For example, if at least one antigen is encoded by RNA (e.g., mRNA), the RNA may be encapsulated in lipid nanoparticles for delivery to the monocytes or phDCs of the present invention. In this embodiment, the encapsulated RNA (e.g., mRNA) is combined with monocytes or phDCs.

[0279] LNPs can contain 3, 4, or 5 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) cofactor lipids. LNPs with five classes of lipids include: (1) ionizable lipids, (2) PEGylated lipids, (3) cholesterol-based lipids, (4) cofactor lipids, and (5) DSPE-PEG-maleimide or DSPN-PEG-azide. DSPE-PEG-maleimide or DSPN-PEG-azide allows for the addition of ligands for targeted delivery.

[0280] (1) Ionizable lipids

[0281] Ionizable lipids facilitate mRNA encapsulation and can be cationic lipids. Cationic lipids provide a positively charged environment at low pH to promote efficient encapsulation of negatively charged mRNAs. 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). In one embodiment, the cationic lipid is SM-102 (9-heptadecyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate; CAS No. 2089251-47-6). In one embodiment, the cationic lipid is MC3 (4-(dimethylamino)-butyric acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecanadien-1-yl ester; CAS No. 1224606-06-7). Other cationic lipids that may be used include those described in Dong et al., 2014.

[0282] (2) PEG-modified lipids

[0283] PEG-modified lipid components offer control over the particle size and stability of nanoparticles. The addition of such components can prevent complex aggregation and provides a means to increase lifetime and enhance LNP delivery to phDC.

[0284] The PEGylated lipids covered include, but are not limited to, polyethylene glycol (PEG) chains of up to 5 kDa covalently attached to lipids having alkyl chains having lengths of C6-C20 (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-dimyristoyl-racemic-glycerol-3-methoxypolyethylene glycol (DMG-PEG); 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol (DSPE-PEG); 1,2-dilauryl-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol (DLPE-PEG); or 1,2-distearyl-racemic-glycerol-polyethylene glycol (DSG-PEG). In a particularly exemplary embodiment, the PEG has a high molecular weight, such as 2000-2400 g / mol. In some embodiments, the 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.

[0285] (3) Cholesterol-based lipids

[0286] The cholesterol component provides stability to the lipid bilayer structure within the nanoparticles. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, N,N-dimethyl-N-ethylcarboxamido-cholesterol, 1,4-bis(3-N-oleenylaminopropyl)piperazine, imidazole cholesterol ester, β-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0287] (4) Lipid-assisted

[0288] The assisting lipid enhances the structural stability of the LNP and facilitates endosome escape. It improves the uptake and release of RNA (e.g., mRNA) loads. The cross-presentation capacity of DCs may be limited by non-specific degradation during endosome maturation. Therefore, LNPs that provide better endosome escape may be useful in some embodiments (e.g., if the antigen is in the form of, for example, a protein, peptide, or mRNA). In one embodiment, the assisting lipid is a non-cationic lipid. In some embodiments, the assisting lipid is a zwitterionic lipid with fusion properties for enhancing load uptake and release. Examples of accessory lipids are 1,2-dioleoyl-SN-glycero-3-phosphate ethanolamine (DOPE); 1,2-distearyl-sn-glycero-3-phosphate choline (DSPC); 1,2-dioleoyl-sn-glycero-3-phosphate-L-serine (DOPS); 1,2-ditransoleoyl-sn-glycero-3-phosphate choline (DPOC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dilauroyl-sn-glycero-3-phosphate choline (DLPC), 1,2-distearylphosphatidylethanolamine (DSPE), and 1,2-dilauroyl-sn-glycero-3-phosphate ethanolamine (DLPE).

[0289] In one embodiment, the lipid nanoparticles comprise cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE. In another embodiment, the lipid nanoparticles comprise SM102, cholesterol, DMG-PEG2000, and DSPC. In some embodiments, monocytes or phDCs can be loaded with different antigens to produce multivalent antigen-specific phDCs. For example, if at least one antigen is encoded by mRNA encapsulated in an LNP, the LNP can carry more than one antigen (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens) encoding the same or different pathogens or the same or different tumors. For example, the LNP can carry multiple mRNA molecules, each encoding a different antigen. The LNP can also carry polycistronic mRNAs that can be translated into more than one antigen. If the LNP carries different mRNA molecules, multiple copies of each mRNA molecule are typically present.

[0290] molar ratio of lipid components

[0291] 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 cofactor lipids is A:B:C:D, where A+B+C+D=100%. In some embodiments (particularly for cKK-E12-based lipids), the molar ratio of cationic lipids to total lipids (i.e., A) in the LNP is 35-45%. In some embodiments, the molar ratio of the PEGylated lipid component to total lipids (i.e., B) is 0.25-2.75%. 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 cofactor lipids to total lipids (i.e., D) is 16-35% (e.g., 16-32%, such as 16%). In one embodiment, the ratio of the components is 35:2.5:46.5:16 (A:B:C:D).

[0292] 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.

[0293] In some embodiments (particularly for SM102-based lipids), the molar ratio of cationic lipids to total lipids in the LNP (i.e., A) is 45-55%. In some embodiments, the molar ratio of the PEGylated lipid component to total lipids (i.e., B) is 0.25-2.75%. 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 accessory lipids to total lipids (i.e., D) is 5-20% (e.g., 7-18%). In one embodiment, the ratio of the components is 50:38.5:1.5:10 (A: B: C: D).

[0294] In one embodiment, the lipid nanoparticles comprise SM102, cholesterol, DMG-PEG2000 (synonymous with DMG-PEG-2K), and DSPC in a ratio of 50:38.5:1.5:10. In another embodiment, the lipid nanoparticles comprise MC3, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16.

[0295] In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the cofactor lipid. In some embodiments, the LNP contains cationic lipids and cofactor lipids in a molar ratio greater than 1. To calculate the actual amount of each lipid to be incorporated into the LNP formulation, the molar amount of the cationic lipid is first 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, the molar amount of each other lipid is calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid. Lipid nanoparticles containing antigens (e.g., in the form of RNA, particularly mRNA encoding at least one antigen) can be provided frozen.

[0296] Size and amount of lipid nanoparticles

[0297] Suitable LNPs can be fabricated in various sizes. In some embodiments, the majority of the purified LNPs, i.e., LNPs greater than about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, have a size of about 50 to 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, substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles have a size of about 70 to 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, greater than about 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the LNPs in the compositions of the present invention have a size of about 85 to 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). In some embodiments, monocytes or phDCs are 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, monocytes or phDCs are combined with 0.1 to 100 µg of encapsulated RNA, particularly mRNA, for example, with 1 to 50 µg, 1 to 25 µg, or 5 to 15 µg of encapsulated RNA, particularly mRNA. In some embodiments, monocytes or phDCs are combined with about 10 µg of encapsulated RNA, particularly mRNA. In some embodiments, phDCs are combined with about 20 µg of encapsulated RNA, particularly mRNA. In one embodiment, 1 µg to 20 µg of mRNA contained or encapsulated in lipid nanoparticles is combined with 1*10 4 Up to 1 * 10 7 A combination of monocytes, particularly if the monocytes are obtained from a PBMC fraction of blood. In one embodiment, 1 µg to 20 µg of mRNA contained or encapsulated in lipid nanoparticles with 1*10 4 Up to 1 * 10 7 A combination of PBMCs containing monocytes. As a reference, a ratio of approximately 0.4 µg of mRNA contained in or encapsulated in lipid nanoparticles can be used with approximately 106 A combination of PBMCs containing monocytes (e.g., obtainable via Ficoll density gradient centrifugation). In the case of using monocytes from (anticoagulated) whole blood, a combination of 1 µg to 20 µg of mRNA contained or encapsulated in lipid nanoparticles with 0.1 to 100 ml of whole blood containing monocytes.

[0298] lipid complex

[0299] At least one antigen (e.g., at least one mRNA encoding at least one antigen) may be contained in the lipid complex. The mixing of RNA (e.g., mRNA) and positively charged liposomes results in the formation of lipid complex particles through spontaneous self-assembly. Liposomes typically contain at least two components: cationic lipids and neutral lipids. Lipid complexes have been extensively described in the art; see, for example, Nanomedicine: Nanotechnology, Biology and Medicine, 2009. In one embodiment, the neutral lipid is an accessory lipid as defined above. In an exemplary embodiment, the cationic lipid is DOTMA, and the neutral lipid is DOPE.

[0300] 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 an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one other lipid is about 2:1. The average diameter of the RNA-lipid complex particles described herein is, in one embodiment, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, the average diameter of the RNA-lipid complex particles is about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, or about 700 nm. In one embodiment, the average diameter of the RNA-lipid complex particles is about 250 nm to about 700 nm. In another embodiment, the average diameter of the RNA-lipid complex particles is about 300 nm to about 500 nm. In an exemplary embodiment, the average diameter of the RNA-lipid complex particles is about 400 nm.

[0301] In one embodiment, the (mRNA) lipid complex particle comprises at least one cationic lipid and at least one neutral lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenoyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In one embodiment, the at least one neutral lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol (Chol), and / or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC). In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenoyl-3-trimethylammonium propane (DOTMA) and the at least one neutral lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE). In one embodiment, the lipid complex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine (DOPE). Spleen-targeting RNA lipid complex particles are described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that lipid complex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells.

[0302] Oligomers, polymers, or lipid compounds containing an oligomeric (alkyleneamine) moiety

[0303] At least one antigen (e.g., at least one mRNA encoding at least one antigen) may be contained in oligomers, polymers, or lipid compounds comprising an oligomeric (alkyleneamine) moiety (e.g., a characteristic oligomeric (alkyleneamine) moiety as described in PCT / EP2014 / 063756). Specifically, at least one antigen (e.g., at least one mRNA encoding at least one antigen) may be contained in oligomers, polymers, or lipid compounds as described in PCT / EP2014 / 063756. A key characteristic of oligomers, polymers, or lipid compounds comprising an oligomeric (alkyleneamine) moiety is that they contain a common structural entity of formula (I):

[0304]

[0305] Such oligomers, polymers, or lipid compounds containing an oligomeric (alkyleneamine) moiety can be selected from:

[0306] a) Oligomers or polymers containing multiple groups of formula (II) as side chains and / or as terminal groups:

[0307]

[0308] The variables are a, b, p, m, n, and R. 2 To R 6 As defined below, each group of formula (II) among a plurality of such groups is defined independently:

[0309] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1.

[0310] p is 1 or 2

[0311] m is 1 or 2; n is 0 or 1 and m + n ≥ 2; and

[0312] R 2 To R 5 Each of these elements is independently selected from: hydrogen; the 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 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl with one C-C double bond; protecting group of amino group; -C(NH)-NH2; poly(ethylene glycol) chain;

[0313] R 6 Selected from: 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 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl having a C-C double bond; a protecting group of an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain and an acceptor ligand, wherein one or more nitrogen atoms shown in formula (II) may be protonated to provide a cationic group of formula (II);

[0314] b) Oligomers or polymers containing multiple groups of formula (III) as repeating units:

[0315]

[0316] The variables are a, b, p, m, n, and R. 2 To R 5Independently defined as follows, for each group of formula (III) among a plurality of such groups, the following groups are defined independently:

[0317] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1.

[0318] p is 1 or 2

[0319] m is 1 or 2; n is 0 or 1 and m + n ≥ 2; and

[0320] R 2 To R 5 Each of these elements is independently selected from: hydrogen; the 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 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl groups having one C-C double bond; protecting groups of amino groups; -C(NH)-NH2 and poly(ethylene glycol) chains;

[0321] Furthermore, one or more of the nitrogen atoms shown in formula (III) may be protonated to provide a cationic group of formula (III); and

[0322] c) Lipid compounds having the structure of formula (IV):

[0323]

[0324] The variables are a, b, p, m, n, and R. 2 To R 6 As defined below:

[0325] a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1.

[0326] p is 1 or 2

[0327] m is 1 or 2; n is 0 or 1 and m + n ≥ 2; and

[0328] R 1 To R 6 Each of these elements is independently selected from: hydrogen; the 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-R7 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl groups having one C-C double bond; protecting groups of amino groups; -C(NH)-NH2; poly(ethylene glycol) chains and acceptor ligands.

[0329] The condition is R 1 To R 6 At least two residues in it are: the group –CH 2 -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 , where R 7 Selected from C3-C18 alkyl or C3-C18 alkenyl having a C-C double bond; and wherein one or more nitrogen atoms shown in formula (IV) may be protonated to provide a cationic lipid compound of formula (IV);

[0330] Preferably, the oligomer, polymer, or lipid compound comprising the oligomeric (alkyleneamine) moiety is selected from a) and b), wherein

[0331] a) is an oligomer or polymer containing multiple groups of formula (IIa) as side chains and / or as terminal groups:

[0332]

[0333] Where a, b, m, n and R 2 To R 6 As defined above, and wherein one or more of the nitrogen atoms shown in formula (IIa) may be protonated to provide a cationic oligomer or polymer structure; and

[0334] b) is an oligomer or polymer containing multiple groups of formula (IIIa) as repeating units:

[0335]

[0336] Where a, b, m, n and R 2 To R 5 As defined above, and in which one or more of the nitrogen atoms shown in formula (IIIa) can be protonated to provide a cationic oligomer or polymer structure.

[0337] Furthermore, lipid compounds containing oligo(alkyleneamine) moieties can be selected from lipid compounds having the structure of formula (IVa):

[0338]

[0339] Where a, b, m, n and R 1 To R 6 As defined above, and in which one or more of the nitrogen atoms shown in formula (IVa) can be protonated to provide a cationic lipid compound.

[0340] For such oligomers, polymers, or lipid compounds containing an oligomeric (alkyleneamine) moiety, n may be 1 in formula (II), (IIa), (III), (IIIa), (IV), or (IVa); or m may be 1 and n may be 1.

[0341] Furthermore, for such oligomers, polymers, or lipid compounds containing an oligomeric (alkyleneamine) moiety, in formulas (II), (IIa), (III), (IIIa), (IV), or (IVa), a can be 1 and b can be 2; or a can be 2 and b can be 1.

[0342] A non-limiting example of such oligomers, polymers, or lipid compounds containing an oligomeric (alkyleneamine) moiety is a cationic lipid prepared as follows: 100 mg of N,N'-bis(2-aminoethyl)-1,3-propanediamine (0.623 mmol) is mixed with 575.07 mg of 1,2-epoxydodecane (3.12 mmol, (N-1) equivalents, where N is twice the number of primary amines plus once the number of secondary amines in each oligomeric (alkyleneamine) moiety), and the mixture is continuously shaken at 80°C for 96 h. Such oligomers, polymers, or lipid compounds are also referred to as lipid compounds "C12-(2-3-2)". Oligomers, polymers, or lipid compounds containing an oligomeric (alkyleneamine) moiety, particularly polymers, can be copolymers, especially statistical copolymers. Such copolymers can be copolymers containing repeating alkyleneamine units of alternating lengths in a statistical / random arrangement (e.g., in contrast to less preferred polymers containing a similar arrangement of repeating alkyleneamine units of non-alternating lengths). The copolymer may be a cationic (e.g., protonated) copolymer. The copolymers to be used are known in the art and are described, for example, in EP 14 19 9439.2, WO 01 / 00708, EP-A1 1 198 489 and CA-A1 2,377,207.

[0343] In particular, the copolymer can be a statistical copolymer comprising a plurality of repeating units (a), which are independently selected from repeating units of the following formulas (a1) and (a2):

[0344]

[0345] and multiple repeating units (b), which are independently selected from the repeating units of the following formulas (b1) to (b4):

[0346]

[0347]

[0348]

[0349]

[0350] The molar ratio of the sum of repeating units (a) to the sum of repeating units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, and one or more of the nitrogen atoms of the repeating units (a) and / or (b) contained in the copolymer can be protonated to provide a cationic copolymer.

[0351] The copolymer can be a statistical copolymer, wherein any repeating unit (a) and any repeating unit (b) are statistically distributed in the copolymer macromolecule. It is typically obtained by copolymerizing a set of monomer mixtures, where the monomers that produce repeating unit (a) in the polymerization reaction produce repeating unit (b) in the polymerization reaction. Preferably, the copolymer is a random copolymer, wherein any repeating unit (a) and any repeating unit (b) are randomly distributed in the polymer macromolecule. Such copolymers can be linear, branched, or dendritic copolymers. As a skilled reader will understand, repeating units of formula (a1), (b1), or (b3) having two valences (i.e., open bonds with adjacent units) result in a copolymer structure that extends linearly. Thus, a linear copolymer may contain repeating units of formula (a1) and one or more types of repeating units of formulas (b1) and (b3), but not repeating units of formulas (a2), (b2), or (b4). As will be further understood, the presence of repeating units of formula (a2), (b2), or (b4) having three valences provides branching points for the copolymer structure. Therefore, the branched copolymer comprises one or more types of repeating units of formula (a2), (b2), and (b4), and may further comprise one or more types of repeating units of formula (a1), (b1), and (b3). Such copolymers may comprise a plurality of repeating units (a) independently selected from repeating units of formula (a1) and (a2) as defined above, and a plurality of repeating units (b) independently selected from repeating units of formula (b1) to (b4) as defined above. Preferably, it is a copolymer comprising a plurality of repeating units (a) independently selected from repeating units of formula (a1) and (a2) as defined above, and a plurality of repeating units (b) independently selected from repeating units of formula (b1) and (b2) as defined above. 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 formula (a1), (b1), and (b3), and particularly comprising repeating units of formula (a2) and one or more types of repeating units of formula (b2) and (b4), and optionally further comprising one or more types of repeating units of formula (a1), (b1), and (b3). Consistent with the above, therefore, more preferably, copolymers are branched copolymers comprising repeating units of formula (a2) and repeating units of formula (b2), and optionally further comprising one or more types of repeating units of formula (a1) and (b1). In the copolymer, the total number of repeating units (a) and repeating units (b) is generally 20 or more, preferably 50 or more, and more preferably 100 or more. Typically, the total number of repeating units (a) and repeating units (b) is 10,000 or less, preferably 5,000 or less, and more preferably 1,000 or less.Furthermore, preferably, repeating units (a) and (b) in the copolymer account for 80 mol% or more of all repeating units in the copolymer, more preferably 90 mol% or more. Further preferably, repeating units (a) selected from (a1) and (a2) and repeating units (b) selected from (b1) and (b2) in the copolymer account for 80 mol% or more of all repeating units in the copolymer, more preferably 90 mol% or more. Most preferably, all repeating units in the copolymer are repeating units (a) or (b), particularly all 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 the copolymer, as measured, for example, by size exclusion chromatography relative to a linear poly(ethylene oxide) standard, is typically 1,000 to 500,000 Da, preferably 2,500 to 250,000 Da, more preferably less than 5,000 to 50,000 Da. The terminal groups of such copolymers typically contain one or more types of groups (c), which are independently selected from groups of formulas (c1) to (c3), preferably groups of formulas (c1) and (c2).

[0352]

[0353] Preferably, the terminal groups in the copolymer consist of one or more types of groups (c), said groups (c) being independently selected from groups of formulas (c1) to (c3), preferably groups of formulas (c1) and (c2). As those skilled in the art will understand, the number of terminal groups depends on the structure of the copolymer. While linear copolymers have only two ends, branched copolymers, particularly dendritic copolymers, contain a greater number of terminal groups. As will be further understood, one or more nitrogen atoms of the terminal groups (c) contained in the copolymer may also be protonated to provide a cationic copolymer. In the copolymer, the molar ratio of the sum of repeating units (a) to the sum of 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, it should be understood that this ratio is generally determined for multiple macromolecules of the copolymer and generally represents the total ratio of the sum of repeating units (a) to the sum of repeating units (b) in the multiple macromolecules. As described above, one or more nitrogen atoms in the copolymer can be protonated to produce a copolymer in cationic form, typically oligocationic or polycationic. It should be understood that the primary, secondary, or tertiary amine groups in the repeating unit (a) or (b) or the terminal group (c) can act as proton acceptors, particularly in water and aqueous solutions, including physiological fluids. Therefore, such copolymers typically have an overall positive charge in aqueous solutions with a pH below 7.5. As described herein, an aqueous solution is a solution in which the solvent contains 50% (vol. / vol.) or more, preferably 80% or 90% or more, and most preferably 100% water. Furthermore, if the compositions are in contact with physiological fluids (including, for example, blood and pulmonary fluids) with a pH below 7.5, they typically contain repeating units (a) and (b) in which nitrogen atoms are 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 can then be calculated, for example, from the Henderson-Hasselbach equation. Any charge can be distributed across several basic centers and is not necessarily attributed to a single point. Typically, in solutions at physiological pH, the copolymers used in compositions comprise repeating units having amino groups in a protonated state and repeating units having amino groups in an unprotonated state. However, as a skilled reader will understand, copolymers can also be provided as a dry salt form containing a copolymer in a cationic form. It will also be understood that the counter-charge (anion) of the positively charged protonated amino groups in compositions comprising copolymers and nucleic acids (particularly mRNA) is typically provided by the anionic portion contained in the nucleic acid. If the positively charged groups are in excess compared to the anionic portion in the nucleic acid, the positive charge can be balanced by other anions, particularly those commonly encountered in physiological fluids, such as Cl or HCO3-. -Consistent with the above, the preferred copolymer is a random copolymer, wherein 80 mol% or more of all repeating units, more preferably all repeating units are formed of a plurality of repeating units (a), which are independently selected from repeating units of formulas (a1) and (a2):

[0354]

[0355]

[0356] and multiple repeating units (b), which are independently selected from the repeating units of the following formulas (b1) and (b2):

[0357]

[0358]

[0359] The molar ratio of the sum of repeating units (a) to the sum of 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; wherein the terminal groups of the copolymer are formed by groups (c), said groups (c) being independently selected from groups of formulas (c1) and (c2):

[0360] Furthermore, one or more nitrogen atoms comprising the repeating unit (a) and / or (b) and / or terminal group (c) in the copolymer can be protonated to provide a cationic copolymer. More preferably, the copolymer is a branched copolymer comprising units (a2) and (b2), optionally together with units (a1) and / or (b1). The preparation of the copolymer is described in EP 4 223 306 A2, which is incorporated herein by reference in its entirety. In principle, lipid compounds are preferred nanoparticles, particularly compared to oligomers, and even more particularly compared to polymers.

[0361] definition

[0362] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0363] When the term "comprising" is used in this specification and claims, it does not exclude other elements. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a set is defined below as including at least a certain number of embodiments, this should also be understood as disclosing a set that preferably consists only of those embodiments.

[0364] For the purposes of this invention, the term "obtained" is considered to be a preferred embodiment of the term "available / capable of obtaining". If, for example, phDC is defined below as obtainable by a particular method, this should also be understood as disclosing a phDC obtained by that method.

[0365] When an indefinite or definite article, such as "a," "one," or "the," is used to refer to a singular noun, this includes the plural form of the noun unless otherwise specified. Unless the context clearly indicates otherwise, the term "or" means "and / or" and is used interchangeably with the term "and / or." In the context of this invention, the terms "about" or "approximately" represent a range of accuracy that, as will be understood by those skilled in the art, still ensures the technical effect of the features under discussion. This term typically indicates a deviation from the indicated value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0366] Furthermore, the terms "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," or "(i)," "(ii)," "(iii)," "(iv)," etc., used in the specification and claims are used to distinguish similar elements and are not necessarily used to describe an order or chronological order. It should be understood that such terms are interchangeable where appropriate, and embodiments of the invention described herein can operate in orders other than those described or illustrated herein.

[0367] In cases where the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” or “(i),” “(ii),” “(iii),” “(iv)” refer to steps of a method or use or measurement, unless otherwise stated, there is no temporal or time interval consistency between these steps; that is, unless otherwise stated in the application described above or below, these steps may be performed simultaneously, or there may be time intervals of seconds, minutes, hours, days, weeks, months, or even years between these steps.

[0368] The term "about" means a quantity, level, value, quantity, frequency, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 30%, 25%, 20%, 15%, 30%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated value. In the context of this invention, the term "about" indicates an accuracy range that, as will be understood by those skilled in the art, still ensures the technical effect of the feature in question. Typically, the term "about" is intended to modify numerical values ​​above and below the stated value by ±10% variance.

[0369] Technical terms are used according to their common sense. If a term conveys a specific meaning, its definition will be given below the text in which it is used.

[0370] This document should be understood as referring to any inflammatory, humoral, or cell-mediated response that occurs in order to eliminate an antigen. Immune responses can be driven by innate or adaptive immunity (also known as acquired immunity). Such responses may include, but are not limited to, antibody production, cytokine secretion, complement activity, and cytolytic activity. In one embodiment, the immune response is a response of the adaptive immune system. In one embodiment, the immune response is a T-cell response. In one embodiment, the immune response is a cytotoxic T-lymphocyte (CTL) response (which is interchangeable with a CD8+ T-cell response). In another embodiment, the immune response includes the secretion of cytokines such as IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IFNγ, and / or TNF-α. In one embodiment, the immune response includes the secretion of IFNγ.

[0371] "Effective" or "effective" refers to the ability to function as intended. For example, an "effective" immune response is one that eliminates the antigen in a subject or provides an acceptable level of immune protection against the antigen in a subject.

[0372] As used herein, the term "antigen" has the common meaning in the relevant field. Therefore, an antigen is defined as a substance recognized by the immune system as foreign or toxic, triggering an immune response. Preferably, the antigen will trigger a T cell response. More preferably, the antigen will trigger a CD8+ and / or CD4+ T cell response. Antigens can be derived from the subject, tumors, cancers, viruses, bacteria, prions, fungi, or parasites. Antigens can also include vaccines (therapeutic or prophylactic; e.g., peptides, proteins, killed pathogens, mRNA, or attenuated pathogens) as well as peptides, polypeptides, and proteins.

[0373] In a preferred embodiment, the antigen is a peptide or protein. In this case, the antigen may contain many amino acids, ranging from small peptides to large proteins, such as 4 to 2000 amino acids or more, 4 to 1800 amino acids, 4 to 1600 amino acids, or 4 to 1400 amino acids. In one embodiment, the antigen (e.g., an mRNA-encoded antigen) 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 (e.g., an mRNA-encoded antigen) contains at least 50 amino acids. In one embodiment, the antigen (e.g., an mRNA-encoded antigen) contains at least 500 amino acids. In one embodiment, the antigen (e.g., an mRNA-encoded antigen) contains at least 1000 amino acids. In one embodiment, the antigen (e.g., an mRNA-encoded antigen) contains 100 to 1500 amino acids. In one embodiment, the antigen (e.g., an mRNA-encoded antigen) comprises 200 to 1300 amino acids. In another embodiment, the antigen (e.g., an mRNA-encoded antigen) comprises 400 to 1300 amino acids.

[0374] As used herein, the term "neo-antigen" refers to a newly formed antigen arising from tumors or cancer cells due to various tumor-specific or cancer-specific alterations, such as genomic mutations, dysregulated RNA splicing, disordered post-translational modifications, and integrated viral open reading frames. Therefore, a neoantigen is an antigen not present in the normal (wild-type) (e.g., human) genome. Neoantigens are recognized as non-self and trigger an immune response independent of central and peripheral tolerance. Neoantigens can be identified using a variety of methods known in the art, such as next-generation sequencing.

[0375] The isolation or generation of tumor-specific neoantigens is a routine procedure for those skilled in the art (see, for example, Lang F, Schrörs B, Löwer M, Türeci Ö, Sahin U. Identification of neo-antigens for individualized therapeutic cancer vaccines. Nat Rev Drug Discov. 2022 Apr;21(4):261-282. doi:10.1038 / s41573-021-00387-y; Xie et al., Neo-antigens: promising targets for cancer therapy, 2023, Sig Transduct Target Ther 8, 9 (2023). https: / / doi.org / 10.1038 / s41392-022-01270-x; Li, J., Xiao, Z., Wang, D. et al., The screening, identification, design and clinical application of tumor-specific neo-antigens for TCR-T cells. Mol Cancer). 22, 141 (2023). In one particular embodiment, at least one tumor-specific neoantigen comprises the sequence in Table 4.

[0376] In some embodiments, a tumor-specific or tumor-associated antigen is first isolated from the patient, and then a nucleic acid sequence encoding the tumor-specific or tumor-associated antigen is loaded into an LNP, and the LNP is combined with a phDC. The antigen-loaded phDC is then used to subsequently test the subject's immune response to at least one tumor-specific or tumor-associated antigen using the methods described herein. In one embodiment, determining the nucleic acid sequence encoding the tumor-specific or tumor-associated antigen involves exon sequencing and / or transcriptome sequencing.

[0377] When antigens are involved, the term “string” as used herein refers to polynucleotides, such as multiple mRNAs, which encode more than one (i.e., two or more) antigens and / or antigenic epitopes in a single molecule and are separated by a linker.

[0378] When antigens are involved, the term "pool," as used herein, refers to multiple, i.e., two or more polynucleotides, such as mRNAs, each encoding an antigen and / or an epitope of an antigen. An mRNA pool can contain mRNAs encoding multiple antigens and / or epitopes in equimolar ratios. Alternatively, an mRNA pool can contain mRNAs encoding multiple antigens and / or epitopes in different specific ratios. Thus, mRNAs encoding some antigens and / or epitopes can be present in higher amounts than mRNAs encoding others, resulting in pools of various mRNAs with different concentrations. For example, in the case where each mRNA is contained in a separate LNP, an LNP pool expressing only one antigen and / or epitope is generated.

[0379] The terms “mRNA vaccine” or “mRNA therapeutic” refer to mRNA-based medicines and are used interchangeably with “preventive mRNA vaccine” or “therapeutic mRNA vaccine,” respectively. Examples of mRNA vaccines (or preventive mRNA vaccines) include two approved mRNA products, Pfizer–BioNTech’s BNT162b2 and Moderna’s mRNA-1273, both of which contain chemically modified uridine bases. mRNA therapeutics primarily refer to mRNA-based medicines used for cancer immunotherapy and are often also referred to in the art as “therapeutic mRNA vaccines.”

[0380] Dendritic cells, also referred to herein 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. The function of DCs is to capture and process antigens. When DCs endocytose antigens, they process them into smaller fragments, typically peptides, which are displayed on the surface of the DC, where they are presented, for example, to antigen-specific T cells via MHC molecules. After antigen uptake, DCs migrate to lymph nodes. During maturation, DCs can be prompted by various signals, including signaling via Toll-like receptors (TLRs), to express co-stimulatory signals that induce the activation and proliferation of homologous effector T cells (Teffs), thereby initiating a T cell-mediated immune response against that antigen. Alternatively, DCs can present antigens to antigen-specific T cells without providing co-stimulatory signals (or simultaneously providing co-inhibitory signals), resulting in inappropriate Teff activation. Such presentation can lead to, for example, death or unresponsiveness of antigen-recognizing T cells, or can induce the generation and / or expansion of regulatory T cells (Tregs). The term "dendritic cell" includes differentiated dendritic cells, immature and mature dendritic cells. These cells can be characterized by expressing certain cell surface markers (e.g., CD11c, class II MHC, and at least low levels of CD80 and CD86), CD11b, CD304 (BDCA4)). In some embodiments, DCs express CD8, CD103, CD1d, etc. Other DCs can be identified by the absence of lineage markers such as CD3, CD14, CD19, CD56, etc. In one embodiment, phDCs are characterized by expressing at least one molecular marker of HLA-DR, CD83, CD86, ICAM, or PLAUR. Furthermore, dendritic cells can be functionally characterized by their ability to stimulate allogeneic responses and mixed lymphocyte responses (MLR).

[0381] The term "AB serum" or "human AB serum" refers to cell culture reagents known in the art for use with some human cell types, which provide growth factors, vitamins, nutrients, and trace elements and transport factors. Human AB serum is collected from healthy male donor volunteers with the AB serotype.

[0382] The term "FBS" refers to a widely used growth supplement for cell culture media. It typically contains high levels of embryonic growth factors.

[0383] The terms "individual" and "subject" are used interchangeably herein. "Individual" refers to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In some embodiments of the invention, "individual" or "subject" is "patient."

[0384] The term "patient" refers to an individual or subject who is receiving treatment or preventative treatment.

[0385] As used herein, "cells capable of secreting IFNγ" refers to cells capable of secreting IFNγ. The term "interferon-γ" or "IFNγ" (interferon gamma, type II interferon) refers to a macrophage activating factor and immunointerferon, primarily produced by immune cells (such as T lymphocytes and natural killer cells) in response to antigens. IFNγ is a dimeric protein composed of two 146-amino acid subunits. IFNγ is a glycoprotein that functionally exists as a homodimer of approximately 45 kDa. On SDS-PAGE, due to differential glycosylation, IFNγ presents as a combination of 25 kDa, 20 kDa, and a small number of 15.5 kDa bands. Functions of IFNγ include antiviral activity, antitumor antiproliferative activity, induction of class I and II MHC and macrophage activation, and enhancement of B lymphocyte immunoglobulin secretion. IFNγ participates in cytokine regulation and also works synergistically with other cytokines. IFNγ activation occurs through the binding of IFNγ receptors I and II and the activation of the JAK-STAT pathway.

[0386] Cells capable of secreting IFNγ can be identified by various methods known in the art, such as the FACS method described above or the ELIspot assay. Several commercial kits are available, for example, from Miltenyi. In one embodiment, cells capable of secreting IFNγ include CD8+ T cells, natural killer cells, γδ T cells, natural killer T cells, CD4+ T cells, group 1 innate lymphocytes, IFN-producing killer dendritic cells, memory CD8+ T cells, and memory CD4+ T cells.

[0387] As mentioned herein, "T cells" include CD8+ T cells, γδ T cells, natural killer T cells, CD4+ T cells, memory CD8+ T cells, and memory CD4+ T cells. In a preferred embodiment, T cells include CD4+ T cells, CD8+ T cells, memory CD4+ T cells, and memory CD8+ T cells.

[0388] As mentioned in this article, CD4+ T cells (also known as T helper cells or Th cells) are activated upon interaction with the antigen-MHC complex and differentiate into specific subtypes primarily dependent on the cytokine environment of the microenvironment. In addition to the classic helper T cells 1 and 2, other subsets were identified, including helper T cells 17, regulatory T cells, follicular helper T cells, and helper T cells 9, each with its own characteristic cytokine profile. For example, Th1 T cells primarily secrete IFNγ, lymphotoxin α (LF α), and IL2, while Th2 T cells primarily secrete IL4, IL5, IL9, IL13, IL10, IL25, and amphiregulin. Th17 T cells primarily secrete IL17A, IL17F, IL21, and IL22. The main effector cytokines of Treg cells include IL10, TGF-β, and IL35. Follicular helper T cells secrete IFNγ, IL4, and IL10. The cytokines mentioned can be used to characterize the immune response according to the method of the present invention.

[0389] As mentioned in this article, CD8+ (cytotoxic) T cells (also known as CTLs) express T cell receptors like CD4+ T cells. However, unlike the CD4 molecule, cytotoxic T cells express the dimerized co-receptor CD8, which is typically composed of a CD8 α chain and a CD8 β chain. CD8+ T cells recognize peptides presented by MHC class I molecules present on all nucleated cells. CD8+ T cells are crucial for immune defense against intracellular pathogens, including viruses and bacteria, as well as for tumor surveillance. When CD8+ T cells recognize their antigens and are activated, they primarily secrete TNF-α and IFN-γ, which have antitumor and antiviral / microbial effects.

[0390] CD8+ and CD4+ T cells can also exist as memory T cells, which maintain their antigen specificity throughout the host's lifespan without further antigen stimulation and can confer immune protection against antigens to the subject. Memory T cells, in particular, express the marker CD45RO, which can be used to characterize the immune response according to the method of the present invention.

[0391] As mentioned herein, “natural killer (NK) cells” are lymphocytes belonging to the same family as T cells, originating from common progenitor cells. However, as cells of the innate immune system, NK cells are classified as group I innate lymphocytes (ILCs; ILCs include NK cells and ILC1, both capable of secreting IFNγ) and respond rapidly to a variety of pathological challenges. NK cells are known for killing virus-infected cells and detecting and controlling early signs of cancer. Activated NK cells secrete a variety of cytokines such as IFN-γ, TNF-α, GM-CSF, IL-10, IL-5, and IL-13, as well as chemokines such as MIP-1α, MIP-1β, IL-8, and RANTES. The mentioned cytokines and chemokines can be used to characterize the immune response according to the methods of the present invention.

[0392] As mentioned in this article, "γδ(γδ)T cells" are the prototype of "unconventional" T cells and represent a relatively small subset of T cells in peripheral blood. γδT cells are defined by the expression of a heterodimeric T cell receptor (TCR) composed of γ and δ chains. This distinguishes them from classic CD4+ T cells and CD8+ T cells that express αβ TCR. γδT cells primarily secrete IFN-γ and TNF-α.

[0393] As mentioned herein, “natural killer T (NKT) cells” are a subset of CD1d-restricted T cells located at the boundary between the innate and adaptive immune systems. NKT cells can be further subdivided into functional subsets that utilize T cell or natural killer (NK) cell-like effector mechanisms to rapidly respond to a variety of glycolipids and stress-related proteins. Due to their major regulatory role in immune responses through the secretion of cytokines, NKT cells are also considered important players in tumor immune surveillance. Upon activation, NKT cells produce IFN-γ, IL-4, TNF-α, IL-5, IL-6, IL-10, IL-13, and TGF-β. Type I NKT cells synthesize growth factors such as IL-2 and GM-CSF, as well as chemokines such as RANTES, MIP-1α, and MIP-1β. These cytokines, growth factors, and chemokines can be used to characterize immune responses according to the methods of the present invention.

[0394] As mentioned in this article, "IFNγ-producing killer dendritic cells" are a subset of dendritic cells (DCs), exhibiting a chimera of NK cells and DCs, namely interferon-producing killer dendritic cells (IKDCs). IKDCs not only secrete type I and type II interferons to effectively recognize and kill tumor cells, but also express MHC-II molecules to present antigens. Therefore, IKDCs are considered important immune surveillance cells for tumors, providing a link between innate and adaptive immunity.

[0395] Cells capable of secreting IFNγ can be isolated from subjects (e.g., mammalian or human subjects) using any suitable method known in the art. For example, cells capable of secreting IFNγ (e.g., T cells) can be isolated from the peripheral blood of subjects (e.g., mammalian or human subjects) by density centrifugation using a stepwise density gradient consisting of a mixture of carbohydrate Ficoll™ and the iodine-containing high-density compound metrizamide. This produces a mononuclear cell population, called peripheral blood mononuclear cells (PBMCs), which have been reduced to erythrocytes and most polymorphonuclear leukocytes or granulocytes, and are mainly composed of lymphocytes and monocytes. T lymphocytes can be isolated from PBMCs by binding the sample to an antibody-coated plastic surface (this is referred to in the art as “panning”) or by treating with specific antibodies and complement to kill unwanted cells. Alternatively, PBMCs can be passed through a steel wool column coated with antibodies and nylon, with different populations being differentially eluted. Furthermore, T lymphocytes can be isolated from PBMCs using flow cytometry or fluorescence-activated cell sorting (FACS). Janeway et al. further described a method for isolating lymphocytes from mammals, particularly humans.

[0396] As used herein, “autologous” means any tissue, cell or sample (possibly modified) taken from the subject and suitable for reintroduction, wholly or partially, into the same (“autologous”) subject.

[0397] As used herein, “allogeneic” means any tissue, cell, or sample (possibly modified) that is taken from a donor (i.e., not the subject) and wholly or partially reintroduced into the subject, and is therefore different from the subject (“allogeneic”). In other words, “allogeneic” means any tissue, cell, or sample that is not derived from the subject themselves.

[0398] As used herein, the term "hematopoietic graft" refers to any graft (e.g., cell population) derived from hematopoietic tissue that is removed from a donor and is suitable for reintroduction, wholly or partially, into the same or different recipient. Hematopoietic grafts include grafts derived from bone marrow, peripheral blood stem cell transplantation (PBSCT), leukocytes, leukocytes, or umbilical cord blood (e.g., umbilical cord blood grafts).

[0399] As used herein, the terms "polynucleotide" or "nucleic acid" are intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinant-derived, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes RNA transcribed in vitro or synthetic RNA. In one embodiment of all aspects of the invention, RNA encoding an antigen (e.g., mRNA) is expressed in monocytes or phDCs. The loaded monocytes or phDCs present the antigen to the subject's immune system.

[0400] The nucleic acids (e.g., mRNA) described herein can be recombinant and / or isolated molecules. In this disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-furanose ribonucleotide. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, mRNA, circular RNA, synthetic RNA, recombinant RNA, self-amplifying 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 aforementioned RNA types, such as "RNA," "mRNA," "self-amplifying RNA," "modified mRNA," or "synthetic RNA," always contain a segment that still retains its function. A "segment" can refer to a portion of a nucleotide sequence, i.e., a sequence shortened at the 5' or 3' end. The fragment of the RNA (e.g., mRNA) sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the residues from the RNA (e.g., mRNA) sequence. The fragment of the RNA (e.g., mRNA) sequence preferably comprises at least 18, particularly at least 24, at least 36, at least 45, at least 60, at least 90, at least 150, or at least 300 consecutive residues from the RNA sequence (e.g., mRNA).

[0401] The alteration can target internal RNA (e.g., mRNA) nucleotides or the addition of non-nucleotide substances to the ends of RNA (e.g., mRNA). This document also considers cases where the nucleotides in the RNA (e.g., mRNA) can be non-standard nucleotides, such as chemically synthesized nucleotides, naturally occurring modified nucleotides, or deoxynucleotides. For the purposes of this disclosure, these altered RNAs (e.g., mRNAs) are considered analogs of naturally occurring RNAs (e.g., mRNAs).

[0402] In some embodiments of the invention, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As known in the art, mRNA typically comprises a 5' untranslated region (5'-UTR), a coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA (e.g., mRNA) is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, wherein the DNA refers to a nucleic acid containing deoxyribonucleotides. In one embodiment, the RNA (e.g., mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription using a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase.

[0403] DNA templates for in vitro transcription can be obtained by cloning nucleic acids (especially cDNA) and introducing them into a suitable in vitro transcription vector. cDNA can be obtained by reverse transcription of RNA.

[0404] In one embodiment, the RNA, particularly the mRNA, described herein may have modified nucleosides. In some embodiments, the RNA, particularly the mRNA, contains modified nucleosides replacing at least one (e.g., each) uridine.

[0405] "Pseudouridine" is an example of a modified nucleoside, which is an isomer of uridine in which uracil is attached to a pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond. Another exemplary modified nucleoside is N1-methyl-pseudouridine. Another exemplary modified nucleoside is 5-methyl-uridine. In some embodiments, one or more uridine residues in the RNA (e.g., mRNA) described herein are replaced by modified uridine residues such as N1-methyl-pseudouridine or 5-methyl-uridine, or combinations thereof. In one embodiment, the RNA contains other modified nucleosides or contains further modified nucleosides, such as modified cytidine. For example, in one embodiment, cytidine in the RNA is partially or completely replaced by 5-methylcytidine, preferably completely replaced. In one embodiment, the RNA contains 5-methylcytidine and one or more selected from pseudouridine, N1-methyl-pseudouridine, and 5-methyl-uridine. In one embodiment, the RNA contains 5-methylcytidine and N1-methyl-pseudouridine. In some embodiments, the RNA comprises 5-methylcytidine replacing each cytidine and N1-methylpseuuridine replacing each uridine. In some embodiments, the RNA (e.g., mRNA) may comprise more than one type of modified nucleoside. In some embodiments, only a subset of uridine residues is modified. In some embodiments, all uridine residues are modified.

[0406] In some embodiments, the modified nucleoside is a modified uridine. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), and uridine. 5-Hydroxyacetic acid (cmo5U), methyl 5-hydroxyacetic acid ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), methyl 5-carboxyhydroxymethyl-uridine ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5 5-Methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauronic acid methyl-uridine (τm5U), 1-tauronic acid methyl-pseudouridine, 5-tauronic acid methyl-2-thio-uridine (τm5s2U) U), 1-Taurate methyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with deoxythymidine nucleobases), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deazo-pseudouridine, 2-thio-1-methyl-1-deazo-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-Dihydrouridine, 5-Methyl-Dihydrouridine (m5D), 2-Thio-Dihydrouridine, 2-Thio-Dihydropseudouridine, 2-Methoxy-uridine, 2-Methoxy-4-Thio-uridine, 4-Methoxy-pseudouridine, 4-Methoxy-2-Thio-pseudouridine, N1-Methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-Methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3Ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thiouridine (inm5S2U), α-Thio-uridine, 2'-O-Methyl-uridine (Um), 5,2'-O-Dimethyl-uridine (m5Um), 2'-O 1-Methyl-pseudouridine (ψm), 2-thio-2'-O-methyluridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um) and 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-methoxycarbonylvinyl)uridine and 5-[3-(1-E-propenylamino)]uridine.

[0407] In some implementations, the modified nucleoside is a modified cytidine. Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudocytidine, 3-methylcytidine (m3C), N4-acetylcytidine (ac4C), 5-formylcytidine (f5C), N4-methylcytidine (m4C), 5-methylcytidine (m5C), 5-halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm5C), 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thio-cytidine (s2C), 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio-1-methyl-1-deazo-pseudocytidine, 1-methyl-1-deazo-pseudocytidine, zablaline, 5-aza-zablaline, 5-methyl -Zebralin, 5-aza-2-thio-zaebralin, 2-thio-zaebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-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-afa-cytidine, 2'-F-cytidine, and 2'-OH-afa-cytidine.

[0408] In some embodiments, the modified nucleoside is adenosine. Exemplary nucleobases and nucleosides having modified adenine include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deadenine, 7-deadenine-8-aza-adenosine, 7-deadenine-2-amino-purine, 7-deadenine-8-aza-2-amino-purine, 7-deadenine-2,6-diamino-purine, 7-deadenine-8-aza-2,6-diamino-purine, 1-methyl-adenosine (m1A), and 2-methyl-adenosine. N6-methyl-adenosine (m2A), 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-glycylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N 6-Threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynor-valylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalyl-carbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenosine, 2-Methylthio-adenine, 2-methoxy-adenine, α-thio-adenine, 2'-O-methyl-adenine (Am), N6,2'-O-dimethyl-adenine (m6Am), N6,N6,2'-O-trimethyl-adenine (n62Am), 1,2'-O-dimethyl-adenine (m1Am), 2'-O-ribosyl-adenine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, 2'-F-ara-adenine, 2'-F-adenine, 2'-OH-ara-adenine, and N6-(19-amino-pentaenodecyl)adenine.

[0409] In some embodiments, the modified nucleoside is a modified guanidine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methylinosine (m1I), wyoside (imG), methylwyoside (mimG), 4-demethyl-wyoside (imG-14), isowyoside (imG2), weitingoside (yW), peroxyweitingoside (O2yW), hydroxyweitingoside (OhyW), incompletely modified hydroxyweitingoside (OhyW*), 7-deazoguanosine, guidingoside (Q), epoxyguidingoside (oQ), galactosylguidingoside (g) alQ), mannosylguanosine (manQ), 7-cyano-7-deazoguanosine (preQo), 7-aminomethyl-7-deazoguanosine (preQ1), archaenoside (G+), 7-deazo-8-azaguanosine, 6-thioguanosine, 6-thio-7-deazoguanosine, 6-thio-7-deazo-8-azaguanosine, 7-methylguanosine (m7G), 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine (m1G), N2-methyl α-Gyroguanosine (m2G), N2,N2-dimethylguanosine (m22G), N2,7-dimethylguanosine (m2,7G), N2,N2,7-trimethylguanosine (m2,2,7G), 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, N2,N2-dimethyl-6-thioguanosine, α-thioguanosine, 2'-O-methylguanosine (Gm), N2-methyl-2'-O-methylguanosine (m2 Gm), N2,N2-dimethyl-2'-O-methylguanosine (m22Gm), 1-methyl-2'-O-methylguanosine (m1Gm), N2,7-dimethyl-2'-O-methylguanosine (m2,7Gm), 2'-O-methylinosine (Im), 1,2'-O-dimethylinosine (m1lm), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thioguanosine, O6-methylguanosine, 2'-F-ara-guanosine and 2'-F-guanosine.

[0410] 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 with a 5'-cap analogue. The term "5'-cap" refers to a structure present at the 5'-terminus of the mRNA molecule and is typically composed of guanosine nucleotides linked to the mRNA via 5'-to-5'-triphosphate bonds. In one embodiment, the guanosine is methylated at the 7-position. The provision of mRNA with a 5'-cap or a 5'-cap analogue can be achieved by in vitro transcription (where the 5'-cap is co-transcribed into the mRNA strand, or by using a capping enzyme to ligate it to the mRNA post-transcriptionally).

[0411] The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule such as mRNA. An untranslated region (UTR) can be located at the 5' (upstream) end of an open reading frame (OPF) (5'-UTR) and / or the 3' (downstream) end of an OPF (3'-UTR). A 5'-UTR (if present) is located at the 5' end upstream of the start codon in a protein-coding region. A 5'-UTR is downstream of a 5'-cap (if present), for example, directly adjacent to the 5'-cap. A 3'-UTR (if present) is located at the 3' end downstream of the stop codon in a protein-coding region, but the term "3'-UTR" preferably does not contain a poly(A) sequence. Therefore, a 3'-UTR is upstream of a poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0412] As used herein, the terms "poly(A) sequence" or "poly-A tail" refer to a continuous or discontinuous sequence of adenosine 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 can be understood in the 3'-UTR of the RNA (e.g., mRNA) described herein. A continuous poly(A) sequence is characterized by a continuous sequence of adenosine residues. Continuous poly(A) sequences are typical in nature. The RNA (e.g., mRNA) according to the invention may have a poly(A) sequence that is posttranscribed by template-independent RNA polymerase to the free 3' end of the RNA (e.g., mRNA), or may have a poly(A) sequence encoded by DNA and transcribed by template-dependent RNA polymerase. A poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on both RNA and protein levels in transfected eukaryotic cells, where the protein is 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 comprises, or consists of, at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 800, at most 400, at most 300, at most 200, or at most 150 A nucleotides. In one embodiment, the poly(A) sequence comprises, at least 300 nucleotides, or consists of, 300 nucleotides. In this context, "consistently composed of" means that the majority of nucleotides in the poly(A) sequence are typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly(A) sequence, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "consisting of" means that all nucleotides in the poly(A) sequence are 100% A nucleotides, based on the number of nucleotides in the poly(A) sequence. The term "A nucleotide" or "A" refers to adenosine monophosphate. In some embodiments, the poly(A) sequence is linked based on a DNA template during RNA transcription (e.g., in the preparation of in vitro transcribed RNA, such as mRNA), which contains repeating dT nucleotides (deoxythymidine monophosphate) in a strand complementary to the coding strand. The DNA sequence encoding the poly(A) sequence (coding strand) is called the poly(A) cassette.

[0413] The term "codon-optimized" refers to altering the codons in the coding region of a nucleic acid molecule to reflect typical codon usage in the host organism, preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this invention, the coding region is preferably codon-optimized for optimal expression in subjects to be treated with the RNA (e.g., mRNA) molecules described herein. Codon optimization is based on the finding that translation efficiency also depends on the frequency of tRNA occurrence in cells. Therefore, the RNA (e.g., mRNA) sequence can be modified such that rare codons are replaced with codons that yield high-frequency tRNA.

[0414] In some embodiments of the 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, wherein 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. Regarding the fact that several codons encode the same amino acid (so-called degeneracy of the genetic code), the codons most favorable for stability (so-called alternative codon usage) can be determined. Depending on the amino acid to be encoded by the RNA, the RNA (e.g., mRNA) sequence has a variety of modification possibilities compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing them with other codons that encode the same amino acid but do not contain A and / or U or contain a lower amount of A and / or U nucleotides.

[0415] In the context of this invention, the term "transcription" refers to the process of transcribing the genetic code in a DNA sequence into RNA (e.g., mRNA). Subsequently, the RNA (e.g., mRNA) can be translated into peptides or proteins.

[0416] According to the present invention, the term "transcription" includes "in vitro transcription," wherein the term "in vitro transcription" refers to the process of synthesizing RNA, particularly mRNA, in vitro in a cell-free system, preferably using a suitable cell extract. Regarding RNA (e.g., mRNA), the term "expression" or "translation" refers to the process of assembling an amino acid sequence in a cell ribosome guided by an mRNA chain to produce a peptide or protein. In one embodiment, at least a portion of the RNA (e.g., mRNA) (e.g., formulated into RNA (e.g., mRNA) (lipid) particles) as described herein is delivered to a monocyte or phDC. In one embodiment, the RNA (e.g., mRNA) is translated by the monocyte or phDC to produce the peptide or protein it encodes. RNA (e.g., mRNA) particles (such as RNA (e.g., mRNA) lipid particles as described herein) can be used to deliver RNA to monocytes or phDCs.

[0417] "Encoding" refers to the intrinsic property of a specific nucleotide sequence in a polynucleotide (e.g., gene, cDNA, RNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules with defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties during biological processes. Therefore, if the translation of RNA (e.g., mRNA) occurs in a cell, the RNA (e.g., mRNA) sequence can encode a protein (e.g., an antigen).

[0418] As used herein, terms such as “reduce,” “effectively reduce,” “reduce,” “inhibit,” or “weaken” refer to the ability to reduce or cause an overall reduction, preferably by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or even more. These terms include complete or substantially complete inhibition, i.e., reduction to zero or substantially zero. Terms such as “increase,” “enhance,” or “exceed” preferably refer to an increase or enhancement 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 even more.

[0419] According to this disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together by peptide bonds. The terms "protein" or "polypeptide" refer to large peptides, particularly peptides having at least about 150 amino acids, but unless otherwise stated, the terms "peptide," "protein," and "polypeptide" are used as synonyms herein.

[0420] A “fragment” can refer to a portion of an antigen (such as a disease-associated antigen), an amino acid sequence, an antigenic protein, or RNA (including the types of RNA listed above, and especially mRNA).

[0421] If the fragment involves an amino acid sequence (e.g., an antigen), the term refers to a portion of the amino acid sequence, specifically a sequence representing the shortening of that amino acid sequence (e.g., the amino acid sequence of an antigen) at the N-terminus and / or C-terminus. A C-terminal shortened fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end. An N-terminal shortened fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end, provided that the truncated open reading frame contains a start codon for initiating translation. The 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 from the amino acid sequence. The amino acid sequence fragment preferably 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 from the amino acid sequence.

[0422] In this document, "variant" refers to 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 it may be 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, one to about 20 amino acid modifications, one to about 10 amino acid modifications, or one to about 5 amino acid modifications compared to the parent.

[0423] In this article, "wild-type," "WT," or "natural" refers to amino acid sequences that exist in nature, including allelic variations. Wild-type amino acid sequences, peptides, or proteins have amino acid sequences that have not been intentionally modified.

[0424] 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 mutants, splicing variants, post-translational modification variants, conformations, isotypes, allele variants, species variants, and species homologs, particularly those naturally occurring variants. The term “variant” specifically includes fragments of amino acid sequences. Preferably, the degree of similarity (or identity) between a given amino acid sequence and an amino acid sequence that is a variant of said 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, can be performed using tools known in the art, preferably using optimal sequence alignment, such as Align, using standard settings, preferably EM-BOSS::needle, Matrix: Blosum62, etc. "Sequence similarity" indicates the percentage of identical amino acids or represents the percentage of conserved amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of identical nucleotides between the sequences. The identity percentage is obtained by determining the number of identical positions at corresponding locations in the sequences to be compared, dividing that number by the number of comparison positions (e.g., the number of positions in the reference sequence), and then multiplying the result by 100.

[0425] Specifically, for example, the percentage of identity between two polynucleotide sequences can be calculated by aligning the two sequences for optimal comparison (e.g., vacancies can be introduced into one or both of the first and second nucleic acid sequences to achieve optimal alignment, and dissimilar sequences can be ignored for comparison purposes). In some embodiments, the length of the sequences aligned for comparison purposes is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference sequence. Nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the two molecules are identical at that position. The percentage of identity between two sequences is a function of the number of common positions in the sequences, taking into account the number of vacancies introduced for optimal alignment of the two sequences and the length of each vacancy. The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. For example, the percentage of identity between two nucleic acid sequences can be determined using methods described in the following literature: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, ed., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., Stockton Press, New York, 1991. For example, the percentage of identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been integrated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4.Alternatively, the percentage of identity between two nucleic acid sequences can be determined using the GAP program in the GCG software package, utilizing the NWSgapdna.CMP matrix. Methods commonly used to determine the percentage of identity between sequences include, but are not limited to, the methods disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988). Techniques for determining identity have been incorporated into publicly available computer programs. Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG software package, Devereux, J. et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, SF et al., J. Molec. Biol., 215, 403 (1990)).

[0426] In one embodiment, the fragment or “variant” is preferably a “functional fragment” or “functional variant.” The terms “functional fragment” or “functional variant” refer to any fragment or variant that exhibits one or more functional properties that are the same as or similar to those of the unfragmented reagent from which it is derived (e.g., an unfragmented amino acid sequence, an unfragmented RNA sequence, an unfragmented mRNA sequence, etc.), i.e., they are functionally identical. For an antigen, a specific function is one or more immunogenic (synonymous with “antigenicity” herein) activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the terms “functional fragment” or “functional variant” specifically refer to a variant molecule or sequence that contains a sequence with one or more amino acid or nucleotide changes compared to the parent molecule or sequence, but still achieves one or more functions of the parent molecule or sequence, such as inducing an immune response (i.e., possessing antigenicity). 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 different embodiments, the function of the functional fragment or 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 the immunogenicity of the parent reagent, molecule, or sequence. However, in other embodiments, the immunogenicity of the functional fragment or variant may be enhanced compared to the parent molecule or sequence.

[0427] As used herein, the term "vaccine" refers to a composition that induces an immune response when administered to a subject. The term "prophylactic" refers to a vaccine administered before the onset of a pathological event or exposure to an infectious agent, such as before infection with an infectious disease or before the onset of a tumor.

[0428] In contrast to vaccination, the term "treatment" or "therapeutic treatment" involves treating a subject (e.g., a mammal, such as a human) or cells to alter the current course of the disease. Therapeutic treatment is administered at the onset of a pathological event or following exposure to an infectious agent.

[0429] The term "disease" or "symptom" refers to any disease or symptom involving an antigen, such as a disease characterized by the presence of an antigen. The disease can be, for example, an infectious disease or a neoplastic disease (cancer). In one embodiment, a disease involving an antigen is a disease involving cells expressing the antigen (preferably on the cell surface).

[0430] The terms "tumor" and "cancer" refer to a disease in which some cells of the body grow uncontrollably and spread to other parts of the body. A tumor can be cancerous or non-cancerous (benign). In the context of this invention, unless otherwise specified, the terms tumor and cancer are used interchangeably. A cancerous tumor spreads to or invades nearby tissues and can move to distant parts of the body to form new tumors (also known as metastasis). A cancerous tumor can also be called a malignant tumor. Many cancers form solid tumors, but blood cancers, such as leukemia, do not form solid tumors. Benign tumors do not spread to or invade nearby tissues. Blood cancers include leukemia, lymphoma, myelodysplastic syndromes (MDS), myelodysplastic disorders (MPD), multiple myeloma, and all its subtypes.

[0431] The term "infectious disease" refers to any illness that can be transmitted between individuals or between organisms and is caused by microbial agents (such as the common cold). Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, fungal diseases, prion diseases, or parasitic diseases caused by viruses, bacteria, fungi, prions, and parasites, respectively. In this regard, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (such as 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.

[0432] "Coronavirus disease 2019," or COVID-19, is a contagious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). SARS-CoV-2, which causes atypical pneumonia, has been designated a Public Health Emergency of International Concern. SARS-CoV-2 (MN908947.3) belongs to the β-coronavirus lineage B. It shares at least 70% sequence similarity with SARS-CoV. Specifically, 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 indicate a disease diagnosed clinically, epidemiologically, or otherwise. Generally, coronaviruses have four structural proteins: envelope (E), membrane (M), nucleocapsid (N), and spike protein (S). The E and M proteins play important functions in viral assembly, and the N protein is essential for viral RNA synthesis. The key glycoprotein S is responsible for viral binding and entry into target cells. The S protein is synthesized as a single-stranded, inactive precursor and is cleaved in the producing cell by a furin-like host protease into two non-covalently bound subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) that recognizes a host cell receptor. The S2 subunit contains a fusion peptide, two heptapeptide repeats, and a transmembrane domain, all essential for mediating viral-host cell membrane fusion through a large conformational rearrangement. SARS-CoV-2 is used to represent all variants of this virus belonging to the domain Riboviria, kingdom Orthornavirae, phylum Pisuviricota, class Pisoniviricetes, order Nidovirales, family Coronaviridae, genus Betacoronavirus, subgenus Sarbecovirus, species Severe Acute Respiratory Syndrome-Associated Coronavirus, and strain 2 of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) according to the ICTV classification.

[0433] The invention will now be described with reference to some specific examples; however, these examples are for illustrative purposes and not intended to be limiting.

[0434] Example

[0435] Example

[0436] Experiment 1: Human phDC production

[0437] Isolation of human peripheral blood mononuclear cells (PBMCs)

[0438] Peripheral blood was collected from healthy human volunteer donors as needed for the experiment. Whole blood was collected into 1:100 5,000 U / mL heparin (McKesson Packaging Services). Platelet-containing peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral whole blood using Isolymph gradient separation (CTLScientific Supply Corp).

[0439] Human PBMC TI processing solution

[0440] The isolated human platelet-containing PBMCs were resuspended in fetal bovine serum (FBS). Then, cells (up to 2*10⁻⁶) were... 7 PBMCs / plates were incubated in the TI chamber at 37°C for 1 hour. This step allows platelet-activated plasma proteins to deposit in the chamber, and platelets to adhere to the coated chamber surface, as confirmed by optical microscopy. Cells were then passed through the TI chamber using an infusion pump at a rate of 0.09 mL / min. After plate passage, cells were collected, and the TI chamber was washed with 100% FBS at 0.49 mL / min, while simultaneously physically agitating the plate surface by tapping or flicking to help detach all adhered cells from the chamber.

[0441] Experiment 2: Detection of ELISpot spike protein in SARS-CoV-2 reactive T cells

[0442] LNP transduction of human phDC containing spike protein mRNA for ELISpot

[0443] PBMCs containing neonatal phDCs from healthy human donors, produced according to the method described in Experiment 1, were passaged at 5*10⁻⁶ cells per well. 5Cells were plated in human IFNγ ELISpot plates, in triplicate for each experimental group. To each well, 62.5–250 ng / well of cKK-E12-based LNPs (cKK-E12, cholesterol, C14-PEG 2000-PE, and DOPE in a ratio of 35:46.5:2.5:16) containing spike protein mRNA (prepared and supplied by Santangelo Laboratory, Emory University; RNA sequence corresponds to SEQ ID NO: 19) were added to 200 μL of RPMI (Gibco) supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen) without phenol red. Positive control wells were treated with overlapping pools of class I and II spike peptides (Miltenyi Biotec, PepTivator SARS-CoV-2 Prot-S Complete). Negative control wells were treated with cKK-E12-based LNPs containing unrelated (e.g., nanoluciferase) protein mRNA at a dose of 62.5–250 ng / well.

[0444] Elispot readings

[0445] After overnight incubation, wells were washed, and IFNγ spots were detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFU) for each experimental condition were quantified using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). As a standard practice, an ELISpot signal was considered positive if it was higher than 50 SFU / million cells, and / or if the experimental group signal was at least 2 times higher than the control (mimic) group signal.

[0446] CD4 and CD8 T cell reduction

[0447] To test the contribution of T cell subsets to cytokine production, CD4 or CD8 T cells can be selectively depleted from PBMCs after plate passage, but before overnight incubation with LNPs in Elispot wells. T cells were depleted using a standard depletion kit (Miltenyi; CellSep).

[0448] result

[0449] Figure 1A Example of phDC ELISpot using PBMCs isolated from a single human donor 4 weeks post-SARS-CoV-2 infection. IFNγ release was significantly increased in the presence of phDCs [spikes] compared to the negative control. This demonstrates the detection of human spike-specific T cells by phDCs transduced via mRNA in Covid-19 convalescent donors.

[0450] Figure 1B Example of phDC ELISpot dose response using PBMCs from a single human donor 4 weeks post-SARS-CoV2 infection. When LNP [spike] antigen was added at 62.5 ng / well, IFNγ release increased to levels exceeding 150 SFU / million. T cell activation was further improved at higher doses, but evidence of a response plateau was shown at 250 ng / well. This demonstrates that phDC LNP spike dose response is detectable down to 62.5 ng / well.

[0451] Figure 2A In the phDC ELISpot, 18 previously vaccinated human donors were screened for the SARS-CoV-2 spike antigen. Donors were divided into two cohorts based on whether they had previously been infected with SARS-CoV-2 (black) or not (blue). Statistical analysis employed an unpaired two-tailed Mann-Whitney U test. This demonstrated that human phDCs transduced with LNP [spike] can distinguish between T-cell responses associated with innate immunity and responses associated with vaccination alone.

[0452] Figure 2B In the phDC ELISpot, 11 previously vaccinated and recovered human donors were screened against the SARS-CoV-2 spike antigen. The dashed line represents the standard threshold cutoff for a positive response (50 SFU / million cells). This indicates that: 1) the strength of the response is generally negatively correlated with the duration of recovery; and 2) the phDC-induced IFNγ response is persistent and can be detected up to one year after Covid infection.

[0453] Figure 2C Prior to incorporation into the standard 18-hr phDC [spike] ELISpot IFNγ assay, PBMCs from convalescent donors passaged for vaccination underwent CD8 or CD4 T cell depletion. CD8 T cell depletion eliminated most of the phDC [spike] response, while CD4 depletion had the least impact. This indicates that the convalescent phDC [spike] IFNγ response is primarily driven by CD8+ T cells.

[0454] Figure 2DExamples of phDC ELISpots were obtained using PBMCs isolated from a single human donor before and six weeks after SARS-CoV-2 infection. Post-infection IFNγ release increased to over the positive threshold of 50 SFU / million cells. This demonstrates that phDC-induced IFNγ T cell responses increase after natural Covid infection, showcasing the feasibility and practicality of longitudinal immune surveillance.

[0455] Experiment 3: MART1 phDC ELISpot assay using LNPs containing MART1 peptide mRNA

[0456] LNP transduction of human phDC containing MART1 peptide mRNA for ELISpot

[0457] PBMCs containing neonatal phDCs from healthy human HLA-A2+ donors, produced according to the method described in Experiment 1 and passaged in 5*10 wells, were used. 5 Cells were seeded in triplicate for each experimental group and stimulated with 125 ng / well of SM102-based LNPs (SM102, cholesterol, DMG-PEG-2K, DSPC, in a ratio of 50:38.5:1.5:10) containing HLA-A2 conjugate immunodominant MART1 peptide (ELAGIGILTV) mRNA in 200 µL of medium (LNPs prepared and supplied by Santangelolaboratory, Emory University, RNA sequence corresponding to SEQ ID NO: 23). The medium consisted of phenol red-free RPMI (Gibco) supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen). Positive control wells were treated with MART1 peptide (Tufts University Peptide Synthesis Core). Negative control wells were treated with 125 ng / well of SM102-based LNPs containing unrelated protein mRNA (e.g., nanoluciferase).

[0458] ELISpot readings

[0459] After overnight incubation, wells were washed, and IFNγ spots were detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFU) for each experimental condition were quantified using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). As a standard practice, an ELISpot signal was considered positive if it was higher than 50 SFU / million cells, and / or if the experimental group signal was at least 2 times higher than the control (mimic) group signal.

[0460] result

[0461] To evaluate the function of phDCs transfected with MART1 peptide mRNA LNP, two separate T cell stimulation assays were performed using three human HLA-A2+ donors. First, phDCs transfected with LNP [MART-1] were co-cultured with MART1-specific DMF5 T cells for 3 days. Compared to the co-culture group with phDCs transfected with LNP [mimicry], a significantly increased IFNγ release was observed in all three donors. Figure 3 A) indicates that antigen-transfected phDCs can stimulate T cells via specific TCR binding. Secondly, the ability of phDCs transfected from the same donor, LNP [MART-1], to naturally bind to endogenous precursor T cells in the phDC IFNγ ELISpot assay was evaluated. Compared to phDCs transfected with LNP [mimicry], the LNP [MART-1] group showed a significantly increased number of IFNγ spots, indicating the initiation of an antigen-specific immune response (…). Figure 3 B).

[0462] Experiment 4 (Hypothetical): Screening for antigens of interest using phDC ELISpot

[0463] LNP transduction of human phDC containing antigenic mRNA for ELISpot

[0464] PBMCs containing newly generated phDCs (e.g., from cancer patients) produced according to the method described in Experiment 1 and passaged in 5*10 wells were used. 5Cells were plated in triplicate for each experimental group and stimulated with an appropriate amount of LNP containing, for example, mRNA encoding a cancer antigen of interest (e.g., a protein or peptide antigen isolated or identified from autologous tumor material) in 200 μL of medium supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen) in phenol red-free RPMI (Gibco). Negative control wells were treated with an equal amount of LNP containing mRNA of an unrelated protein (e.g., nanoluciferase).

[0465] ELISpot readings

[0466] After overnight incubation, wells were washed, and IFNγ spots were detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFU) for each experimental condition were quantified using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). As a standard practice, an ELISpot signal was considered positive if it was higher than 50 SFU / million cells, and / or if the experimental group signal was at least 2 times higher than the control (mimic) group signal.

[0467] result

[0468] A positive ELISpot IFNγ signal will identify autologous tumor antigens to which the patient's T cells are responsive.

[0469] Experiment 5 (Hypothetical): Using the phDC ELISpot assay to prescreen prostate cancer patients for phDC vaccination The

[0470] LNP transduction of human phDC containing PSMA (prostate-specific membrane antigen) protein mRNA for ELISpot

[0471] PBMCs containing neonatal pHDCs, generated via plate passage from high-risk prostate cancer patients and produced according to the method described in Experiment 1, were processed at 5 x 10⁻⁶ cells per well. 5Cells were seeded in triplicate for each experimental group and stimulated with an appropriate amount of LNP containing mRNA encoding PSMA protein in 200 μL of medium consisting of phenol red-free RPMI (Gibco) supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen). Negative control wells were treated with an equal amount of LNP containing mRNA of an unrelated protein (e.g., nanoluciferase).

[0472] ELISpot readings

[0473] After overnight incubation, wells were washed, and IFNγ spots were detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFU) for each experimental condition were quantified using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). As a standard practice, an ELISpot signal was considered positive if it was higher than 50 SFU / million cells, and / or if the experimental group signal was at least 2 times higher than the control (mimic) group signal.

[0474] result

[0475] Positive ELISpot IFNγ signaling will identify prostate cancer patients with pre-existing reactivity to PSMA. These patients can then be selected for clinical trials using phDC vaccination or other vaccination strategies that use PSMA as an antigen, with the aim of activating and amplifying their anti-PSMA T-cell responses, thereby delaying or preventing their cancer recurrence.

[0476] Experiment 6 (Hypothetical): Detection of SARS-CoV-2 booster vaccine in pre-screened high-risk individuals using phDC ELISpot. Vaccination

[0477] LNP transduction of human phDC containing spike protein mRNA for ELISpot

[0478] PBMCs containing neonatal phDCs, generated by plate passage from previously infected or vaccinated individuals at high risk of severe SARS-CoV-2 pathology, produced according to the method described in Experiment 1, were distributed at 5*10⁶ per well. 5Cells were plated in triplicate for each experimental group and stimulated with an appropriate amount of LNP containing mRNA encoding the spike protein in 200 μL of medium consisting of phenol red-free RPMI (Gibco) supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen). Negative control wells were treated with an equal amount of LNP containing mRNA of an unrelated protein (e.g., nanoluciferase).

[0479] ELISpot readings

[0480] After overnight incubation, wells were washed, and IFNγ spots were detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFU) for each experimental condition were quantified using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). As a standard practice, an ELISpot signal was considered positive if it was higher than 50 SFU / million cells, and / or if the experimental group signal was at least 2 times higher than the control (mimic) group signal.

[0481] result

[0482] Negative or low ELISpot IFNγ signaling will identify high-risk individuals with low responsiveness to the spike protein and therefore weakened protection against SARS-CoV-2 infection. These individuals can then be selected for clinical trials of phDC booster vaccination or other SARS-CoV-2 booster vaccination strategies, with the goal of activating and amplifying their antiviral T-cell responses, thereby enhancing their protective immunity.

[0483] Experiment 7 (Hypothetical): Monitoring Cancer Patients' Response to Immunotherapy Using the phDC ELISpot Assay

[0484] LNP transduction of human phDC containing cancer protein or peptide mRNA for ELISpot

[0485] PBMCs containing newly generated phDCs, obtained from cancer patients undergoing immunotherapy (anti-cancer vaccines, immune checkpoint inhibitors, etc.) via lamina propagation, were longitudinally analyzed using ELISpot throughout the entire treatment course from the start of treatment. Cells were generated according to the method described in Experiment 1, at a density of 5*102 5Cells were seeded in triplicate per well for each experimental group and stimulated with an appropriate amount of LNP containing mRNA encoding a patient-responsive cancer antigen (e.g., for cancer vaccination, the antigen used for vaccination; for immune checkpoint blockade therapy, the patient-responsive antigen, such as that identified as described in Experiment 4) in 200 μL of medium supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen) without phenol red (Gibco). Negative control wells were treated with an equal amount of LNP containing mRNA of an unrelated protein (e.g., nanoluciferase).

[0486] ELISpot readings

[0487] After overnight incubation, wells were washed, and IFNγ spots were detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFU) for each experimental condition were quantified using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). As a standard practice, an ELISpot signal was considered positive if it was higher than 50 SFU / million cells, and / or if the experimental group signal was at least 2 times higher than the control (mimic) group signal.

[0488] result

[0489] ELISpot IFNγ signaling can be used longitudinally to monitor a patient's immune response to their individual cancer immunotherapy. If the signal is considered low (i.e., does not increase after therapy, or initially increases and then decreases over time), this finding may help in making treatment decisions, such as increasing the therapy dose, repeating cancer antigen screening to verify whether the tumor immunogenicity profile has changed during treatment, or selecting a different treatment modality for the patient.

[0490] Experiment 8 (Hypothetical): Using phDC ELISpot assay as a method for isolating antigen-responsive T cells

[0491] LNP transduction of human phDC containing cancer protein or peptide mRNA for ELISpot

[0492] PBMCs containing newly generated phDCs from cancer patients prior to receiving adoptive T-cell therapy (e.g., neoantigen-targeted CAR-T) were passaged via ELISpot to identify TCRs that recognize specific antigens (i.e., MHC-peptide complexes). Cells were generated at a density of 5 x 10⁻⁶ cells / cells. 5 Cells were seeded in triplicate per well for each experimental group and stimulated with an appropriate amount of LNP containing mRNA encoding the target cancer antigen of interest in 200 μL of medium consisting of phenol red-free RPMI (Gibco) supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen). Negative control wells were treated with an equal amount of LNP containing mRNA of an unrelated protein (e.g., nanoluciferase).

[0493] Simultaneously, an identical reference plate was established in the tissue culture plate, which had corresponding wells that would match the ELISpot wells.

[0494] ELISpot readings

[0495] After overnight incubation, wells were washed, and IFNγ spots were detected using biotinylated anti-human IFNγ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates according to the manufacturer's protocol (MAbTech, 3420-2AST-2). Spot-forming units (SFU) for each experimental condition were quantified using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany). As a standard practice, an ELISpot signal was considered positive if it was higher than 50 SFU / million cells, and / or if the experimental group signal was at least 2 times higher than the control (mimic) group signal.

[0496] result

[0497] ELISpot IFNγ screening will identify wells with signals above a threshold, which will correspond to the same wells that exhibit robust T-cell responses to the corresponding antigens screened in those wells. These corresponding wells will be matched between the ELISpot and a reference plate for subsequent single-cell sequencing analysis. Considering the costs associated with single-cell sequencing, the phDC ELISpot will facilitate the identification of a smaller range of highly reactive antigen-specific T cells, which can subsequently be isolated from the reference plate and sent to downstream combined scRNA (single-cell) and scTCR analysis. TCR sequences associated with cells producing IFNγ transcripts will be identified as having target antigen specificity and can be used for downstream transgenic cell therapy preparations (e.g., novel CAR-T).

[0498] Experiment 9 (Hypothetical): In-depth characterization of antigen-reactive T cells in phDC ELISpot assay

[0499] LNP transduction of human phDC containing antigenic mRNA for in-depth characterization of T cell responses

[0500] PBMCs containing newly generated phDCs, generated by plate passage according to the method described in Experiment 1 (e.g., from cancer patients monitored before treatment (e.g., in Experiment 5) or throughout treatment (e.g., in Experiment 7)), were prepared at 5*10⁻⁶ cells per well. 5 Cells were plated in multiple identical plates, with three copies for each experimental group. Cells in each plate were identically stimulated in 200 μL of a medium containing an appropriate amount of LNPs encoding, for example, a cancer antigen of interest (e.g., a protein or peptide antigen isolated or identified from autologous tumor material from a patient; or a protein or peptide antigen being administered by the patient as an anticancer vaccine). The medium consisted of phenol red-free RPMI (Gibco) supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen). Negative control wells were treated with an equal amount of LNPs containing unrelated protein mRNA (e.g., nanoluciferase).

[0501] In-depth analysis of antigen-reactive T cells

[0502] After overnight incubation, all plates were washed and plate 1 was processed in the standard manner as described above (Experiments 2-7) for ELISpot analysis. Once reactive wells were identified using ELISpot assays on plate 1, cells from the same wells in other identically created replicate plates could be further analyzed to assess the identity and phenotype of antigen-reactive T cells responsible for producing IFNγ. Such analyses can be performed as follows:

[0503] - Multicolor flow cytometry, in which cells are gated by intracellular IFNγ staining and additional markers are analyzed (CD4, CD8 and gdTCR for T cell subset grouping; T cell differentiation markers such as CD25, CD27, CD44, CD45RA, CD62L, CXCR3, CXCR5, CCR6 and CCR7; other cytokines and degranulation markers such as TNFα, IL-17, granzyme and CD107a; T cell activation and exhaustion markers such as CD69, OX40, CD40L, CD200, PD-1, 4.1BB, LAG3 and TIM3).

[0504] - Flow cytometry / mass spectrometry analysis, such as CyTOF; similarly, allows for multiple biomarker analysis of IFNγ-producing cells identified by ELISpot assay.

[0505] - Single-cell RNASeq, in which T cells activated by a single antigen can be specifically identified by the expression of IFNγ, and the gene expression characteristics of such cells can be analyzed.

[0506] result

[0507] In-depth analysis of T cells that produce a detectable response in an ELISpot assay can provide additional information for clinical decision-making. For example, in cancer patients, it may be desirable to know whether the cells responding in an ELISpot assay have an “exhausted” phenotype or a phenotype associated with a good clinical outcome; in an infection / vaccination setting, it may be desirable to know whether the T cell response is biased towards cellular (CD8) immunity or antibody (CD4) immunity, and what other cytokines are involved (e.g., whether TNFα or IL-17 is also produced).

[0508] Experiment 10: Tumor neoantigen phDC ELISpot assay using LNPs containing tumor neoantigen mRNA

[0509] animal

[0510] C57Bl / 6J and B6.SJL-Ptprc a Pepc b / BoyJ (B6.SJL CD45.1) mice were purchased from Jackson Laboratory. Mice were housed and housed at the Yale Animal Resource Center in accordance with institutional guidelines. Age- and sex-matched mice at least 7 weeks old were used for all experiments. Experiments were conducted according to animal protocols approved by the Yale Institutional Animal Care and Use Committee and consistent with the National Institutes of Animal Healthcare Guidelines. Mice were kept in a specific pathogen-free environment with free access to food and water. The animal facility was accredited by the Association for Assessment of Laboratory Animal Care.

[0511] Cell culture

[0512] Unless otherwise stated, RPMI 1640, Dulbecco modified Eagle medium (DMEM), OptiMEM, DMEM / F12, and medium supplements were obtained from nvitrogen, Carlsbad, CA. Complete RPMI 1640 (cRPMI) medium was prepared by supplementing RPMI 1640 with 10% heat-inactivated fetal bovine serum (FBS) (Gemini Foundation), 10 mM Hepes, 2 mM 1-glutamine, 1 mM sodium pyruvate, and 1% penicillin / streptomycin. All cell lines were cultured at 37°C in 5% CO2. MC38 (Kerafast) was cultured in cRPMI supplemented with a final concentration of 1 mg / ml geneticin (Gibco). All cell lines were routinely tested for mycoplasma using standard polymerase chain reaction methods at the time of freezing of each cell batch. Cells were used for experiments within two passages of thawed and frozen aliquots.

[0513] Isolation of mouse PBMCs

[0514] Unless otherwise specified, peripheral blood was collected from mice via the superficial temporal vein or the posterior orbital plexus into containers containing 1:100 heparin (5000 U / ml; McKesson Packaging Services). Platelet-containing PBMCs were isolated from peripheral whole blood using Lympholyte M (Cedarlane Labs) gradient separation.

[0515] Transfer to Immunization Room

[0516] This miniaturized ECP device, called a transimmune (TI) chamber, was designed and manufactured by Transimmune AG in collaboration with the Fraunhofer Institute for Biomedical Engineering in Saarland, Germany, for Dr. Edelson's laboratory. The sterile polystyrene TI chamber has external dimensions of 25 x 75 mm, a flow path of 18 x 66 mm, and a flow channel height of 290 ± 15 µm.

[0517] IVT mRNA synthesis

[0518] For IVT, plasmids were linearized using Not-I HF (New England Biolabs). The linearized template was purified by sodium acetate precipitation (ThermoFisher Scientific) and rehydrated with nuclease-free water. In vitro transcription was performed using the HiScribe T7 Kit (NEB) according to the manufacturer's instructions, including N1-methyl-pseudouridine substitution. The resulting RNA was treated with DNase I (Aldevron) to remove the template and was purified. The RNA was capped with a Cap-1 structure using guanylate transferase and 2'-O-methyltransferase (Aldevron). Enzyme-promoted polyA tailing (Aldevron) was performed. mRNA concentration was measured using Nanodrop. The mRNA stock solution concentration was 1–3 mg / ml. The mRNA product was analyzed by capillary electrophoresis to ensure purity.

[0519] LNP [mRNA] formulation

[0520] mRNA was diluted in 10 mM citrate buffer (pH 3) to generate an aqueous phase. To prepare the organic phase, ionizable lipids, cholesterol, PEG lipids, and cofactor lipids were added to 100% ethanol. The molar ratios and suppliers of the lipid components used in each LNP formulation were as previously disclosed. cKK-E12 was purchased from Organicx, and SM102 from BroadPharm. Cholesterol was from Sigma, and C14PEG2K, DMG-PEG, DOPE, and DSPC were from Avanti. All in vivo formulations used a mass ratio of 20 (ionizable lipids:mRNA). Using an Ignite benchtop system (Precision NanoSystems Inc.) with a microfluidic cartridge, the two phases were mixed at a 3:1 aqueous-to-organic phase flow rate of 12 mL / min to generate LNPs encapsulating mRNA (LNP[mRNA]). The LNPs were then diluted and concentrated using a 100 kDa MWCO centrifuge filter (MilliporeSigma). After sterile filtration, particle size was determined by dynamic light scattering on a Zetasizer Nano ZS (Malvern). RiboGreen assay (Invitrogen) was performed to calculate the encapsulation percentage and concentration of mRNA. After characterizing the size and concentration, the LNP [mRNA] solution was mixed with sucrose to a final concentration of 5% (w / v), aliquoted, and stored at -80°C until use.

[0521] MC38 neoantigens were identified using cross-referenced whole-exome and bulk RNA sequencing data, along with reference reports that utilized neoantigen prediction workflows and empirical experiments to identify and validate specific MC38 neoantigens. Potential antigens that were co-present (with the same mutation in our MC38 lineage), expressed (TPM value >1), and demonstrated predicted and / or empirical immunogenicity through at least two peer-reviewed reports were selected. This explains a total of 12 neoantigens and one endogenous retroviral antigen (ERV) p15E. Each antigen was encoded into a CKK-E12 LNP, as described above. Additionally, these 13 antigens were divided into two categories: antigens that had been predicted and empirically validated (9 of which, including p15E) and antigens that had only been predicted by other laboratories (4 of which). Antigens from each of these two categories were combined into multi-mRNA neoantigen strings, designated 8x+p15E and 4x, respectively. The sequences of the neoantigens can be found below.

[0522] Table 4: Overview of tumor neoantigen sequences and SEQ ID NO. NA = nucleic acid, AA = amino acid.

[0523]

[0524] PhDC generation and transfection with LNP [mRNA]

[0525] The detailed procedure for generating phDCs has previously been described in visual form (Ventura et al., PMID: 31157760). In short, isolated PBMCs suspended in FBS are passed through a transimmunoassay (TI) plate chamber at a rate of 0.09 ml / min. After plate passage, cells are washed and cultured in tissue-free plates at 0.5–2 x 10⁻⁶ cells / min. 6 / cm 2 Cell densities within the specified range were cultured overnight in RPMI supplemented with 15% autologous plasma. Immediately after cell plating, antigens (i.e., soluble proteins / peptides, LNPs [mRNA]) were added to the plated cells.

[0526] ELISpot measurement

[0527] Analyze IFN-γ secretion following stimulation with LNP [antigen]-phDC in the IFN-γ ELISpot assay, following the manufacturer's recommendations (Mabtech; #3321-4APW-2). Incubate the negative control with an LNP [mimic] (e.g., encoded nanoluciferase). Each assay should be repeated at least three times.

[0528] For tumor antigen assays, PBMCs are derived from tumor-bearing mice expressing the target antigen or neoantigen. Typically, tumor-bearing mice are euthanized 11 days after tumor inoculation, but assays are performed between 2 and 50 days post-inoculation. Terminal bloodletting is routinely performed on 30 tumor-bearing mice, yielding approximately 30 ml of peripheral blood. (Approximately 5-10 x 10⁻⁶) 6 A separate PBMC will be passed down through the board to generate a phDC.

[0529] PBMCs containing newly generated phDCs (500,000 PBMCs / well) passaged in slabs were typically stimulated for 18 h at 37°C with LNP [mRNA] (125 ng mRNA / well) in the presence of OvernightMedia (RPMI (Gibco) supplemented with 15% autologous human plasma and 1% penicillin / streptomycin / L-glutamine (Invitrogen)). The final standard concentration of LNP [mRNA] used was 0.625 µg / mL. However, in some experiments, the concentration of cKK-E12-based LNP (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione)) could range from 62.5–250 ng / well. Cells were subsequently removed, and the filters were washed five times with PBS solution containing 0.5% FBS. According to the manufacturer's protocol, spot detection was performed using biotinylated anti-human IFN-γ mAb, streptavidin-ALP, and BCIP / NBT-plus substrates. For analysis, dried filter plates were imaged using an ELISpot reader and ELISpot 6.0 iSpot software (Autoimmun Diagnostika GmbH, Strasburg, Germany) or ImmunoSpot (CTL). Results were compared with simulated control samples from the same donor at the same time point.

[0530] To test the contribution of T cell subsets to cytokine production and improve assay sensitivity, CD4+ or CD8+ T cells were selectively depleted from PBMCs after plate passage but before overnight incubation with LNPs in Elispot wells. T cells were depleted using a standard positive selection depletion kit (Miltenyi; CellSep).

[0531] In the ELISPOT assay, a positive response is considered to be achieved if at least two of the following three classic criteria are met:

[0532] 1) Background (simulation) greater than 2.5x;

[0533] 2) Greater than 50 SFU / 1x10 6 ;

[0534] 3) The background subtraction result is greater than 50 SFU / 1E6.

[0535] statistics

[0536] Data are presented as mean ± standard error of the mean (sem) or mean ± standard deviation (sd). All statistics were performed using Prism (GraphPad software). Analysis of variance (ANOVA) and Tukey's multiple comparison test were used to compare multiple groups, and two-tailed Student's t-tests were used to compare two groups within the parametric data. A p-value < 0.05 was considered significant. Statistical significance was expressed as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0537] Biorender is used to generate all schematics.

[0538] result

[0539] Figure 5 This paper compares antigen-specific T-cell responses in PBMC populations with (B) and without (A) CD4+ T-cell depletion. Positive responses to more antigens were recorded after CD4+ T-cell depletion. Positive responses were defined according to the three criteria mentioned above. Therefore, CD4+ T-cell depletion increases the sensitivity of the assay.

[0540] Figure 6 The results of ELIspot assays using phDCs loaded with LNPs carrying multiple mRNA constructs (“strings”) or multiple individual mRNAs (“pools”) encoding different tumor neoantigen epitopes are presented. The results indicate that while antigen strings can elicit responses, these responses are not additive, likely due to a dilution effect when multiple antigens are expressed together in a single string. These data highlight the importance of antigen dosing and configuration when designing multivalent immunotherapies.

[0541] Figure 7 The results of the ELIspot assay for detecting the response to the tumor-associated LCMV-derived antigen GP33 are shown, comparing the full-length LCMV GP protein (gp) and the GP33 peptide (gp33). The response was significant. The assay was more sensitive to the shorter GP33 peptide containing the GP33 epitope than to the full-length GP protein.

[0542] Another preferred embodiment of the present invention relates to:

[0543] 1. A method for testing an immune response in a subject to at least one antigen in vitro, comprising:

[0544] - Provides a mixture containing antigen-specific phDCs and cells capable of secreting interferon-γ (IFNγ);

[0545] - Determine the activation of the cells capable of secreting IFNγ.

[0546] 2. The method according to embodiment 1, wherein activation of the cells capable of secreting IFNγ indicates an immune response of the subject to the at least one antigen.

[0547] 3. The method according to embodiment 1 or 2, wherein the antigen-specific phDC is obtained by at least the following steps:

[0548] - To subject monocytes to physical forces;

[0549] - Add at least one antigen.

[0550] 4. The method according to embodiment 1 or 2, wherein the antigen-specific phDC is obtained by at least the following steps:

[0551] - Combine monocytes with at least one antigen;

[0552] - subject the mixture of the monocyte and the at least one antigen to physical forces.

[0553] 5. The method according to embodiment 1 or 2, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ is obtained by at least the following steps:

[0554] - Expose the sample containing monocytes and cells capable of secreting IFNγ to physical forces;

[0555] - Add at least one antigen.

[0556] 6. The method according to embodiment 1 or 2, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ is obtained by at least the following steps:

[0557] - Combine a sample containing monocytes and cells capable of secreting IFNγ with at least one antigen;

[0558] - subject the mixture of the sample containing monocytes and cells capable of secreting IFNγ with the at least one antigen to physical force.

[0559] 7. The method according to any one of embodiments 3 to 6, wherein the mononuclear cells are autologous to the subject.

[0560] 8. The method according to any one of embodiments 1 to 7, wherein the cells capable of secreting IFNγ are autologous to the subject.

[0561] 9. The method according to any one of embodiments 3 to 6 or 8, wherein the mononuclear cells are allogeneic to the subject.

[0562] 10. The method according to embodiment 7, wherein the cells capable of secreting IFNγ are allogeneic to the subject.

[0563] 11. The method according to embodiment 10, wherein the cells capable of secreting IFNγ are obtained from a cell line or donor, optionally obtaining a blood sample, graft, or tissue sample obtained from the donor.

[0564] 12. The method according to embodiment 9, wherein the mononuclear cells are obtained from a donor, optionally obtaining a blood sample, graft, or tissue sample obtained from the donor.

[0565] 13. The method according to embodiment 11 or 12, wherein the graft is a cell graft, optionally a hematopoietic cell graft or a bone marrow graft.

[0566] 14. The method according to any one of embodiments 11 to 13, wherein the subject has received or is designated to receive a graft from the donor in the future.

[0567] 15. The method according to any one of embodiments 1 to 14, wherein the at least one antigen comprises nucleic acid, peptide, protein, cell extract or apoptotic cell.

[0568] 16. The method according to any one of embodiments 1 to 15, wherein the at least one antigen comprises at least one DNA, optionally at least one genomic DNA or cDNA.

[0569] 17. The method according to any one of embodiments 1 to 15, wherein the at least one antigen comprises at least one RNA, optionally at least one mRNA.

[0570] 18. The method according to embodiment 17, wherein the at least one mRNA is contained in a preventive vaccine or a therapeutic vaccine.

[0571] 19. The method according to embodiment 17 or 18, wherein the at least one mRNA is a modified mRNA.

[0572] 20. The method according to embodiment 19, wherein the modified mRNA comprises at least one modified nucleoside.

[0573] 21. The method according to embodiment 20, wherein the at least one modified nucleoside is a modified uridine, and optionally all uridine residues in the at least one mRNA are replaced with modified uridine.

[0574] 22. The method according to embodiment 21, wherein the modified uridine is N1-methylpseudouridine.

[0575] 23. The method according to any one of embodiments 17 to 22, wherein the at least one mRNA is partially modified with N1-methylpseuuridine.

[0576] 24. The method according to any one of embodiments 17 to 22, wherein the at least one mRNA is completely modified with N1-methylpseuuridine.

[0577] 25. The method according to any one of embodiments 17 to 24, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen, bacterial antigen, fungal antigen, prion antigen, or parasitic antigen.

[0578] 26. The method according to any one of embodiments 17 to 25, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen.

[0579] 27. The method according to any one of embodiments 17 to 26, wherein the at least one mRNA comprises a coding sequence encoding a coronavirus antigen.

[0580] 28. The method according to any one of embodiments 17 to 27, wherein the at least one mRNA comprises a coding sequence encoding a SARS-CoV-2 antigen, optionally, the at least one mRNA comprises a nucleotide sequence corresponding to SEQ ID NO: 19 (as used in the examples) or a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19.

[0581] 29. The method according to any one of embodiments 17 to 24, wherein the at least one mRNA comprises a coding sequence encoding a tumor-associated antigen.

[0582] 30. The method according to embodiment 29, wherein the tumor-associated antigen is associated with solid tumors or with hematologic malignancies.

[0583] 31. The method according to any one of embodiments 1 to 30, wherein the at least one antigen is contained in nanoparticles.

[0584] 32. The method according to any one of embodiments 1 to 31, wherein the at least one antigen is contained in lipid nanoparticles.

[0585] 33. The method according to embodiment 32, wherein the lipid nanoparticles comprise cKK-E12 lipid, SM-102 lipid, or MC3 lipid.

[0586] 34. The method according to any one of embodiments 1 to 33, wherein the subject has a disease associated with the at least one antigen, has a disease associated with the at least one antigen, is at risk of recurrence of a disease associated with the at least one antigen, or is at risk of developing a disease associated with the at least one antigen in the future.

[0587] 35. The method according to any one of embodiments 17 to 24 or 29 to 34, wherein the subject has a tumor and the at least one mRNA is derived from the tumor.

[0588] 36. The method according to any one of embodiments 16 or 31 to 34, wherein the subject has a tumor and the at least one DNA is derived from the tumor.

[0589] 37. The method according to any one of embodiments 3 to 36, wherein the physical force is a shear force.

[0590] 38. The method according to embodiment 37, wherein the shear force is generated by a flow chamber, a bag, or a combination of a flow chamber and a bag.

[0591] 39. The method according to embodiment 38, wherein platelets and / or plasma components are present in the flow chamber, bag, or combination of the flow chamber and bag.

[0592] 40. The method according to any one of embodiments 1 to 39, wherein the activation of the cells capable of secreting IFNγ is determined by ELIspot, FACS, lymphocyte proliferation assay, calcium response measurement and / or ELISA.

[0593] 41. The method according to any one of embodiments 1 to 40, wherein the activation of the cells capable of secreting IFNγ is measured by determining one or more of IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17 and TNF-α.

[0594] 42. The method according to any one of embodiments 1 to 41, wherein the activation of the cells capable of secreting IFNγ is measured by measuring IFNγ.

[0595] 43. The method according to any one of embodiments 1 to 42, wherein subjects are selected for clinical trials, therapies, or vaccinations based on the measured activation of the cells capable of secreting IFNγ.

[0596] 44. The method according to implementation plan 43, wherein the therapy includes cancer treatment or desensitization therapy.

[0597] 45. The method according to any one of embodiments 1 to 44, wherein the cells capable of secreting IFNγ include CD8+ T cells, natural killer cells, γδ T cells, natural killer T cells, CD4+ T cells, group 1 innate lymphocytes, IFN-producing killer dendritic cells, memory CD8+ T cells and memory CD4+ T cells.

[0598] 46. ​​The method according to any one of embodiments 1 to 45, wherein the cells capable of secreting IFNγ include T cells.

[0599] 47. The method according to any one of embodiments 1 to 46, wherein the cell capable of secreting IFNγ is a CD8+ T cell or a CD4+ T cell, optionally wherein the T cell is rich in CD8+ T cells or CD4+ T cells.

[0600] 48. The method according to any one of embodiments 1 to 47, wherein the cells capable of secreting IFNγ do not include CD4+ T cells.

[0601] 49. The method according to any one of embodiments 1 to 47, wherein the cells capable of secreting IFNγ do not include CD8+ T cells.

[0602] 50. The method according to any one of embodiments 1 to 48, wherein the method is performed at multiple time points to analyze the immune response of the subject over time.

[0603] 51. The method according to any one of embodiments 1 to 8, 10, 11 or 14 to 50, wherein the antigen-specific phDC is autologous to the subject.

[0604] 52. The method according to any one of embodiments 1 to 6, 8, 9, 12 or 14 to 50, wherein the antigen-specific phDC is allogeneic to the subject.

[0605] 53. A screening method for identifying immunogenic antigens in vitro, comprising:

[0606] - Provides a mixture containing antigen-specific phDCs and cells capable of secreting IFNγ;

[0607] - Determine the activation of the cells capable of secreting IFNγ.

[0608] 54. The screening method according to embodiment 53, wherein activation of the cells capable of secreting IFNγ indicates an immunogenic antigen.

[0609] 55. The screening method according to embodiment 53 or 54, wherein the activation of the cells capable of secreting IFNγ is associated with the immunogenicity of the antigen.

[0610] 56. The method according to any one of embodiments 53 to 55, wherein the antigen-specific phDC is obtained by at least the following steps:

[0611] - To subject monocytes to physical forces;

[0612] - Add at least one antigen.

[0613] 57. The method according to any one of embodiments 53 to 55, wherein the antigen-specific phDC is obtained by at least the following steps:

[0614] - Combine monocytes with at least one antigen;

[0615] - subject the mixture of the monocyte and the at least one antigen to physical forces.

[0616] 58. The method according to any one of embodiments 53 to 55, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ is obtained by at least the following steps:

[0617] - Expose the sample containing monocytes and cells capable of secreting IFNγ to physical forces;

[0618] - Add at least one antigen.

[0619] 59. The method according to any one of embodiments 53 to 55, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ is obtained by at least the following steps:

[0620] - Combine a sample containing monocytes and cells capable of secreting IFNγ with at least one antigen;

[0621] - subject the mixture of the sample containing monocytes and cells capable of secreting IFNγ with the at least one antigen to physical force.

[0622] 60. The method according to any one of embodiments 56 to 59, wherein the mononuclear cells are obtained from the subject.

[0623] 61. The method according to any one of embodiments 53 to 60, wherein the cells capable of secreting IFNγ are obtained from the subject.

[0624] 62. The method according to embodiment 60, wherein the cells capable of secreting IFNγ are allogeneic to the subject.

[0625] 63. The method according to embodiment 62, wherein the cells capable of secreting IFNγ are obtained from a cell line or donor, optionally obtaining a blood sample, graft, or tissue sample obtained from the donor.

[0626] 64. The method according to embodiment 63, wherein the graft is a cell graft, optionally a hematopoietic cell graft or a bone marrow graft.

[0627] 65. The method according to any one of embodiments 60 to 64, wherein the subject has received or is designated to receive a graft from the donor in the future.

[0628] 66. The method according to any one of embodiments 56 to 65, wherein the at least one antigen comprises nucleic acid, peptide, protein, cell extract or apoptotic cell.

[0629] 67. The method according to any one of embodiments 56 to 66, wherein the at least one antigen comprises at least one DNA, optionally at least one genomic DNA or cDNA.

[0630] 68. The method according to any one of embodiments 56 to 66, wherein the at least one antigen comprises at least one RNA, optionally at least one mRNA.

[0631] 69. The method according to embodiment 68, wherein the at least one mRNA is contained in a preventive vaccine or a therapeutic vaccine.

[0632] 70. The method according to embodiment 68 or 69, wherein the at least one mRNA is a modified mRNA.

[0633] 71. The method according to embodiment 70, wherein the modified mRNA comprises at least one modified nucleoside.

[0634] 72. The method according to embodiment 71, wherein the at least one modified nucleoside is a modified uridine, and optionally all uridine residues in the at least one mRNA are replaced with modified uridine.

[0635] 73. The method according to embodiment 72, wherein the modified uridine is N1-methylpseudouridine.

[0636] 74. The method according to any one of embodiments 68 to 73, wherein the at least one mRNA is partially modified with N1-methylpseuuridine.

[0637] 75. The method according to any one of embodiments 68 to 73, wherein the at least one mRNA is completely modified with N1-methylpseuuridine.

[0638] 76. The method according to any one of embodiments 68 to 75, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen, bacterial antigen, fungal antigen, prion antigen, or parasitic antigen.

[0639] 77. The method according to any one of embodiments 68 to 76, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen.

[0640] 78. The method according to any one of embodiments 68 to 77, wherein the at least one mRNA comprises a coding sequence encoding a coronavirus antigen.

[0641] 79. The method according to any one of embodiments 68 to 78, wherein the at least one mRNA comprises a coding sequence encoding a SARS-CoV-2 antigen, and optionally, the at least one mRNA comprises a nucleotide sequence corresponding to SEQ ID NO: 19 or a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19.

[0642] 80. The method according to any one of embodiments 68 to 75, wherein the at least one mRNA comprises a coding sequence encoding a tumor-associated antigen.

[0643] 81. The method according to embodiment 80, wherein the tumor-associated antigen is associated with solid tumors or with hematologic malignancies.

[0644] 82. The method according to any one of embodiments 56 to 81, wherein the at least one antigen is contained in nanoparticles.

[0645] 83. The method according to any one of embodiments 56 to 82, wherein the at least one antigen is contained in lipid nanoparticles.

[0646] 84. The method according to embodiment 83, wherein the lipid nanoparticles comprise cKK-E12 lipid, SM-102 lipid or MC3 lipid.

[0647] 85. The method according to any one of embodiments 60 to 84, wherein the subject has a disease associated with the at least one antigen, already has a disease associated with the at least one antigen, is at risk of recurrence of a disease associated with the at least one antigen, or is at risk of developing a disease associated with the at least one antigen in the future.

[0648] 86. The method according to any one of embodiments 60 to 75 or 80 to 84, wherein the subject has a tumor and the at least one mRNA is derived from the tumor.

[0649] 87. The method according to any one of embodiments 67 or 82 to 85, wherein the subject has a tumor and the at least one DNA is derived from the tumor.

[0650] 88. The method according to any one of embodiments 56 to 87, wherein the physical force is a shear force.

[0651] 89. The method according to embodiment 88, wherein the shear force is generated by a flow chamber, a bag, or a combination of a flow chamber and a bag.

[0652] 90. The method according to embodiment 89, wherein platelets and / or plasma components are present in the flow chamber, the bag, or a combination of the flow chamber and the bag.

[0653] 91. The method according to any one of embodiments 53 to 90, wherein the activation of the cells capable of secreting IFNγ is determined by ELIspot, FACS, lymphocyte proliferation assay, calcium response measurement and / or ELISA.

[0654] 92. The method according to any one of embodiments 53 to 91, wherein the activation of the cells capable of secreting IFNγ is measured by determining one or more of IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17 and TNF-α.

[0655] 93. The method according to any one of embodiments 53 to 92, wherein the activation of the cells capable of secreting IFNγ is measured by measuring IFNγ.

[0656] 94. The method according to any one of embodiments 53 to 93, wherein the cells capable of secreting IFNγ include CD8+ T cells, natural killer cells, γδ T cells, natural killer T cells, CD4+ T cells, group 1 innate lymphocytes, IFN-producing killer dendritic cells, memory CD8+ T cells, and memory CD4+ T cells.

[0657] 95. The method according to any one of embodiments 53 to 94, wherein the cells capable of secreting IFNγ include T cells.

[0658] 96. The method according to any one of embodiments 53 to 95, wherein the cell capable of secreting IFNγ is a CD8+ T cell or a CD4+ T cell, optionally wherein the T cell is rich in CD8+ T cells or CD4+ T cells.

[0659] 97. The method according to any one of embodiments 53 to 95, wherein the cells capable of secreting IFNγ do not include CD4+ T cells.

[0660] 98. The method according to any one of embodiments 53 to 95, wherein the cells capable of secreting IFNγ do not include CD8+ T cells.

[0661] 99. The method according to any one of embodiments 53 to 98, wherein the antigen-specific phDC is autologous to the subject.

[0662] 100. An antigen that can be identified by a screening method according to any one of embodiments 53 to 99.

[0663] 101. A pharmaceutical composition comprising the antigen as described in embodiment 100.

[0664] 102. The pharmaceutical composition according to embodiment 101, which optionally comprises phDC that is autologous to the subject.

[0665] 103. Methods for determining whether a subject is suitable for a particular protocol, including:

[0666] - Provides a mixture containing antigen-specific phDCs and cells capable of secreting IFNγ;

[0667] - Determine the activation of the cells capable of secreting IFNγ;

[0668] The activation of the cells that secrete IFNγ indicates whether the subject is suitable or unsuitable for the specific protocol.

[0669] 104. The method according to embodiment 103, wherein the antigen-specific phDC is obtained by at least the following steps:

[0670] - To subject monocytes to physical forces;

[0671] - Add at least one antigen.

[0672] 105. The method according to embodiment 103, wherein the antigen-specific phDC is obtained by at least the following steps:

[0673] - Combine monocytes with at least one antigen;

[0674] - subject the mixture of the monocyte and the at least one antigen to physical forces.

[0675] 106. The method according to embodiment 103, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ is obtained by at least the following steps:

[0676] - Expose the sample containing monocytes and cells capable of secreting IFNγ to physical forces;

[0677] - Add at least one antigen.

[0678] 107. The method according to embodiment 103, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ is obtained by at least the following steps:

[0679] - Combine a sample containing monocytes and cells capable of secreting IFNγ with at least one antigen;

[0680] - subject the mixture of the sample containing monocytes and cells capable of secreting IFNγ with the at least one antigen to physical force.

[0681] 108. The method according to any one of embodiments 104 to 107, wherein the mononuclear cells are autologous to the subject.

[0682] 109. The method according to any one of embodiments 103 to 107, wherein the cells capable of secreting IFNγ are autologous to the subject.

[0683] 110. The method according to any one of embodiments 104 to 107 or 109, wherein the mononuclear cells are allogeneic to the subject.

[0684] 111. The method according to embodiment 108, wherein the cells capable of secreting IFNγ are allogeneic to the subject.

[0685] 112. The method according to embodiment 111, wherein the cells capable of secreting IFNγ are obtained from a cell line or donor, optionally obtaining a blood sample, graft, or tissue sample obtained from the donor.

[0686] 113. The method according to embodiment 110, wherein the mononuclear cells are obtained from a donor, optionally obtaining a blood sample, graft, or tissue sample obtained from the donor.

[0687] 114. The method according to embodiment 112 or 113, wherein the graft is a cell graft, optionally a hematopoietic cell graft or a bone marrow graft.

[0688] 115. The method according to any one of embodiments 112 to 114, wherein the subject has received or is designated to receive a graft from the donor in the future.

[0689] 116. The method according to any one of embodiments 104 to 115, wherein the at least one antigen comprises nucleic acid, peptide, protein, cell extract or apoptotic cell.

[0690] 117. The method according to any one of embodiments 104 to 116, wherein the at least one antigen comprises at least one DNA, optionally at least one genomic DNA or cDNA.

[0691] 118. The method according to any one of embodiments 104 to 116, wherein the at least one antigen comprises at least one RNA, optionally at least one mRNA.

[0692] 119. The method according to embodiment 118, wherein the at least one mRNA is contained in a preventive vaccine or a therapeutic vaccine.

[0693] 120. The method according to embodiment 118 or 119, wherein the at least one mRNA is a modified mRNA.

[0694] 121. The method according to embodiment 120, wherein the modified mRNA comprises at least one modified nucleoside.

[0695] 122. The method according to embodiment 121, wherein the at least one modified nucleoside is a modified uridine, and optionally all uridine residues in the at least one mRNA are replaced with modified uridine.

[0696] 123. The method according to embodiment 122, wherein the modified uridine is N1-methylpseudouridine.

[0697] 124. The method according to any one of embodiments 118 to 123, wherein the at least one mRNA is partially modified with N1-methylpseuuridine.

[0698] 125. The method according to any one of embodiments 118 to 123, wherein the at least one mRNA is completely modified with N1-methylpseuuridine.

[0699] 126. The method according to any one of embodiments 118 to 125, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen, bacterial antigen, fungal antigen, prion antigen, or parasitic antigen.

[0700] 127. The method according to any one of embodiments 118 to 126, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen.

[0701] 128. The method according to any one of embodiments 118 to 127, wherein the at least one mRNA comprises a coding sequence encoding a coronavirus antigen.

[0702] 129. The method according to any one of embodiments 118 to 128, wherein the at least one mRNA comprises a coding sequence encoding a SARS-CoV-2 antigen, optionally, the at least one mRNA comprises a nucleotide sequence corresponding to SEQ ID NO: 19 (as used in the examples) or a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19.

[0703] 130. The method according to any one of embodiments 118 to 125, wherein the at least one mRNA comprises a coding sequence encoding a tumor-associated antigen.

[0704] 131. The method according to embodiment 130, wherein the tumor-associated antigen is associated with solid tumors or with hematologic malignancies.

[0705] 132. The method according to any one of embodiments 104 to 131, wherein the at least one antigen is contained in nanoparticles.

[0706] 133. The method according to any one of embodiments 104 to 132, wherein the at least one antigen is contained in lipid nanoparticles.

[0707] 134. The method according to embodiment 133, wherein the lipid nanoparticles comprise cKK-E12 lipid, SM-102 lipid, or MC3 lipid.

[0708] 135. The method according to any one of embodiments 104 to 134, wherein the subject has a disease associated with the at least one antigen, already has a disease associated with the at least one antigen, is at risk of recurrence of a disease associated with the at least one antigen, or is at risk of developing a disease associated with the at least one antigen in the future.

[0709] 136. The method according to any one of embodiments 118 to 115 or 130 to 135, wherein the subject has a tumor and the at least one mRNA is derived from the tumor.

[0710] 137. The method according to any one of embodiments 119 or 132 to 135, wherein the subject has a tumor and the at least one DNA is derived from the tumor.

[0711] 138. The method according to any one of embodiments 104 to 137, wherein the physical force is a shear force.

[0712] 139. The method according to embodiment 138, wherein the shear force is generated by a flow chamber, a bag, or a combination of a flow chamber and a bag.

[0713] 140. The method according to embodiment 139, wherein platelets and / or plasma components are present in the flow chamber, the bag, or a combination of the flow chamber and the bag.

[0714] 141. The method according to any one of embodiments 103 to 140, wherein the activation of the cells capable of secreting IFNγ is determined by ELIspot, FACS, lymphocyte proliferation assay, calcium response measurement and / or ELISA.

[0715] 142. The method according to any one of embodiments 103 to 141, wherein the activation of the cells capable of secreting IFNγ is measured by determining one or more of IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17 and TNF-α.

[0716] 143. The method according to any one of embodiments 103 to 142, wherein the activation of the cells capable of secreting IFNγ is measured by measuring IFNγ.

[0717] 144. The method according to any one of embodiments 103 to 143, wherein the cells capable of secreting IFNγ include CD8+ T cells, natural killer cells, γδ T cells, natural killer T cells, CD4+ T cells, group 1 innate lymphocytes, IFN-producing killer dendritic cells, memory CD8+ T cells and memory CD4+ T cells.

[0718] 145. The method according to any one of embodiments 103 to 144, wherein the cells capable of secreting IFNγ include T cells.

[0719] 146. The method according to any one of embodiments 103 to 145, wherein the cell capable of secreting IFNγ is a CD8+ T cell or a CD4+ T cell, optionally wherein the T cell is rich in CD8+ T cells or CD4+ T cells.

[0720] 147. The method according to any one of embodiments 103 to 146, wherein the cells capable of secreting IFNγ do not include CD4+ T cells.

[0721] 148. The method according to any one of embodiments 103 to 146, wherein the cells capable of secreting IFNγ do not include CD8+ T cells.

[0722] 149. The method according to any one of embodiments 103 to 109, 111, 112 or 114 to 148, wherein the antigen-specific phDC is autologous to the subject.

[0723] 150. The method according to any one of embodiments 103 to 107, 110, 113 or 114 to 148, wherein the antigen-specific phDC is allogeneic to the subject.

[0724] 151. The method according to any one of embodiments 1 to 150, wherein the specific protocol is a clinical trial, a therapy, or a vaccination.

[0725] 152. The method according to implementation plan 151, wherein the therapy includes cancer treatment or desensitization therapy.

[0726] 153. A method for identifying and / or isolating T cells capable of recognizing at least one antigen from a subject, comprising:

[0727] - Provides a mixture containing antigen-specific phDCs and T cells;

[0728] - Determine the activation of the T cells;

[0729] - Identify and / or isolate one or more T cells from the T cell population that recognize antigens presented by antigen-specific phDCs.

[0730] 154. The method according to embodiment 153, wherein activation of the T cells indicates one or more T cells that recognize one or more antigens presented by the antigen-specific phDCs.

[0731] 155. The method according to embodiment 153 or 154, wherein the antigen-specific phDC is obtained by at least the following steps:

[0732] - To subject monocytes to physical forces;

[0733] - Add at least one antigen.

[0734] 156. The method according to embodiment 153 or 154, wherein the antigen-specific phDC is obtained by at least the following steps:

[0735] - Combine monocytes with at least one antigen;

[0736] - subject the mixture of the monocyte and the at least one antigen to physical forces.

[0737] 157. The method according to embodiment 153 or 154, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ is obtained by at least the following steps:

[0738] - Expose the sample containing monocytes and cells capable of secreting IFNγ to physical forces;

[0739] - Add at least one antigen.

[0740] 158. The method according to embodiment 153 or 154, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ is obtained by at least the following steps:

[0741] - Combine a sample containing monocytes and cells capable of secreting IFNγ with at least one antigen;

[0742] - subject the mixture of the sample containing monocytes and cells capable of secreting IFNγ with the at least one antigen to physical force.

[0743] 159. The method according to any one of embodiments 155 to 158, wherein the mononuclear cells are autologous to the subject.

[0744] 160. The method according to any one of embodiments 1 to 7, wherein the T cells are autologous to the subject.

[0745] 161. The method according to any one of embodiments 153 to 160, wherein the at least one antigen comprises nucleic acid, peptide, protein, cell extract or apoptotic cell.

[0746] 162. The method according to any one of embodiments 153 to 161, wherein the at least one antigen comprises at least one DNA, optionally at least one genomic DNA or cDNA.

[0747] 163. The method according to any one of embodiments 153 to 160, wherein the at least one antigen comprises at least one RNA, optionally at least one mRNA.

[0748] 164. The method according to embodiment 163, wherein the at least one mRNA is contained in a preventive vaccine or a therapeutic vaccine.

[0749] 165. The method according to embodiment 163 or 164, wherein the at least one mRNA is a modified mRNA.

[0750] 166. The method according to embodiment 164, wherein the modified mRNA comprises at least one modified nucleoside.

[0751] 167. The method according to embodiment 165, wherein the at least one modified nucleoside is a modified uridine, and optionally all uridine residues in the at least one mRNA are replaced with modified uridine.

[0752] 168. The method according to embodiment 167, wherein the modified uridine is N1-methylpseudouridine.

[0753] 169. The method according to any one of embodiments 163 to 168, wherein the at least one mRNA is partially modified with N1-methylpseuuridine.

[0754] 170. The method according to any one of embodiments 163 to 168, wherein the at least one mRNA is completely modified with N1-methylpseuuridine.

[0755] 171. The method according to any one of embodiments 163 to 170, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen, bacterial antigen, fungal antigen, prion antigen, or parasitic antigen.

[0756] 172. The method according to any one of embodiments 163 to 171, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen.

[0757] 173. The method according to any one of embodiments 163 to 172, wherein the at least one mRNA comprises a coding sequence encoding a coronavirus antigen.

[0758] 174. The method according to any one of embodiments 163 to 173, wherein the at least one mRNA comprises a coding sequence encoding a SARS-CoV-2 antigen, optionally, the at least one mRNA comprises a nucleotide sequence corresponding to SEQ ID NO: 19 (as used in the examples) or a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19.

[0759] 175. The method according to any one of embodiments 163 to 170, wherein the at least one mRNA comprises a coding sequence encoding a tumor-associated antigen.

[0760] 176. The method according to embodiment 175, wherein the tumor-associated antigen is associated with solid tumors or with hematologic malignancies.

[0761] 177. The method according to any one of embodiments 153 to 176, wherein the at least one antigen is contained in nanoparticles.

[0762] 178. The method according to any one of embodiments 163 to 177, wherein the at least one antigen is contained in lipid nanoparticles.

[0763] 179. The method according to embodiment 178, wherein the lipid nanoparticles comprise cKK-E12 lipid, SM-102 lipid, or MC3 lipid.

[0764] 180. The method according to any one of embodiments 153 to 179, wherein the subject has a disease associated with the at least one antigen, already has a disease associated with the at least one antigen, is at risk of recurrence of a disease associated with the at least one antigen, or is at risk of developing a disease associated with the at least one antigen in the future.

[0765] 181. The method according to any one of embodiments 155 to 180, wherein the physical force is a shear force.

[0766] 182. The method according to embodiment 181, wherein the shear force is generated by a flow chamber, a bag, or a combination of a flow chamber and a bag.

[0767] 183. The method according to embodiment 182, wherein platelets and / or plasma components are present in the flow chamber, the bag, or a combination of the flow chamber and the bag.

[0768] 184. The method according to any one of embodiments 153 to 183, wherein the activation of the T cells is determined by ELIspot, FACS, lymphocyte proliferation assay, calcium response measurement and / or ELISA.

[0769] 185. The method according to any one of embodiments 153 to 183, wherein the activation of the T cells is measured by determining one or more of IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17 and TNF-α.

[0770] 186. The method according to any one of embodiments 153 to 183, wherein the activation of the T cells is measured by determining IFNγ.

[0771] 187. The method according to any one of embodiments 153 to 186, wherein the T cells include CD8+ T cells, γδ T cells, CD4+ T cells, memory CD8+ T cells, and memory CD4+ T cells.

[0772] 188. The method according to any one of embodiments 153 to 187, wherein the T cell is a CD8+ T cell or a CD4+ T cell, optionally wherein the T cell is rich in CD8+ T cells or CD4+ T cells.

[0773] 189. The method according to any one of embodiments 153 to 188, wherein the T cells do not comprise CD4+ T cells.

[0774] 190. The method according to any one of embodiments 153 to 188, wherein the T cells do not contain CD8+ T cells.

[0775] 191. The method according to any one of embodiments 180 to 190, wherein the disease is a tumor.

[0776] 192. The method according to any one of embodiments 180 to 191, wherein the disease is leukemia.

[0777] 193. The method according to embodiment 191 or 192, wherein the at least one mRNA is derived from the tumor or leukemia.

[0778] 194. The method according to embodiment 191 or 192, wherein the at least one DNA is derived from the tumor.

[0779] 195. The method according to any one of embodiments 153 to 194, wherein CD8+ or CD4+ T cells are identified and / or isolated.

Claims

1. A method for testing an immune response in a subject to at least one antigen in vitro, comprising: - Provides a mixture containing antigen-specific phDCs and cells capable of secreting interferon-γ (IFNγ); - Determine the activation of the cells capable of secreting IFNγ; The activation of the cells capable of secreting IFNγ indicates an immune response of the subject to at least one antigen.

2. A screening method for identifying immunogenic antigens in vitro, comprising: - Provides a mixture containing antigen-specific phDCs and cells capable of secreting IFNγ; - Determine the activation of the cells capable of secreting IFNγ; The method described herein includes screening for at least one antigen; and The activation of cells capable of secreting IFNγ indicates an immunogenic antigen.

3. The method according to claim 1 or 2, wherein the antigen-specific phDC can be obtained through at least the following steps: - To subject monocytes to physical forces; - Add at least one antigen; Alternatively, you can obtain it through the following steps: - Combine monocytes with at least one antigen; - subject the mixture of the monocyte and the at least one antigen to physical forces.

4. The method according to any one of claims 1 to 3, wherein the mixture comprising antigen-specific phDCs and cells capable of secreting IFNγ can be obtained by at least the following steps: - Expose the sample containing monocytes and cells capable of secreting IFNγ to physical forces; - Add at least one antigen; Alternatively, you can obtain it through the following steps: - Combine a sample containing monocytes and cells capable of secreting IFNγ with at least one antigen; - subject the mixture of the sample containing monocytes and cells capable of secreting IFNγ with the at least one antigen to physical force.

5. The method according to any one of claims 1 to 4, wherein the monocytes, antigen-specific phDCs and / or the cells capable of secreting IFNγ are autologous to the subject.

6. The method according to any one of claims 1 to 5, wherein the at least one antigen comprises at least one mRNA.

7. The method according to claim 6, wherein the at least one mRNA is completely modified with N1-methylpseuuridine.

8. The method according to claim 6 or 7, wherein the at least one mRNA comprises a coding sequence encoding a viral antigen, bacterial antigen, fungal antigen, prion antigen, parasitic antigen, or tumor-associated antigen.

9. The method according to any one of claims 6, 7 or 8, wherein the at least one mRNA comprises a coding sequence encoding a SARS-CoV-2 antigen, and optionally, the at least one mRNA comprises a nucleotide sequence corresponding to SEQ ID NO: 19 or a nucleotide sequence that is at least 75%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO:

19.

10. The method according to any one of claims 1 to 9, wherein the at least one antigen is contained in nanoparticles, optionally in lipid nanoparticles.

11. The method according to any one of claims 1 to 8 or 10, wherein the subject has a tumor, and the at least one antigen comprises at least one mRNA derived from the tumor.

12. The method according to any one of claims 1 to 11, wherein the activation of the cells capable of secreting IFNγ is determined by ELIspot.

13. The method according to any one of claims 1 to 12, wherein the activation of the cells capable of secreting IFNγ is measured by measuring IFNγ.

14. The method according to any one of claims 1 to 13, wherein the cells capable of secreting IFNγ include CD8+ T cells, natural killer cells, γδ T cells, natural killer T cells, CD4+ T cells, group 1 innate lymphocytes, IFN-producing killer dendritic cells, memory CD8+ T cells, and memory CD4+ T cells.

15. The method according to any one of claims 1 to 14, wherein the cells capable of secreting IFNγ include CD8+ T cells, CD4+ T cells, memory CD8+ T cells and / or memory CD4+ T cells.

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