SARS-COV-2 immunodominant peptide constructions and uses thereof

Abstract

The present application provides methods and compositions for treating and / or preventing COVID-19 by inducing an immune response against the identified SARS-COV-2 immunodominant peptides.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims the benefit of U.S. Provisional Application No. 63 / 113,024, filed on November 12, 2020; the entire contents of the said application are incorporated in whole by reference into this application. [Background technology]

[0002] Coronavirus disease 2019 (or COVID-19) is a global pandemic caused by infection with the Severe Acute Respiratory Syndrome (SARS)-CoV-2 (SARS-CoV-2) virus, which has claimed more than 500,000 lives worldwide and affected millions of people. SARS-CoV-2 is the seventh coronavirus known to infect humans; SARS-CoV, MERS-CoV, and SARS-CoV-2 can cause severe illness, while HKU1, NL63, OC43, and 229E are associated with mild symptoms. To develop effective vaccines and therapies, it is necessary to understand how the adaptive immune response recognizes and eliminates the virus, and how the interaction between the virus and the immune system influences the pathogenesis of the disease. To date, most efforts have focused on B cell-mediated antibody responses to the virus. However, how cytotoxic CD8+ T cells recognize and eliminate infected cells is not well understood.

[0003] CD8 T cells play a crucial role in defense against various pathogens, and much evidence demonstrates that this is also true for SARS-CoV-2. Closely related SARS-CoV studies have revealed that in animal models, CD8 T cells provide protection during the acute phase of infection, and that memory CD8 T cells persist longer than humoral responses in humans after the acute phase of infection. During SARS-CoV-2 infection, the magnitude of the CD8 T cell response correlates with a milder disease course, suggesting a protective role. In particular, SARS-CoV-2-responsive T cells have been observed in the absence of antiviral antibodies in asymptomatic patients who have cleared the virus after exposure to SARS-CoV-2, further demonstrating the protective role of T cells.

[0004] Current efforts in SARS-CoV-2 vaccine development primarily focus on generating neutralizing antibodies against the S protein. The majority of vaccines in clinical development (and all US Ph II / III candidates) use only the S protein as the antigen. As a result, these vaccine candidates lack most of the antigens recognized by CD8 T cells during natural infection. Indeed, careful tracking of T cell responses in patients vaccinated with SARS-CoV-2 S protein vaccines has revealed that CD8 T cell responses are variable and weak. Next-generation vaccines incorporating a broader range of antigens are needed to better engage the CD8 T cell response, in the hope of providing more robust and sustained protection against SARS-CoV-2. To date, SARS-CoV-2 vaccines in development have resulted in weak and variable CD8 responses after vaccination (Mateus et al. (2021) Science 374:eabj9853). [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] This invention is at least in part based on the discovery of SARS-CoV-2 immunodominant peptides. Importantly, some of these immunogenic peptides can induce a T cell response across a wide range of patients, for example, when they are linked, or when they are positioned within a polypeptide, or when they are combined with other peptides on a construct (e.g., a nucleic acid vector construct, e.g., a nucleic acid vector construct that maximizes the size of the vector for the efficacy of the T cell response by packing an optimal immunodominant epitope inside for antigen expression in cells). [Means for solving the problem]

[0006] In some embodiments, the immunogenic polypeptide comprises at least two peptide epitopes selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F. In certain embodiments, the at least two peptide epitopes are present in linked order within the immunogenic polypeptide, and optionally, therefore, at least one immunodominant epitope is present as two or more copies.

[0007] We further provide a number of embodiments that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in some embodiments, at least one immunodominant epitope exists as two or more copies (e.g., two copies, three copies, four copies, five copies, six copies, or more, or any range in between, e.g., two copies of one immunodominant epitope, three copies of another immunodominant epitope, a single copy of a third immunodominant epitope, etc.). In some embodiments, the immunogenic polypeptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more of the peptide epitopes, wherein the immunogenic polypeptide comprises at least one, two, and / or three immunogenic peptide epitopes for each of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and HLA-B*07. The immunogenic polypeptide includes a dominant epitope (for example, one immunodominant epitope for each of the listed HLAs, two immunodominant epitopes for each of the listed HLAs, three immunodominant epitopes for each of the listed HLAs, or any comprehensive range in between, for example, one immunodominant epitope for each of HLA-A*02 and HLA-A*03, plus two immunodominant epitopes for each of HLA-A*01 and HLA-A*11, and three immunodominant epitopes for each of HLA-A*24 and HLA-A*07, etc.). In some embodiments, the immunogenic polypeptide includes three peptide epitopes derived from each of Tables 1A, 1B, 1C, 1D, 1E, and 1F. In some embodiments, the immunogenic polypeptide further includes linkers between the peptide epitopes. In a particular embodiment, the linker comprises at least three amino acids for each of the peptide epitopes, wherein the at least three amino acids are sequential with each of the peptide epitopes. In a particular embodiment, the linker is a proteasome cleavage motif.In some embodiments, the immunogenic polypeptide further optionally comprises one or more full-length SARS-CoV-2 proteins or one or more protein fragments thereof, selected from the group consisting of Orf1ab, M, N, Orf3a, and S, wherein the fragments of the one or more full-length SARS-CoV-2 proteins include SARS-CoV-2 proteins that do not encode a functional SARS-CoV-2 protein. In some embodiments, the immunogenic polypeptide further optionally comprises a ribosome stop / restart segment, an IRES segment, and / or a post-translational cleavage segment, wherein the post-translational cleavage segment is a P2A segment. In some embodiments, the immunogenic polypeptide comprises any one of the amino acid sequences provided in the tables described herein (e.g., Table 1G or Table 1I).

[0008] In a particular embodiment, the immunogenic polypeptide comprises at least two peptide fragments, each of which comprises at least two peptide epitopes, where the at least two peptide epitopes in each peptide fragment are derived from the same protein of SARS-CoV-2.

[0009] As described above, a number of embodiments are provided that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in some embodiments, the at least two peptide fragments are derived from the N protein, M protein, ORF1a / b protein, or ORF3a protein of SARS-CoV-2. In some embodiments, the immunogenic polypeptide comprises up to six of the peptide fragments. In some embodiments, the immunogenic polypeptide further comprises one or more full-length SARS-CoV-2 proteins or one or more protein fragments thereof, selected from the group consisting of Orf1ab, M, N, Orf3a, and S, wherein the one or more full-length SARS-CoV-2 protein fragments include SARS-CoV-2 proteins that do not encode a functional SARS-CoV-2 protein. In some embodiments, the immunogenic polypeptide further optionally comprises a ribosome arrest / restart segment, an IRES segment, and / or a post-translational cleavage segment, wherein the post-translational cleavage segment is a P2A segment. In some embodiments, the immunogenic polypeptide comprises any one of the amino acid sequences provided in the tables described herein (e.g., Table 1H or Table 1J). In some embodiments, the immunogenic polypeptide can induce a T cell response in vitro and / or in vivo, optionally, wherein the T cell response is measured by a tetramer staining assay, a T cell activation assay, a CD137 staining assay, an intracellular IFN-gamma (IFNg) staining assay, a cytokine release assay, and / or a T cell proliferation assay.

[0010] Numerous embodiments are provided that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the immunogenic peptide is optionally derived from the SARS-CoV-2 protein, where the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In another embodiment, the SARS-CoV-2 protein is selected from the group consisting of orf1a / b, S protein, N protein, M protein, orf3a, and orf7a. In yet another embodiment, the immunogenic peptide can induce a T cell response in a subject. In some embodiments, the immunogenic peptide comprises a peptide epitope selected from Tables 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0011] In yet another embodiment, an immunogenic composition [the immunogenic composition comprises at least one immunogenic peptide as described in this application (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, The immunogenic composition is provided which optionally comprises 1) one or more full-length SARS-CoV-2 proteins or one or more protein fragments thereof, selected from the group consisting of Orf1ab, M, N, Orf3a, and S, wherein the one or more full-length SARS-CoV-2 protein fragments include SARS-CoV-2 proteins that do not encode functional SARS-CoV-2 proteins, and / or 2) an adjuvant.

[0012] Numerous embodiments are provided that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the immunogenic composition can induce a T cell response in vitro and / or in vivo. In another embodiment, the T cell response is measured by a tetramer staining assay, a T cell activation assay, a CD137 staining assay, an intracellular IFNg staining assay, a cytokine release assay, and / or a T cell proliferation assay.

[0013] In yet another embodiment, a composition is provided comprising an immunogenic peptide containing at least two peptide epitopes selected from Tables 1A, 1B, 1C, 1D, 1E, and / or 1F, as well as an MHC molecule.

[0014] We further provide a number of embodiments that can be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the MHC molecule is an MHC polymer, optionally, where the MHC polymer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule.In yet another embodiment, the MHC molecule comprises an MHC alpha chain which is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and HLA-B*07, where the HLA allele is HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 allele, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, ​​HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 allele, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 allele, HLA-A*2402, The alleles are selected from the group consisting of HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 alleles, and HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles.The sequences, characteristics, structural information, functional information, and binding partners of these and other HLA alleles are well known in the art (see, for example, the World Wide Web at hla.alleles.org / nomenclature / index.html, hla.alleles.org / data / hla-a.html, and hla.alleles.org / data / hla-b.html).

[0015] In another embodiment, a stable MHC-peptide complex is provided, in which the MHC molecule contains a peptide epitope selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0016] We further provide a number of embodiments that can be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the MHC molecule is an MHC polymer, optionally, where the MHC polymer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule.In yet another embodiment, the MHC molecule comprises an MHC alpha chain which is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and HLA-B*07, where the HLA allele is HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 allele, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, ​​HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 allele, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 allele, HLA-A*2402, The alleles are selected from the group consisting of HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 alleles, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles. In yet another embodiment, the peptide epitope and the MHC molecule are covalently bonded, and / or the alpha and beta chains of the MHC molecule are covalently bonded.In another embodiment, the stable MHC-peptide complex optionally includes a detectable label, where the detectable label is a fluorescent dye molecule.

[0017] In yet another embodiment, an immunogenic composition is provided comprising a stable MHC-peptide complex and an adjuvant as described in this application.

[0018] In yet another embodiment, an isolated nucleic acid encoding the immunogenic polypeptide described in this application, or a complement thereof, optionally wherein the isolated nucleic acid is DNA, RNA, mRNA, cDNA, self-replicating, cyclized, concatemerized, wherein the isolated nucleic acid includes an internal ribosome entry site (IRES) including an expression promoter, including a 5' untranslated region (5'UTR) and / or 3'UTR derived from a gene of interest (e.g., hemoglobin A), and / or an autocleaved 2A peptide (e.g., P2A or T2A).

[0019] In another embodiment, a vector comprising the isolated nucleic acid described in this application is provided. In some embodiments, the vector is an expression vector.

[0020] A basic nucleic acid encoding the immunogenic polypeptide described in this application, or a vector containing the same, may be used in any aspect or embodiment of the present invention where the immunogenic polypeptide is desired (for example, the nucleic acid encodes and produces the immunogenic polypeptide). The nucleic acid is an immunogenic composition for the immunogenic polypeptide encoded and produced, and not for the nucleic acid itself.

[0021] In yet another embodiment, there is provided a cell, a) comprising the isolated nucleic acid described in this application, b) comprising the vector described in this application, and / or c) producing one or more immunogenic polypeptides described in this application, and / or presenting one or more stable MHC-peptide complexes described in this application on its cell surface, optionally wherein the cell is genetically engineered.

[0022] In yet another embodiment, a binding substructure is provided that specifically binds to an immunogenic polypeptide and / or a stable MHC-peptide complex described in this application, optionally wherein the binding substructure is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain (optionally further comprising an intracellular transmembrane domain and an effector domain).

[0023] In yet another embodiment, a device or kit is provided comprising a) one or more immunogenic polypeptides described in this application and / or b) one or more stable MHC-peptide complexes described in this application, wherein the device or kit optionally comprises a reagent for detecting that a) and / or b) bind to a T cell receptor.

[0024] In another embodiment, a method is provided for detecting T cells bound to a stable MHC-peptide complex, comprising: a) contacting a sample containing T cells with the stable MHC-peptide complex described in this application; and b) detecting the binding of T cells to the stable MHC-peptide complex, and optionally further measuring the proportion of stable MHC-peptide-specific T cells bound to the stable MHC-peptide complex.

[0025] Numerous embodiments are provided that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the sample comprises peripheral blood mononuclear cells (PBMCs). In another embodiment, the T cells are CD8+ T cells. In yet another embodiment, the detection and / or measurement steps are performed using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blotting, or intracellular flow assay. In yet another embodiment, the sample comprises T cells that have come into contact with or are suspected of coming into contact with one or more SARS-CoV-2 proteins or fragments thereof.

[0026] In yet another embodiment, a method is provided for determining whether a subject has exposure to and / or protection from SARS-CoV-2: a) incubating a cell population, including T cells, obtained from the subject with an immunogenic polypeptide as described in this application, or a stable MHC-peptide complex as described in this application; and b) detecting the presence or level of responsiveness, where the presence or level of responsiveness compared to a control level indicates that the subject has exposure to and / or protection from SARS-CoV-2.

[0027] In yet another embodiment, a method is provided for predicting the clinical outcome of a subject infected with SARS-CoV-2: a) measuring the presence or level of responsiveness between T cells obtained from the subject and one or more immunogenic polypeptides or one or more stable MHC-peptide complexes described in this application; and b) comparing the presence or level of responsiveness with the presence or level of responsiveness of a control, wherein the control is obtained from a subject with good clinical outcomes, where the presence or higher level of responsiveness in the subject compared to the control indicates that the subject has good clinical outcomes.

[0028] In another embodiment, a method is provided for evaluating the effectiveness of a SARS-CoV-2 therapy, comprising: a) measuring the presence or level of responsiveness between T cells obtained from a subject and one or more immunogenic peptides or stable MHC-peptide complexes described herein, in a first sample obtained from the subject before providing at least a portion of the SARS-CoV-2 therapy to the subject; and b) measuring the presence or level of responsiveness between one or more immunogenic polypeptides or stable MHC-peptide complexes described herein and T cells obtained from the subject, in a second sample obtained from the subject after providing at least a portion of the SARS-CoV-2 therapy, wherein the presence or higher level of responsiveness in the second sample compared to the first sample indicates that the therapy is effective in treating SARS-CoV-2 in the subject.

[0029] Numerous embodiments are provided that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the level of responsiveness is optionally indicated by a) the presence of binding and / or b) T cell activation and / or effector function, where the T cell activation or effector function is T cell proliferation, injury, or cytokine release. In another embodiment, the method optionally further includes a step of repeating steps a) and b) at a later time point, where the subject is receiving treatment to improve a SARS-CoV-2 infection between the first time point and the later time point. In yet another embodiment, the T cell binding, activation, and / or effector function are detected using fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blotting, or intracellular flow assay. In yet another embodiment, the control level is a reference figure. In another embodiment, the control level is the level of a subject not exposed to SARS-CoV-2.

[0030] In yet another embodiment, a method is provided for preventing and / or treating SARS-CoV-2 infection in a subject, wherein the method comprises the step of administering to the subject a therapeutically effective amount of an immunogenic composition comprising and / or encoding one or more immunogenic polypeptides or cells as described in this application.

[0031] A number of embodiments are provided that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the immunogenic composition comprises a nucleic acid encoding an immunogenic polypeptide described herein (e.g., an immunogenic polypeptide comprising at least two peptide epitopes selected from Tables 1A, 1B, 1C, 1D, 1E, and / or 1F). In one embodiment, the immunogenic peptide is optionally derived from the SARS-CoV-2 protein, where the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In one embodiment, the SARS-CoV-2 protein is selected from the group consisting of orf1a / b, S protein, N protein, M protein, orf3a, and orf7a. In one embodiment, the immunogenic polypeptide can induce a T cell response in a subject. In one embodiment, the immunogenic composition comprises two or more immunogenic polypeptides. In yet another embodiment, the immunogenic composition further comprises an adjuvant. In yet another embodiment, the immunogenic composition can induce a T cell response in a subject. In yet another embodiment, the administered immunogenic composition induces an immune response to SARS-CoV-2 in the subject. In yet another embodiment, the administered immunogenic composition induces a T cell immune response to SARS-CoV-2 in the subject. In yet another embodiment, the T cell immune response is a CD8+ T cell immune response.

[0032] In yet another embodiment, a method is provided for identifying peptide-binding molecules or antigen-binding fragments thereof that bind to the peptide epitope of the at least one immunogenic polypeptide described in this application, including: a) providing a cell presenting the peptide epitope of the at least one immunogenic polypeptide described in this application among MHC molecules on the surface of the cell, optionally wherein the cell comprises a nucleic acid encoding and expressing the at least one immunogenic polypeptide; b) measuring the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to the peptide epitope in the MHC molecules on the cell; and c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to the peptide epitope in the MHC molecules.

[0033] Numerous embodiments are provided that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, step a) comprises contacting MHC molecules on the surface of a cell with a peptide epitope selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F. In another embodiment, step a) comprises transfecting the cell with a nucleic acid encoding an immunogenic polypeptide described herein (for example, as a basic nucleic acid encoding an immunogenic polypeptide described herein, such as including a heterosequence encoding a peptide epitope selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F, or as a vector containing such basic nucleic acid).

[0034] In another embodiment, a method is provided for identifying peptide-binding molecules or antigen-binding fragments thereof that bind to the peptide epitope of the at least one immunogenic polypeptide described in this application, including: a) providing a stable MHC-peptide complex in which the MHC molecule contains the peptide epitope of the at least one immunogenic polypeptide described in this application; b) determining the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to the stable MHC-peptide complex; and c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to the stable MHC-peptide complex.

[0035] We further provide a number of embodiments that can be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the MHC molecule is an MHC polymer, optionally, where the MHC polymer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule.In yet another embodiment, the MHC molecule comprises an MHC alpha chain which is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and HLA-B*07, where the HLA allele is HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 allele, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, ​​HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 allele, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 allele, HLA-A*2402, The alleles are selected from the group consisting of HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 alleles, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles. In yet another embodiment, the peptide epitope and the MHC molecule are covalently bonded, and / or the alpha and beta chains of the MHC molecule are covalently bonded.In another embodiment, the stable MHC-peptide complex optionally includes a detectable label, where the detectable label is a fluorescent dye molecule. In yet another embodiment, the plurality of candidate peptide-binding molecules include one or more T cell receptors (TCRs), or one or more antigen-binding fragments of TCRs. In yet another embodiment, the plurality of candidate peptide-binding molecules include at least 2, 5, 10, 100, and 10. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or more, a variety of candidate peptide-binding molecules. In another embodiment, the plurality of candidate peptide-binding molecules include one or more candidate peptide-binding molecules obtained from a sample from the subject or from a population of subjects; or the plurality of candidate peptide-binding molecules include one or more candidate peptide-binding molecules that contain mutations in a parent scaffold peptide-binding molecule obtained from a sample from the subject. In yet another embodiment, the subject or population of subjects is a) not infected with SARS-CoV-2 and / or has recovered from COVID-19, or b) infected with SARS-CoV-2 and / or has COVID-19. In yet another embodiment, the subject or population of subjects is vaccinated with one or more immunogenic polypeptides, wherein the immunogenic polypeptides include peptide epitopes selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F. In another embodiment, the subject is a mammal, optionally, wherein the mammal is a human, a primate, or a rodent. In yet another embodiment, the subject is an HLA-transgenic mouse and / or a human TCR transgenic mouse. In yet another embodiment, the sample comprises T cells. In yet another embodiment, the sample comprises peripheral blood mononuclear cells (PBMCs) or CD8+ memory T cells. In yet another embodiment, the antigen-binding fragment of the TCR is a single-chain TCR (scTCR).

[0036] In another embodiment, a peptide-binding molecule or its antigen-binding fragment identified according to the method of this application is provided, optionally wherein the binding substructure is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-stranded TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain.

[0037] In yet another embodiment, a method is provided for treating SARS-CoV-2 infection in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of genetically engineered T cells expressing a TCR identified by the method of this application.

[0038] In yet another embodiment, a method is provided for treating SARS-CoV-2 infection in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of genetically engineered T cells expressing a TCR that binds to a peptide epitope of at least one immunogenic polypeptide described in this application.

[0039] In another embodiment, a method is provided for treating SARS-CoV-2 infection in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of genetically engineered T cells expressing a TCR that binds to a stable MHC-peptide complex containing a peptide epitope of at least one immunogenic polypeptide described in this application within an MHC molecule.

[0040] We further provide a number of embodiments that can be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the MHC molecule is an MHC polymer, optionally, where the MHC polymer is a tetramer. In another embodiment, the MHC molecule is an MHC class I molecule.In yet another embodiment, the MHC molecule comprises an MHC alpha chain which is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and HLA-B*07, where the HLA allele is HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 allele, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, ​​HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 allele, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 allele, HLA-A*2402, The alleles are selected from the group consisting of HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 alleles, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles. In yet another embodiment, the peptide epitope and the MHC molecule are covalently bonded, and / or the alpha and beta chains of the MHC molecule are covalently bonded.In another embodiment, the stable MHC-peptide complex optionally includes a detectable label, where the detectable label is a fluorescent dye molecule. In yet another embodiment, the T cells are isolated from a) the subject, b) a donor not infected with SARS-CoV-2, or c) a donor recovered from COVID-19.

[0041] In yet another embodiment, a method is provided for preventing or treating SARS-CoV-2 infection in a subject, wherein the method comprises the step of injecting antigen-specific T cells into the subject, wherein the antigen-specific T cells are prepared by: a) stimulating PBMCs or T cells derived from the subject in cells encoding and / or presenting an immunogenic polypeptide as described in this application, a nucleic acid encoding an immunogenic polypeptide as described in this application, a stable MHC-peptide complex comprising a peptide epitope of at least one immunogenic polypeptide as described in this application, or a peptide of at least one immunogenic polypeptide as described in this application in an MHC molecule on the cell surface (for example, derived from a construct expressing an immunogenic polypeptide as described in this application); and b) growing antigen-specific T cells in vitro, optionally isolating the PBMCs or T cells from the subject before stimulating the PBMCs or T cells.

[0042] Numerous embodiments are provided that may be applied to any aspect of the present invention and / or combined with any other embodiments described herein. For example, in one embodiment, the T cells are naive T cells, central memory T cells, or effector memory T cells. In another embodiment, the T cells are CD8+ memory T cells. In yet another embodiment, the agent is placed in contact with the peptide epitope, immunogenic polypeptide, stable MHC-peptide complex, T cell receptor, and / or T cells under conditions and for a time suitable for the formation of at least one immune complex between them. In yet another embodiment, the peptide epitope, immunogenic polypeptide, stable MHC-peptide complex, and / or T cell receptor are expressed by cells, and the cells are grown and / or isolated in one or more steps. In another embodiment, the subject is a mammal, optionally, where the mammal is a human, primate, or rodent.

[0043] This patent application document includes at least one drawing drawn in color. The Office relating to this application will provide a copy of this patent or the publication of the patent application containing the color drawing upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0044] [Figure 1] Figure 1 shows a typical polyepitope vaccine construct encoding an immunodominant epitope (e.g., an embodiment of an immunogenic polypeptide). [Figure 2] Figure 2 shows a typical next-generation SARS-CoV-2 vaccine construct (e.g., an embodiment of an immunogenic polypeptide). [Figure 3] Figure 3 shows the response of memory CD8 T cells from COVID-19 patients to various vaccine constructs. This data represents the average of two patients (shown as individual dots). [Figure 4]Figure 4 shows that the selected vaccine construct exhibits superior CD8 T cell activation compared to the S protein alone. This data was obtained by testing the responsiveness of memory CD8 T cells derived from nine patients to the SARS-CoV-2 vaccine construct. [Figure 5] Figures 5A-5C show typical 19-epitope structures. Figure 5A shows a typical 19-epitope structure with a 3aa linker. Figure 5B shows a typical 19-epitope structure with a KAA linker. Figure 5C shows a typical 19-epitope structure without a linker. [Figure 6] Figures 6A-6C show typical 27-epitope constructs. Figure 6A shows a typical 27-epitope construct with a 3aa linker. Figure 6B shows a typical 27-epitope construct with a KAA linker. Figure 6C shows a typical 27-epitope construct without a linker. [Figure 7] Figure 7 shows a typical fragment construct (for example, an embodiment of at least two peptide fragments, each containing at least two peptide epitopes). [Figure 8] Figure 8 shows representative fragment constructs fused to the S protein segment and P2A. [Figure 9]Figures 9A–9E show fusion structures of representative polyepitope constructs. Figure 9A shows a representative 19-epitope construct with a 3aa linker fused to the S protein segment and P2A. Figure 9B shows a representative 27-epitope construct with a 3aa linker fused to the S protein segment and P2A. Figure 9C shows representative 19-epitope constructs contained within 14 fragments derived from the N, Orf1a, M, Orf3a, and S proteins of SARS-CoV-2. Each fragment is encoded by a nucleic acid sequence ranging from 27 to 195 nucleotides and contains one or more discovered epitopes covering a total of 19 known epitopes. The constructs of the 14 fragments are designed to potentially include any immunogenic epitopes that can be presented from the same proteasome fragment as the discovered epitopes, and potentially include undiscovered epitopes that can be presented on MHC molecules other than those known to bind to and present the discovered epitopes. Figure 9D shows a representative polypeptide construct contained within the fragments that span the entire (all) SARS-CoV-2 M, N, and Orf3a proteins, but are represented by discontinuous fragments, as well as the S protein fragment, combined with 17 epitopes from the SARS-CoV-2 Orf1ab and S proteins. This construct is a combination of a polyepitope construct and a fragment-based construct. For example, the 5' portion of the construct is a concatemer in which 17 epitopes from the SARS-CoV-2 spike and orf1ab proteins are linked together by three surrounding native amino acids. The 3' portion of the construct is a concatemer of a fragment that combines the N, Orf3a, and M proteins, and also contains one fragment of the S protein. To avoid the production of functional proteins (for example, the Orf3a protein is known to have immunosuppressive activity), the N, Orf3a, and M proteins were divided into 2-3 fragments and dispersed in the sequence by alternating the order of each fragment.All 27 discovered epitopes are represented in this construct. The entire N, Orf3a, and M regions of the SARS-CoV-2 genome are included because the N, Orf3a, and M proteins, when scaled to the size of these regions, are the most epitope-rich. While not theoretically bound, this region is considered a biological driver that triggers the processing and presentation of immunogenic epitopes, and therefore likely contains epitopes that can be presented by many different MHCs, potentially increasing the number of patients who can respond to this construct. The designation B.1.617.2_S.PP in the figure refers to the PP-stabilized form of the S protein derived from the delta variant (B.1.617.2). The S protein fragment included in this representative construct originates from the SARS-CoV-2 delta variant. Figure 9E shows a representative polypeptide construct contained within a fragment that spans the entire (all) SARS-CoV-2 M, N, and Orf3a proteins, but is represented by a discontinuous fragment, combined with fragments derived from the SARS-CoV-2 Orf1ab and S proteins. B.1.617.2_S.PP_EpiFrag-M / N / ORF3a contains the same 3' N, Orf3a, M, and S fragments as the Epi-M / N / ORF3a construct described in Figure 9D, but differs in that the 5' 17epi fragment portion is replaced with fragments from the Orf1ab and S proteins surrounding the 17 epitope from Figure 9D. These are the same fragments as those from the 14_fragment epitope. The name B.1.617.2_S.PP in the figure refers to the PP-stabilized form of the S protein derived from the delta variant (B.1.617.2). The S protein fragment included in this representative construct is derived from the SARS-CoV-2 delta variant. The representative sequences shown in Figures 9C-9E do not include additional sequences such as linkers. For example, the 3AA linker is unnecessary because the larger fragment provides space for the linker to the identified epitope.Since unidentified epitopes are not defined, the absence of a linker ensures that the epitope is not directly at the end of the segment. The segment is then directly positioned without a P2A site. One exception is the polyepitope portion in Figure 9D, which includes a 3AA linker. [Figure 10] Figure 10 shows that a memory T cell (Tmem) pool derived from SARS-CoV-2 patients recognizes the vaccine construct shown in Figure 9C. Fourteen fragment mRNA constructs (shown in Figure 9C) were introduced into monoallelic HEK293 T cells, modified to express a single MHC-I molecule, using lipid nanoparticles. Memory T cells isolated from recovered SARS-CoV-2 patients were co-cultured with monoallelic HEK293 T cells treated with the constructs, and the responsiveness to the constructs was tested by measuring interferon-gamma release using ELISA. [Figure 11] Figure 11 shows that TCRs that recognize specific epitopes of SARS-CoV-2 respond to dendritic cells treated with LNPs containing the 27 polyepitope constructs shown in Figure 6A. Monocyte-derived dendritic cells (moDCs) were isolated from blood collected in the United States in 2019, prior to the SARS-CoV-2 outbreak. The moDCs were treated with LNPs containing the mRNA of the constructs shown in Figure 6A. T cells specific to each of the epitopes shown in Table 1A or 1F were co-cultured with LNP-treated moDCs, and T cell responsiveness was measured by flow cytometry staining for activation-induced markers (AIMs) CD69 and CD137. Thus, the epitopes contained within these constructs are processed and adequately presented, inducing epitope-specific responses from T cells. [Figure 12]Figure 12 shows that the TCR that recognizes the SARS-CoV-2 epitope utilizes a common TRAV gene. The frequencies of specific clones that recognize the YLQ, KLW, and SPR epitopes are shown from Tables 1A and F. The shared dominant TRAV gene is highlighted. See also Ferretti et al. (2020) Immunity 53: 1095-1107, particularly Figure 4 and the materials and methods section, for further support demonstrating that the common TRAV gene is utilized by the TCR. [Figure 13] Figure 13 shows a representative, non-exclusive example of an in vitro vaccine model. [Figure 14] Figure 14 shows that pulsing with the SARS-CoV-2 immunodominant peptide induces peptide-specific T cell proliferation from the naive T cell population in the in vitro vaccine model shown in Figure 13. As shown, peptide-specific TCRs were detected using MHC-peptide tetramer staining. [Figure 15] Figure 15 shows that the vaccine construct induces peptide-specific T cell proliferation from a population of naive T cells in the in vitro vaccine model shown in Figure 13. [Figure 16]Figures 16A–16C show that known V regions (Figure 12) corresponding to epitopes known to be presented by the tested MHCs are dominant among the top-growing TCR clones from the in vitro vaccine model treated with the construct in Figure 9C, but not among the TCR clones from the untreated control. Figure 16A shows the TRAV genes of the grown clones, highlighting known TRAV genes corresponding to TCRs known to bind to the YLQ peptide at background-subtracted frequencies exceeding the maximum values ​​in the control samples. Figures 16B and 16C show the TRAV genes of the grown clones responding to the vaccine construct in Figure 9C after restimulation with known peptides listed in Tables 1A and F. For the tested MHCs, TRAV genes corresponding to TCRs known to bind to the peptides in the construct in Figure 9C are highlighted at background-subtracted frequencies exceeding the maximum values ​​in the control samples. Therefore, these constructs induce a response from naive T cells in an in vitro vaccine model that replicates the immune response to natural infection by SARS-CoV-2. [Modes for carrying out the invention]

[0045] [Detailed description of the invention] This invention is based, at least in part, on the discovery of SARS-CoV-2 virus-specific immunogenic polypeptide constructs (which can be used, for example, as vaccines). A systematic and comprehensive investigation was conducted to accurately map T cell targets recognized by convalescent COVID-19 patients. Surprisingly, this study revealed a limited set of highly immunodominant peptide antigens (including some that appear to be universally recognized) that are repeatedly recognized across a wide range of patients.

[0046] To aid in the design of such vaccines containing immunogenic peptides, a comprehensive genome-wide analysis was performed on targets recognized by CD8 T cells after SARS-CoV-2 infection. The landscape of targets recognized on six of the most common HLA alleles (HLA-A:02, HLA-A01, HLA-A03, HLA-A11, HLA-A24, and HLA-B07) was mapped – >85% of individuals were mapped to a set that broadly expresses at least one of these alleles. Core sets of immunodominant epitopes for each allele, repeatedly recognized in the majority of patients with appropriate HLA alleles, were identified and validated. These epitopes were found to be robustly presented on MHC molecules, efficiently recognized by the naive CD8 T cell repertoire across a wide range of patients, and likely to be protective (based on the significant contribution of these epitopes to the overall CD8 T cell response [which is likely protective]). In addition to precisely identifying epitopes presented on specific HLA alleles, patterns of T cell responses to the SARS-CoV-2 genome were also revealed. Regions of the genome that were repeatedly recognized across the entire HLA allele—including the N, M, and ORF3a proteins, as well as parts of ORF1ab—were found, and these regions represent attractive vaccine antigens likely to generate CD8 T cell responses constrained to further alleles. CD8 T cell responses to specific segments of the SARS-CoV-2 genome that are cross-reactive with endemic beta-coronaviruses were also identified. These regions are of particular interest because vaccines targeting them may be able to boost any existing CD8 T cells that are responsive to endemic beta-coronaviruses, potentially leading to a more robust response.

[0047] This data has enabled the design of vaccine candidates containing a broad set of CD8 T cell antigens to more accurately replicate innate immunity against SARS-CoV-2. Specifically, the designed vaccines include polyepitope vaccines and fragment-based vaccines.

[0048] In polyepitope vaccines, multiple immunodominant CD8 T cell epitopes were ligated to express them as a single polyprotein. This approach has the advantage of providing the maximum density of validated T cell epitopes. Variants were designed across a range of 6–29 epitopes. A key design feature of polyepitope vaccines is the ability to maximize the efficient processing and presentation of the desired immunodominant epitope while minimizing any non-natural junctional epitopes. Polyepitopes containing optimal proteasome cleavage sequences were also designed [derived from natural protein sequences (amino acids directly surrounding each epitope in the full-length protein, which have been validated based on our screening data to result in efficient presentation) or synthetic proteasome cleavage sequences were used as linkers between epitopes]. We also used bioinformatics techniques to select the optimal epitope sequence (selecting the best sequence from millions of test variants) to minimize the generation of predicted high-affinity junction epitopes.

[0049] For fragment-based vaccines, we concatenated a set of longer genomic segments derived from SARS-CoV-2 that were identified as immunodominant across a broad range of HLA alleles. This approach has the advantage of including a longer range of the SARS-CoV-2 protein sequence and increases the potential to encode additional CD8 T cell epitopes presented by other HLA alleles that we did not investigate. For these vaccines, we concatenated 2–6 segments.

[0050] Both classes of vaccines are highly modular in terms of broader context and delivery. They can act as mono-vaccines focused on boosting the CD8 T cell response, or they can be co-introduced with antigens designed to generate a neutralizing antibody response. In the latter case, they may be expressed along with other antigens following a ribosome reactivation site, such as a P2A sequence. Some of these designs include an optimized S protein (containing two proline mutations designed to enhance the stability of the protein), followed by a P2A sequence and a polyepitope or polyfragment protein. These can be delivered using mRNA, DNA, viral vectors, or as purified proteins.

[0051] As one example related to specific epitopes of the construct, it was determined in this application that the CD8+ T cell response is dominated by a small number (3-8) highly antigenic (immunodominant) epitopes in SARS-CoV-2 that are shared among patients with the same HLA type. These epitopes are primarily specific to SARS-CoV-2 (i.e., do not occur in "common cold" coronaviruses), are invariant among viral isolates, and are often targeted by multiple clonal types within each patient. Across the six HLA types studied, at least 29 shared epitopes were identified. Notably, only about 10% of these epitopes (3 out of 29) reside in the S protein (i.e., about 90% of SARS-CoV-2 immunodominant epitopes are located outside the S protein), which highlights the need for a new class of vaccines designed to induce a broader CD8+ T cell response. Indeed, none of the mutations in the UK, South Africa, Brazil, or the delta variant occurred among these 29 epitopes, and when patients infected with SARS-CoV-2 were analyzed, some HLA types did not produce any spike protein epitopes (for example, no epitopes were identified in the spike protein in screening data from 5 A*01:01 patients and 5 A*11:01 patients). Notably, it was determined that 94% of the screened patients recognized at least one of the three most dominant epitopes for a given HLA, and 53% of the patients had T cells that recognized all three most dominant epitopes for a given HLA. Further confirmatory analysis in 18 additional A*02:01 patients revealed the repeated presence of memory CD8+ T cells specific to the top six identified A*02:01 epitopes, and single-cell sequencing revealed that patients often possessed >5 different T cell clones targeting each epitope, but that the same T cell receptor Va and Vb regions were primarily used to recognize these epitopes, even across a wide range of patients.T cells targeting the majority (27 out of 29) of these immunodominant epitopes do not cross-react with endemic coronaviruses that cause the common cold, and these epitopes do not occur in highly mutant regions. These results provide a useful tool for better understanding the CD8+ T cell response in COVID-19, and have important implications for designing and developing vaccines.

[0052] Accordingly, the present invention relates in part to identified immunogenic polypeptide constructs, compositions comprising these immunogenic polypeptide constructs alone or together with MHC molecules, stable MHC-peptide complexes, methods for diagnosing, prognosing, and monitoring the T cell response to SARS-CoV-2, and methods for preventing and / or treating SARS-CoV-2 infection by administering immunogenic compositions comprising and / or encoding identified immunogenic polypeptide constructs.

[0053] I. Definition For convenience, certain terms used in this specification, the examples, and the appended claims are set forth herein.

[0054] The articles "a" and "an" are used in this application to refer to one or more (i.e., at least one) grammatical objects of these articles. For example, "an element" means one element or two or more elements.

[0055] As used in this application, the term “administer” means to provide a drug or pharmaceutical composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0056] The term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, such as cytokine production and cytotoxicity. In addition, the term "immune response" also includes immune responses indirectly enabled by T cell activation, such as antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages).

[0057] Conventional T cells (also known as Tconv or Teff) possess effector functions (e.g., cytokine secretion, cytotoxic activity, anti-self recognition, etc.) and enhance the immune response by expressing one or more T cell receptors. Tcon or Teff is generally defined as any non-Treg T cell population, such as naive T cells, activated T cells, memory T cells, resting Tcon, or Tcon differentiated towards the Th1 or Th2 lineage, for example. In some embodiments, Teff is a subset of non-Treg T cells. In some embodiments, Teff is CD4+ Teff or CD8+ Teff, such as CD4+ helper T lymphocytes (e.g., Th0, Th1, Tfh, or Th17) and CD8+ cytotoxic T lymphocytes. As further described in this application, cytotoxic T cells are CD8+ T lymphocytes. "Naive Tcon" refers to CD4+ T lymphocytes differentiated in the bone marrow. +Naive T cells are T cells that have successfully undergone the positive and negative processes of central selection in the thymus but have not yet been activated by antigen exposure. Naive T cells are generally characterized by the surface expression of L-selectin (CD62L), the absence of activation markers such as CD25, CD44, or CD69, and the absence of memory markers such as CD45RO. Therefore, naive T cells are thought to be quiescent and non-dividing, requiring interleukin-7 (IL-7) and interleukin-15 (IL-15) for homeostatic survival (see at least WO 2010 / 101870). The presence and activity of such cells are undesirable in situations that suppress the immune response. Unlike Tregs, Tcons are not anergic and may proliferate in response to antigen-based T cell receptor activation (Lechler et al. (2001) Philos. Trans. R. Soc. Lond. Biol. Sci. 356:625-637). In tumors, exhausted cells may exhibit anerergic characteristics.

[0058] The term "vaccine" refers to a pharmaceutical composition that induces an immune response to an antigen of interest. Such vaccines can also confer protective immunity to a target.

[0059] A “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of preferred vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. A preferred vector is one capable of autonomously replicating and / or expressing the nucleic acid to which it is ligated. A vector capable of directing gene expression (the vector is operably ligated to the gene) is referred to in this application as an “expression vector.” Generally, expression vectors useful in recombinant DNA technology are often in the form of “plasmids” (which generally refer to a circular double-stranded DNA loop, and in the form of these vectors, are not bound to a chromosome). Herein, since plasmids are the most commonly used form of vectors, “plasmids” and “vectors” are used interchangeably. However, as will be understood by those skilled in the art, the present invention is intended to include other forms of expression vectors that perform equivalent functions and which may subsequently become known in the art.

[0060] The term "immunotherapy agent" can refer to any molecule, peptide, antibody, or other drug that can stimulate the host's immune system to produce an immune response to viral infection in a target. Various immunotherapy agents are useful in the compositions and methods described in this application.

[0061] "Isolated protein" refers to a protein that, when isolated from cells or produced by recombinant DNA technology, substantially does not contain other proteins, cellular substances, separation media, and culture media, or when chemically synthesized, substantially does not contain chemical precursors or other chemical substances. An "isolated" or "purified" protein, or a biologically active portion thereof, substantially does not contain cellular substances or other contaminating proteins derived from the cells or tissues from which the antibody, polypeptide, peptide or fusion protein is derived, or when chemically synthesized, substantially does not contain chemical precursors or other chemical substances. The phrase "substantially free of cellular substances" includes the preparation of a biomarker polypeptide, or a fragment thereof, which separates the protein from the cellular components of the cells from which the protein is isolated or the cells that recombinantly produce the protein. In one embodiment, the phrase "substantially free of cellular substances" includes the preparation of a biomarker protein, or a fragment thereof, having less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as "contaminating protein"), more preferably less than about 20% of non-biomarker protein, even more preferably less than about 10% of non-biomarker protein, and most preferably less than about 5% of non-biomarker protein. When an antibody, polypeptide, peptide or fusion protein, or a fragment thereof, such as a biologically active fragment thereof, is recombinantly produced, it is also substantially free of culture media, i.e., the culture media represents less than about 20%, more preferably less than about 10%, and most preferably 5% of the volume of the protein preparation.

[0062] As used herein, the term "isotype" refers to the class of an antibody encoded by a heavy chain constant region gene (e.g., IgM, IgG1, IgG2C, etc.).

[0063] As used herein, the term "K D" is intended to refer to the dissociation equilibrium constant of a specific antibody-antigen interaction. The binding affinity of antibodies encompassed by the present invention may be measured or determined by a standard antibody-antigen assay, e.g., a competitive assay, a saturation assay, or a standard immunoassay, e.g., ELISA or RIA.

[0064] A “Kit” is any product (e.g., a package or container) comprising at least one reagent (e.g., a probe or small molecule for specifically detecting and / or influencing the expression of a marker encompassed by the Invention). The Kit may be advertised, distributed, or sold as a unit for carrying out the methods encompassed by the Invention. The Kit may contain one or more reagents necessary to represent a composition useful in the methods encompassed by the Invention. In certain embodiments, the Kit may further include a reference criterion (e.g., a protein-coding nucleic acid that does not affect or regulate signaling pathways that control cell proliferation, division, migration, survival, or apoptosis). Those skilled in the art can envision many such control proteins, for example, but not limited to, common molecular tags (e.g., green fluorescent protein and beta-galactosidase), unclassified proteins in any pathway encompassing cell proliferation, division, migration, survival, or apoptosis, or ubiquitous housekeeping proteins, etc. The reagents in the kit may be provided in individual containers, or as a mixture of two or more reagents in a single container. Furthermore, the kit may include explanatory materials describing the use of the compositions.

[0065] The terms "prevent," "preventing," "prevention," and "prophylactic treatment" all refer to reducing the likelihood of developing a disease, disability, or symptom in a subject (who does not currently have the disease, disability, or symptom, but is at risk of developing it or is prone to developing it).

[0066] The term "prognosis" includes predictions regarding the possible course and outcome of a viral infection, or the likelihood of recovery from the disease. In some embodiments, statistical algorithms are used to predict the prognosis of a viral infection in an individual. For example, such prognosis may include surgery, the onset of a clinical subtype of the viral infection, the occurrence of one or more clinical factors, or recovery from the disease.

[0067] The term “sample” as used to detect or determine the presence or level of at least one biomarker is typically brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, feces (e.g., feces), tears, and any other bodily fluids (e.g., as defined above, “body fluids”), or tissue samples (e.g., biopsies), e.g., small intestine, large intestine samples, or surgically resected tissue. In certain embodiments, the method encompassed by the present invention further includes the step of obtaining the sample from the individual before detecting or determining the presence or level of at least one marker in the sample.

[0068] The term "low molecular weight" is a term used in the art and includes molecules with a molecular weight of less than approximately 1000 or less than approximately 500. In one embodiment, the low molecular weight is not composed solely of peptide bonds. In another embodiment, the low molecular weight is not an oligomer. Exemplary low molecular weight compounds that may be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, organic low molecular weights (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compound is a low molecular weight organic non-peptide compound. In a further embodiment, the low molecular weight is not biosynthetic.

[0069] The term "specific binding" refers to the binding of an antibody to a specific antigen. Typically, a binding assay, such as one using the antigen of interest as the analyte and the antibody as the ligand, is used. (登録商標) When measured using surface plasmon resonance (SPR) technology in an assay instrument, the antibody was approximately 10 -7 Less than M, for example, approximately 10 -8 M, 10 -9 M or 10 -10 Affinity (K) less than M or even smaller values D The antibody binds to the given antigen with an affinity at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0- times greater than its affinity for binding to non-specific antigens (e.g., BSA, casein) other than the given antigen or closely related antigens. In this application, the terms “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen.” Selective binding is a relative term referring to the ability of an antibody to distinguish the binding of one antigen from the binding of another antigen.

[0070] The term "subject" refers to any healthy animal, mammal, or human, or any animal, mammal, or human suffering from a viral infection (e.g., SARS-CoV-2 infection). The term "subject" is interchangeable with "patient."

[0071] In the context of this application, "percent identity" between amino acid sequences is synonymous with "percent homology," which is determined using an algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, (1990)), modified by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, (1993)). This described algorithm is incorporated into the NBLAST and XBLAST programs by Altschul et al. (J. Mol. Biol. 215:403-410, (1990)). A BLAST nucleotide search is performed using the NBLAST program, with a score of 100 and a word length of 12, to obtain nucleotide sequences homologous to the polynucleotides described in this application. A BLAST protein search is performed using the XBLAST program, with a score of 50 and a word length of 3, to obtain amino acid sequences homologous to the reference polypeptide. To obtain a gapped alignment for comparison, Gapped BLAST is used, as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, (1997)). When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) are used.

[0072] As used in this application, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent, etc., that is involved in transporting or carrying a compound of interest from one organ or part of the body to another organ or part of the body, and that encapsulates the substance.

[0073] "Transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g., mRNA, hnRNA, cDNA, or an analog of such RNA or cDNA) that is complementary or homologous to all or part of a mature mRNA produced by the transcription of a biomarker nucleic acid, and, if any, the normal post-transcriptional processing (e.g., splicing) of the RNA transcript, and the reverse transcription of the RNA transcript.

[0074] The term "T cell" includes CD4 + T cells and CD8 + This includes T cells. The term T cell also includes both T helper 1 T cells and T helper 2 T cells. The term "antigen-presenting cell" includes specialized antigen-presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), as well as other antigen-presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

[0075] The term “T cell receptor” or “TCR” should be understood to encompass the complete TCR, as well as its antigen-binding moiety or antigen-binding fragment. In some embodiments, the TCR is an intact or full-length TCR, e.g., an αβ-type TCR or a γδ-type TCR. In some embodiments, the TCR is a shorter antigen-binding moiety than the full-length TCR, but which binds to a specific peptide bound to an MHC molecule, e.g., an MHC-peptide complex. In some cases, the antigen-binding moiety or fragment of the TCR contains only a portion of the structural domain of a full-length or intact TCR, but can still bind to a peptide epitope (e.g., an MHC-peptide complex) to which a full-length TCR binds. In some cases, the antigen-binding moiety includes a variable domain of the TCR (e.g., a variable α chain and a variable β chain of the TCR) sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chain of the TCR contains complementarity determining regions (CDRs) that are involved in the recognition of peptides, MHCs, and / or MHC-peptide complexes.

[0076] The term “therapeutic effect” refers to a local or systemic effect in animals, particularly mammals, and more specifically humans, caused by a pharmaceutically active substance. Therefore, the term means any substance intended for use in the diagnosis, cure, alleviation, treatment, or prevention of disease, or in improving desirable physical or mental development and condition in animals or humans. The term “therapeutic effective dose” means the amount of substance that produces any desired local or systemic effect in a reasonable benefit / risk ratio applicable to any treatment. In certain embodiments, the therapeutic effective dose of a compound depends on its therapeutic index, solubility, etc. For example, a particular compound discovered by the methods encompassed by the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0077] The terms “therapeutic effective dose” and “effective dose,” as used in this application, mean the amount of a compound, material, or composition containing a compound, as encompassed by the present invention, that is effective in producing any desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment. The toxicity and therapeutic efficacy of the compound in question shall be determined by standard pharmaceutical methods (e.g., LD50) in cell culture or experimental animals. 50 and ED 50 It may be determined by a method for determining the therapeutic index. Compositions exhibiting a large therapeutic index are preferred. In some embodiments, LD 50 The lethal dose may be measured, for example, and the amount of the drug may decrease by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to when the drug is not administered. Similarly, ED 50 (That is, the concentration that can inhibit the symptom by up to half) may be measured, and for example, compared to no administration of the drug, the concentration of the drug may increase by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. Also, similarly, IC 50The following can be measured, for example, that the response to the drug may increase by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more, compared to a case where the drug is not administered. In some embodiments, the T cell immune response in the assay may increase by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even more than 100%. In another embodiment, the viral load may be reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%.

[0078] "Treating" a disease in a subject, or "treating" a subject having a disease, means subjecting the subject to pharmaceutical treatment, such as the administration of a drug, so that at least one symptom of the disease is reduced or prevented from worsening.

[0079] The term "body fluid" refers to fluids that are excreted or secreted from the body, as well as fluids that are not normally excreted or secreted from the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, cerumen and earwax, Cowper's fluid or bulbourethral gland fluid, chyle, ointment, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menstrual fluid, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous fluid, vomit).

[0080] The term "coding region" refers to a region of a nucleotide sequence that contains codons, which are translated into amino acid residues, while the term "noncoding region" refers to a region of a nucleotide sequence that is not translated into amino acids (e.g., the 5' and 3' untranslated regions).

[0081] The term "complementary" refers to a broad concept of sequence complementarity between regions of two nucleic acid chains, or between two regions of the same nucleic acid chain. It is known that an adenine residue in a first nucleic acid region can form a specific hydrogen bond ("base pairing") with a residue in a second nucleic acid region that is antiparallel to the first region, if that residue is thymine or uracil. Similarly, it is known that a cytosine residue in a first nucleic acid chain can form a specific base pairing with a residue in a second nucleic acid region that is antiparallel to the first region, if that residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or different nucleic acid if the two regions are antiparallel and at least one nucleotide residue in the first region can base pair with a residue in the second region. Preferably, the first region includes a first portion, and the second region includes a second portion, so that when the first and second portions are arranged antiparallel, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95%, of the nucleotide residues in the first portion can base pair with the nucleotide residues in the second portion. More preferably, all of the nucleotide residues in the first portion can base pair with the nucleotide residues in the second portion.

[0082] As used in this application, with respect to activated immune cells, the term "co-stimulate" means that the co-stimulating molecule can provide a second, non-activating receptor-mediated signal ("co-stimulating signal") that induces proliferation or effector function. For example, the co-stimulating signal may result in cytokine secretion in a T cell that has received a T cell-receptor-mediated signal. For example, an immune cell that has received a cell-receptor-mediated signal via an activating receptor is referred to in this application as an "activated immune cell."

[0083] The term “determining a suitable treatment regimen for the subject” is interpreted to mean determining a treatment regimen for the subject (i.e., a single therapy or combination of therapies to be used to prevent and / or treat a viral infection in the subject) to be initiated, modified, and / or terminated based on, or essentially, or at least partially, the results of the analysis according to the present invention. Examples include initiating adjuvant therapy postoperatively to reduce the risk of recurrence, or, as another example, changing the dosage of a particular chemotherapy. The determination may be based on the personal characteristics of the subject to be treated, in addition to the results of the analysis according to the present invention. In most cases, the attending physician or doctor will actually determine the appropriate treatment regimen for the subject.

[0084] As used in this application, the term “adjuvant” refers to a substance that, when administered before, together with, or after the administration of an antigen, accelerates, prolongs, and / or enhances the quality and / or intensity of the immune response to the antigen compared to the administration of the antigen alone. Adjuvants can increase the intensity and duration of the immune response induced by vaccination.

[0085] As used in this application, “homologous” refers to the similarity of nucleotide sequences between two regions of the same nucleic acid chain, or between regions of two different nucleic acid chains. A region is homologous in a given position if the positions of nucleotide residues in both regions are occupied by the same nucleotide residues. A first region is homologous to a second region if at least one position of nucleotide residues in each region is occupied by the same residue. The homology between two regions is expressed as the percentage of the positions of nucleotide residues in the two regions that are occupied by the same nucleotide residues. For example, a region having the nucleotide sequence 5'-ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region includes a first portion, and the second region includes a second portion, so that at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95%, of the positions of nucleic acid residues in each portion are occupied by the same nucleotide residues. More preferably, the positions of all nucleotide residues in each of the aforementioned regions are occupied by the same nucleotide residue.

[0086] The term "immune cells" refers to cells that play a role in the immune response. Immune cells are cells of hematopoietic origin, and include lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0087] The term "SARS-CoV-2" or "Severe Acute Respiratory Syndrome Coronavirus 2" refers to the pathogen that causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 was identified as a pandemic by the World Health Organization (WHO) on March 11, 2020. In order to assist the process by which the virus enters host cells, SARS-CoV-2 binds to highly expressed ACE2 receptors in the lower respiratory tract, upper esophagus and stratified epithelial cells, such as type II alveolar cells (AT2) in the lungs, as well as other cells such as absorptive enterocytes from the ileum and colon, bile duct cells, cardiomyocytes, renal proximal tubular cells, and bladder urothelial cells. Therefore, patients infected with this virus not only experience respiratory problems such as pneumonia leading to acute respiratory distress syndrome (ARDS), but also suffer from heart, kidney, and gastrointestinal disorders.

[0088] There is no specific treatment to eradicate the SARS-CoV-2 virus in patients. Treatment approaches for other beta-coronaviruses, such as SARS-CoV or MERS-CoV, can be used. Some of these approaches include lopinavir / ritonavir, chloroquine, and hydroxychloroquine. In some cases, aerosol inhalation of interferon-alpha twice overnight may be used. In some cases, the combination of interferon-alpha with ribavirin has been commonly used for coronaviruses (e.g., MERS-CoV). The combination of interferon and steroids has also been found to accelerate lung repair and increase oxygen survival levels. However, inconsistent results have been shown with interferon-alpha treatment.

[0089] SARS-CoV-2 is an enveloped, unsegmented, positive-sense RNA virus belonging to the sarbecovirus, ortho-corona virinae subfamily, which is widely distributed in humans and other mammals. Its diameter is approximately 65–125 nm, it contains a single strand of RNA, and it has crown-like spikes on its outer surface. SARS-CoV-2 is a novel β-coronavirus following the previously identified SARS-CoV and MERS-CoV (which caused lung failure and potentially fatal respiratory infections, primarily outbreaks in Guangdong, China, and Saudi Arabia).

[0090] The genome size of SARS-CoV-2 varied from 29.8kb to 29.9kb, and its genomic structure followed genetic characteristics specific to known CoVs. More than two-thirds of the 5' genome contained orf1a / b, which encodes the orf1a / b polyprotein, while the 3' one-third consisted of genes encoding four major structural proteins, including the spike (S) glycoprotein, small envelope (E) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein. Furthermore, SARS-CoV-2 also contains six accessory proteins encoded by the ORF3a, ORF6, ORF7a, ORF7b, and ORF8 genes (Khailany et al. (2020) Gene Rep 19:100682).

[0091] The ORF1ab gene is the largest gene segment of the coronavirus and constitutes two ORFs, namely ORF1a and ORF1b. By contributing to ribosome frameshift events, it produces two large duplicate polyproteins, pp1a (ORF1a polyprotein) and pp1ab (ORF1ab polyprotein). These polyproteins are supplemented by protease enzymes, namely papain-like proteases (PLpro) and serine Mpro (chymotrypsin-like protease (3CLpro)), encoded by nsp3 and nsp5. Subsequently, cleavage occurs between pp1a and pp1ab, resulting in nonstructural proteins (nsps) 1-11 and 1-16, respectively. These nsp play important roles in many processes within the virus and host cells. Representative sequences of the orf1a polyprotein and orf1ab polyprotein are shown in Table 1K below.

[0092] ORF3a is one of the accessory proteins encoded by the SARS-CoV-2 genome. Recent studies have shown that the functional domains of the SARS-CoV-2 ORF3a protein are associated with toxicity, infectivity, ion channel formation, and viral release (Issa et al. (2020) mSystems 5:e00266-20). Representative sequences of ORF3a are shown in Table 1K below.

[0093] ORF7a is an accessory protein encoded in another SARS-CoV-2 genome, primarily localized in the Golgi apparatus but also found on the cell surface, and is composed of type I transmembrane proteins. In the SARS-CoV genome, ORF7a overlaps with ORF7b, and thus ORF7a and ORF7b share a transcriptional regulatory sequence (TRS). In some embodiments, ORF7a has a 15-amino acid (aa) N-terminal signal peptide, an 81-aa luminal domain, a 21-aa transmembrane domain, and a 5-aa cytoplasmic tail (Taylor et al. (2015) J. Virol. 89:11820-11833). Representative sequences of ORF7a are shown in Table 1K below.

[0094] The spike protein, or S glycoprotein, is a transmembrane protein with a molecular weight of approximately 150 kDa found on the outer portion of the virus. The S protein has a receptive protein (RBD) located in the S1 subunit of the virus, which facilitates the virus's entry into the host cell by binding to its receptor (ACE2) on the host cell. The S protein forms a homotrimer that protrudes onto the surface of the virus and is attracted to angiotensin-converting enzyme 2 (ACE2), which is expressed in lower respiratory tract cells, thereby facilitating the binding of the enveloped virus to the host cell. This glycoprotein is cleaved into two subunits, S1 and S2, by a furin-like protease in the host cell. The S1 portion constitutes the receptor-binding domain and is responsible for determining the host viral domain and cell directivity, while S2 functions to mediate viral fusion during propagation within the host cell. Representative sequences of the S glycoprotein are shown in Table 1K below.

[0095] The nucleocapsid known as the N protein is a structural component of CoV, localized in the endoplasmic reticulum-Golgi region, and structurally binds to the nucleic acid material of the virus. Because this protein binds to RNA, it is involved in processes related to the viral genome, the viral replication cycle, and the host cell's cellular response to viral infection. The N protein is also highly phosphorylated and is suggested to undergo structural changes that enhance its affinity for viral RNA. Representative sequences of the N glycoprotein are shown in Table 1K below.

[0096] Another important part of this virus is the membrane, or M protein, which is the most structurally structured protein and plays a role in determining the shape of the viral envelope. This protein can bind to all other structural proteins. Binding to the M protein facilitates the stabilization of the nucleocapsid, or N protein, and promotes the completion of viral assembly by stabilizing the N protein-RNA complex inside the inner virion. Representative sequences of the M protein are shown in Table 1K below.

[0097] The final component is the envelope, or E protein, which is the smallest protein in the SARS-CoV-2 structure and plays a role in the production and maturation of this virus.

[0098] The genomic information of SARS-CoV-2 is publicly available, for example, from the NCBI Severe Acute Respiratory Syndrome Coronavirus 2 database (available on the World Wide Web at ncbi.nlm.nih.gov / sars-cov-2 / ) and the NGDC Genome Warehouse (available at bigd.big.ac.cn / gwh / ), along with epidemiological data on sequenced isolates. There is a known and clear correspondence between the amino acid sequence of a particular protein and the nucleotide sequence that can encode that protein, as defined by the genetic code (shown below). Similarly, there is a known and clear correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

[0099] [Table 1]

[0100] An important and well-known feature of the aforementioned genetic code is its redundancy, which allows for the use of two or more coding nucleotide triplets for most of the amino acids used to synthesize proteins (as described above). Therefore, several different nucleotide sequences can code for a given amino acid sequence. Such nucleotide sequences are considered functionally homogeneous because they result in the production of the same amino acid sequence in all organisms (however, some organisms can translate certain sequences more efficiently than others). Furthermore, occasionally, methylated variants of purines or pyrimidines may be found in a given nucleotide sequence. Such methylation does not affect the coding relationship between the trinucleotide codon and its corresponding amino acid.

[0101] In consideration of the above, polypeptide amino acid sequences may be derived using nucleotide sequences of DNA or RNA encoding a biomarker nucleic acid (or any part thereof), while using the genetic code for translating DNA or RNA into amino acid sequences. Similarly, for polypeptide amino acid sequences, corresponding nucleotide sequences that may encode the polypeptide can be inferred from the genetic code (this generates multiple nucleic acid sequences for any given amino acid sequence due to its redundancy). Therefore, the description and / or disclosure in this application relating to nucleotide sequences encoding a polypeptide should be considered to include the description and / or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, the description and / or disclosure of polypeptide amino acid sequences in this application should be considered to include the description and / or disclosure of all possible nucleotide sequences that may encode the amino acid sequence.

[0102] II. Peptides and constructs In certain embodiments, methods and compositions for treating and / or preventing COVID-19 are provided in this application, mediated by induction of an immune response to SARS-CoV-2 by administration of an identified SARS-CoV-2 immunodominant peptide or a nucleic acid encoding an identified SARS-CoV-2 immunodominant peptide.

[0103] In some embodiments, immunogenic polypeptides comprising at least two peptide epitopes or at least two peptide fragments (each of which comprises at least two peptide epitopes) are provided in this application. Various embodiments relating to the peptide epitopes of these immunogenic polypeptides are described below (for example, the peptide epitopes described with respect to an immunodominant peptide may also be constituent peptide epitopes of the immunogenic polypeptide).

[0104] Certain exemplary immunogenic polypeptides (e.g., constructs) are shown in Figures 1, 2, 5A–5C, 6A–6C, 7, 8, 9A, and 9B. As can be seen, the immunogenic polypeptide may contain multiple (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29) peptide epitopes (e.g., peptide epitopes derived up to Table 1A-1F), may contain a fragment of the SARS-CoV-2 protein having at least two such epitopes, and may also have additional segments (e.g., an S protein segment and / or a ribosome arrest / restart segment, an IRES segment and / or a post-translational cleavage segment, optionally, where the post-translational cleavage segment is a P2A fragment). In some embodiments, fragments of the SARS-CoV-2 protein containing the epitope of interest are divided by size, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 1, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190,195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 419, 420, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1005, 1010, 1015, 1020, 1025, 1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090, 1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130,1135, 1140, 1145, 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1271 amino acids (AA) between, or any comprehensive range between, e.g., 8-1271, 30-40, 15-30, 15-40, 30-112, 8-38, 8-96, 8-104, 8-107, The fragments may be defined by 8-112, 8-88, 8-87, 8-85, 8-66, 8-55, 222-275, 222-419, 275-419, 222-1271, etc. In some embodiments, the smallest fragment is an 8AA epitope. In some embodiments, a representative 14-fragment vaccine construct for non-epitope fragments is in the range of 30-40AA, and the smallest fragment among the 14 fragments = [3+9+3]epitope[3aa + epitope + 3aa]. In some embodiments, the largest fragment among the fragments covering the protein is 112AA. In some embodiments, the S protein (1271AA) is covered by a 38-amino acid fragment. In some embodiments, the N protein (419 AA) is covered by one or more fragments of 96 AA, 104 AA, and / or 107 AA (e.g., N_Frag1 = 96 AA, N_Frag2 = 104 AA, and / or N_Frag3 = 107 AA). In some embodiments, the Orf3a protein (275 AA) is covered by one or more fragments of 85 AA, 87 AA, and / or 88 AA (e.g., 3a_Frag1 = 88 AA, 3a_Frag2 = 87 AA, and / or 3a_Frag3 = 85 AA). In some embodiments, the M protein (222 AA) is covered by one or more fragments of 55 AA and / or 66 AA (e.g., M_Frag1 = 66 and / or M_Frag2 = 55).

[0105] In certain embodiments, the SARS-CoV-2 immunodominant peptide comprises a peptide epitope selected from Tables 1A, 1B, 1C, 1D, 1E, and / or 1F (e.g., consisting of). The peptide epitopes described herein may be combined with MHC molecules (e.g., specific HLA molecules having specific alpha-chain alleles). For example, as further described in the Examples, the peptides in Table 1A are HLA-A*02 Serum type (e.g., HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, Identify the peptides in Table 1C in association with the MHC of the alpha chain having the serotype encoded by HLA-A*0260 and / or HLA-A*274 alleles; identify the peptides in Table 1C in association with the MHC of the alpha chain having the HLA-A*03 serotype (e.g., serotype encoded by HLA-A*0301, HLA-A*0302, HLA-A*0305, and / or HLA-A*307); identify the peptides in Table 1B in association with the MHC of the alpha chain having the HLA-A*01 serotype (e.g., HLA-A*0101, HLA-A*0102, HLA-A*0103, and / Identify the peptides in Table 1D in association with the MHC of the alpha chain having HLA-A*11 serotypes (e.g., HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, and / or HLA-A*1119 alleles); identify the peptides in Table 1E in association with the MHC of the alpha chain having HLA-A*24 serotypes (e.g., HLA-A*2402, Identify the alpha chain MHC in association with the serotype encoded by the HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, and / or HLA-A*2458 alleles;Furthermore, the peptides in Table 1F are identified in association with the MHC of the alpha chain having an HLA-A*07 serotype (e.g., serotype encoded by the HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and / or HLA-B*0721 allele). In some embodiments, the SARS-CoV-2 immunodominant peptide is derived from a SARS-CoV-2 protein selected from Table 1K. In some embodiments, one or more SARS-CoV-2 immunodominant peptides are administered alone or in combination with an adjuvant.

[0106] In certain embodiments, a composition comprising one or more SARS-CoV-2 immunogenic peptides and adjuvants described in this application is provided in this application.

[0107] [Table 2]

[0108] [Table 3]

[0109] [Table 4]

[0110] [Table 5]

[0111] [Table 6]

[0112] [Table 7]

[0113] Table 1G [ka] [ka] [ka] [ka] [ka] [ka]

[0114] Table 1H [ka]

[0115] Table 1I [ka] [ka] [ka] *Note: In Figure 2 (continued), the 27th epitope fragment labeled #2 is a more accurate illustration of the construct labeled "27_epitope_3aa_linker" on page 35. The construct illustrations in Figure 2 are earlier versions of the construct illustrations shown in Figure 2 (continued), and each of these construct illustrations is an earlier version of the accurate construct illustration provided in Figures 5-9. [ka] [ka]

change

[0116] Table 1J

change

[0117] Table 1K

change

change

change

change

change

change

change

change

change

[0118] Table 2 [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] >YP_009724389 (ORF1a / b protein of SARS-CoV-2) >YP_009724390 (SARS-CoV-2 S protein) >YP_009724397 (SARS-CoV-2 N protein) >YP_009724391 (orf3a protein of SARS-CoV-2) > YP_009724393.1 (SARS-CoV-2 M protein) > YP_009724395.1 (orf7a protein of SARS-CoV-2) Table 2 includes nucleic acid sequences, or portions thereof, that have at least approximately 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity, or any comprehensive range between, for example, 85-99% identity, with nucleic acids encoding polypeptides listed in Table 1A-1K over their entire length. Such nucleic acid sequences may encode polypeptides having one or more functions of full-length peptides or polypeptides as further described in this application, and may also represent RNA nucleic acid molecules (e.g., thymine substituted with uredines).

[0119] In some embodiments, the application provides orf1a / b polypeptides and / or nucleic acids encoding orf1a / b polypeptides. An orf1a / b polypeptide is a polypeptide comprising an amino acid sequence corresponding to the amino acid sequence of an orf1a / b polyprotein and / or a portion of the orf1a / b amino acid sequence of sufficient length to induce an orf1a / b-specific immune response. In certain embodiments, the orf1a / b polypeptide also comprises amino acids that do not correspond to the aforementioned amino acid sequence (for example, a fusion protein comprising an orf1a / b amino acid sequence and an amino acid sequence corresponding to a non-orf1a / b protein or polypeptide). In some embodiments, the orf1a / b polypeptide comprises only the amino acid sequence corresponding to an orf1a / b polyprotein or fragment thereof.

[0120] In some embodiments, the orf1a / b polypeptide is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, It has an amino acid sequence consisting of, or comprising, 3500, 4000, 4500, 5000, 5500, 6000, 6500, or 7000 consecutive amino acids. In some embodiments, the consecutive amino acids are identical to the amino acid sequence of orf1a / b described in Table 1K. In some embodiments, the orf1a / b polypeptide consists of, or comprises, one or more peptide epitopes selected from the group consisting of orf1a / b peptide epitopes listed in Tables 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0121] In some embodiments, the application provides S protein polypeptides and / or nucleic acids encoding S protein polypeptides. An S protein polypeptide is a polypeptide comprising an amino acid sequence corresponding to the amino acid sequence of an S protein polyprotein and / or a portion of the S protein amino acid sequence of sufficient length to induce an S protein-specific immune response. In certain embodiments, the S protein polypeptide also includes amino acids that do not correspond to the aforementioned amino acid sequence (e.g., a fusion protein comprising an S protein amino acid sequence and an amino acid sequence corresponding to a non-S protein or polypeptide). In some embodiments, the S protein polypeptide comprises only the amino acid sequence corresponding to an S protein polyprotein or a fragment thereof.

[0122] In a particular embodiment, the S protein polypeptide is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, The S polypeptide has an amino acid sequence consisting of, or comprising, 900, 950, 1000, 1050, 1100, 1150, 1200, or 1250 consecutive amino acids. In some embodiments, the consecutive amino acids are identical to the amino acid sequence of the S protein described in Table 1K. In some embodiments, the S polypeptide consists of, or comprises, one or more peptide epitopes selected from the group consisting of S peptide epitopes listed in Tables 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0123] In some embodiments, the application provides N protein polypeptides and / or nucleic acids encoding N protein polypeptides. An N protein polypeptide is a polypeptide comprising an amino acid sequence corresponding to the amino acid sequence of an N protein polyprotein and / or a portion of the N protein amino acid sequence of sufficient length to induce an N protein-specific immune response. In certain embodiments, the N protein polypeptide also includes amino acids that do not correspond to the aforementioned amino acid sequence (e.g., a fusion protein comprising an N protein amino acid sequence and an amino acid sequence corresponding to a non-N protein or polypeptide). In some embodiments, the N protein polypeptide comprises only the amino acid sequence corresponding to an N protein polyprotein or a fragment thereof.

[0124] In a particular embodiment, the N protein polypeptide has at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, The N polypeptide has an amino acid sequence consisting of, or comprising, 260, 270, 280, 290, or 300 consecutive amino acids. In some embodiments, the consecutive amino acids are identical to the amino acid sequence of the N protein described in Table 1K. In some embodiments, the N polypeptide consists of, or comprises, one or more peptide epitopes selected from the group consisting of N peptide epitopes listed in Tables 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0125] In some embodiments, the application provides M protein polypeptides and / or nucleic acids encoding M protein polypeptides. An M protein polypeptide is a polypeptide comprising an amino acid sequence corresponding to the amino acid sequence of an M protein polyprotein and / or a portion of the M amino acid sequence of sufficient length to induce an M protein-specific immune response. In certain embodiments, the M protein polypeptide also includes amino acids that do not correspond to the aforementioned amino acid sequence (e.g., a fusion protein comprising an M protein amino acid sequence and an amino acid sequence corresponding to a non-M protein or polypeptide). In some embodiments, the M protein polypeptide comprises only the amino acid sequence corresponding to an M protein polyprotein or a fragment thereof.

[0126] In a particular embodiment, the M protein polypeptide has an amino acid sequence that essentially consists of, or comprises, at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 consecutive amino acids from the M protein amino acid sequence listed in Table 1K. In some embodiments, the sequence of amino acids is identical to the amino acid sequence of the M protein described in Table 1K. In some embodiments, the M polypeptide essentially consists of one or more peptide epitopes selected from the group consisting of M peptide epitopes listed in Tables 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0127] In some embodiments, the application provides orf3a polypeptides and / or nucleic acids encoding orf3a polypeptides. An orf3a polypeptide is a polypeptide comprising an amino acid sequence corresponding to the amino acid sequence of an orf3a polyprotein and / or a portion of the orf3a amino acid sequence of sufficient length to induce an orf3a-specific immune response. In certain embodiments, the orf3a polypeptide also comprises amino acids that do not correspond to the aforementioned amino acid sequence (e.g., a fusion protein comprising an orf3a amino acid sequence and an amino acid sequence corresponding to a non-orf3a protein or polypeptide). In some embodiments, the orf3a polypeptide comprises only the amino acid sequence corresponding to an orf3a polyprotein or fragment thereof.

[0128] In a particular embodiment, the orf3a polypeptide is at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, It has an amino acid sequence consisting of, or comprising, 260 or 270 consecutive amino acids. In some embodiments, the consecutive amino acids are identical to the amino acid sequence of the orf3a protein described in Table 1K. In some embodiments, the orf3a polypeptide consists of, or comprises, one or more peptide epitopes selected from the group consisting of orf3a peptide epitopes listed in Tables 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0129] In some embodiments, the application provides orf7a polypeptides and / or nucleic acids encoding orf7a polypeptides. An orf7a polypeptide is a polypeptide comprising an amino acid sequence corresponding to the amino acid sequence of an orf7a polyprotein and / or a portion of the orf7a amino acid sequence of sufficient length to induce an orf7a-specific immune response. In certain embodiments, the orf7a polypeptide also comprises amino acids that do not correspond to the aforementioned amino acid sequence (e.g., a fusion protein comprising an orf7a amino acid sequence and an amino acid sequence corresponding to a non-orf7a protein or polypeptide). In some embodiments, the orf7a polypeptide comprises only the amino acid sequence corresponding to an orf7a polyprotein or fragment thereof.

[0130] In certain embodiments, the orf7a polypeptide has an amino acid sequence consisting of, or comprising, at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, or 120 consecutive amino acids, as described in Table 1K. In some embodiments, the consecutive amino acids are identical to the amino acid sequence of the orf7a protein described in Table 1K. In some embodiments, the orf7a polypeptide essentially comprises one or more peptide epitopes selected from the group consisting of orf7a peptide epitopes listed in Table 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0131] As is well known to those skilled in the art, polypeptides having substantial sequence similarity may evoke identical or very similar immune responses in host animals. Accordingly, in some embodiments, derivatives, equivalents, variants, fragments, or variants of the SARS-CoV-2 immunogenic peptides described herein, or fragments thereof, may be suitable for the methods and compositions provided herein.

[0132] In some embodiments, this application provides variations or derivatives of SARS-CoV-2 immunogenic polypeptides. Modified polypeptides may have a modified amino acid sequence, for example, by conservative substitution, but are still considered functional equivalents that elicit an immune response in reaction to the unmodified protein antigen. As used in this application, the term “conservative substitution” refers to the substitution of an amino acid residue by another, biologically similar residue. It is well known in the art that amino acids within the same conservative group can typically be substituted for one another without substantially affecting the function of the protein. According to certain embodiments, a derivative, equivalent, variant, or variant of the ligand-binding domain of a SARS-CoV-2 immunogenic peptide is a polypeptide that is at least 85% homologous to the sequence of the SARS-CoV-2 immunogenic peptide or a fragment thereof described in this application. In some embodiments, the homology is at least 90%, at least 95%, at least 98%, or greater.

[0133] The immunogenic peptides encompassed by the present invention may include peptide epitopes derived from the SARS-CoV-2 protein, such as those listed in Table 1A, 1B, 1C, 1D, 1E, and / or 1F. In some embodiments, the immunogenic peptide is 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In some embodiments, the amino acid sequence of the peptide is modified and may include conserved or non-conserved mutations. The peptide may contain at most 1, 2, 3, 4, or more mutations. In some embodiments, the peptide may contain at least 1, 2, 3, 4, or more mutations.

[0134] In some embodiments, peptides may be chemically modified. For example, peptides may be mutated to alter their properties (e.g., detectability, stability, in vivo distribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, function, etc.). N-methylation is an example of methylation that may occur in peptides encompassed by the present invention. In some embodiments, peptides may be modified by methylation of free amines, such as by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0135] Chemical modifications may include polymers, polyethers, polyethylene glycol, biopolymers, amphoteric polymers, polyamino acids, fatty acids, dendrimers, Fc regions, monosaturated carbon chains such as palmitic acid or myristoleic acid, or albumin. Chemical modifications of peptides by the Fc region may result in fusion Fc-peptides. Examples of polyamino acids include polyamino acid sequences in which single amino acids repeat (e.g., polyglycine), polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination thereof. In some embodiments, peptides encompassed by the present invention may be modified to increase the stability and / or half-life of the peptide. In some embodiments, the half-life of peptides encompassed by the present invention may be extended by utilizing attachments of hydrophobic substructures, for example, attachments to the N-terminus, C-terminus, or internal amino acids. In other embodiments, the peptide may include post-translational modifications (e.g., methylation and / or amidation) that may affect the serum half-life. In some embodiments, a simple carbon chain may be conjugated to the fusion protein or peptide (e.g., by myristoylation and / or palmitylation). In some embodiments, the simple carbon chain may allow the fusion protein or peptide to be easily separated from the unconjugated substance. Methods that may be used to separate the fusion protein or peptide from the unconjugated substance include, but are not limited to, solvent extraction and reverse-phase chromatography. Lipid-soluble substructures may extend the half-life via reversible binding to serum albumin. The conjugated substructure may be a lipid-soluble substructure that extends the peptide's half-life via reversible binding to serum albumin. In some embodiments, the lipid-soluble substructure may be cholesterol or cholesterol derivatives, such as cholestanes, cholestanes, cholestadienes, and oxysterols.In some embodiments, the peptide may be conjugated to myristic acid (tetradecanoic acid) or a derivative thereof. In other embodiments, the peptide may be coupled (e.g., conjugated) to an agent that modifies its half-life. Examples of agents that modify the half-life include, but are not limited to, polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, amphoteric water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine, and serine, water-soluble polymers containing glycine, glutamic acid, and serine, Fc regions, fatty acids, palmitic acid, or molecules that bind to albumin. In some embodiments, spacers or linkers may be coupled to the peptide and act as spacers or linkers, for example, to facilitate conjugation or fusion to another molecule, and to facilitate cleavage of the peptide from such conjugated or fused molecule, such as 1, 2, 3, 4, or more amino acid residues. In some embodiments, the fusion protein or peptide may be conjugated to, for example, other substructures that can modify or alter the properties of the peptide.

[0136] Peptides may be conjugated to agents used in imaging, research, therapy, theranostics, pharmaceuticals, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, peptides may be conjugated or fused with detectable agents, such as fluorescent dye molecules, near-infrared dyes, contrast agents, nanoparticles, metal-containing nanoparticles, metal chelates, X-ray contrast agents, PET agents, metals, radioisotopes, dyes, radionuclide chelators, or other suitable substances that may be used for imaging. In some embodiments, detectable substructures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more may be linked to the peptide. Non-limited examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye does not readily quench with biological tissues and fluids. In some embodiments, the fluorescent dye molecule is a fluorescent agent that emits electromagnetic waves at wavelengths between 650 nm and 4000 nm, and such electromagnetic waves are used to detect such an agent. Non-exclusive examples of fluorescent dyes that may be used as conjugate molecules include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, ZQ800, or indocyanine green (ICG).In some embodiments, cyanine dyes (e.g., Cy7, Cy5.5, and Cy5) are often used as near-infrared dyes. Additional non-limiting examples of fluorescent dyes for use as conjugate molecules encompassed by the present invention include acradine orange or yellow, Alexa Fluors (e.g., Alexa Fluor). 790, 750, 700, 680, 660, and 647) and any derivatives thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO dyes and any derivatives thereof, auramine-rhodamine stains and any derivatives thereof, bensantrhone, biman, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)nathacene, bisbenzimide, brainbow, calcein, carbodyfluorescein and any derivatives thereof, 1-chloro-9,10-bis(phenylethynyl)anthracene and any derivatives thereof, DAPI, DiOC6, DyLight Fluors and any derivatives thereof, epicoconone, ethidium bromide, FlAsH-EDT2, Fluo dyes and any derivatives thereof, FluoProbe and any derivatives thereof, fluorescein and any derivatives thereof, Fura and any derivatives thereof, GelGreen and any derivatives thereof, GelRed and any derivatives thereof, fluorescent proteins and any derivatives thereof, such as m-isoform proteins such as mCherry and any derivatives thereof, hetamethine dye and any derivatives thereof, Hoechst stain, iminocoumarin, Indian yellow, indo-1 and any derivatives thereof, laurdan, Lucifer yellow and any derivatives thereof, luciferin and any derivatives thereof, luciferase and any derivatives thereof, mercocyanin and any derivatives thereof, Nile dye and any derivatives thereof, perylene, phloxine, phyco dye (phyco Examples include dyes and any derivatives thereof, propium iodide, pyranine, rhodamine and any derivatives thereof, ribogreen, RoGFP, rubrene, stilbene and any derivatives thereof, sulforhodamine and any derivatives thereof, SYBR and any derivatives thereof, synapto-pHluorin, tetraphenyl butadiene, tetrasodium tris, Texas Red, Titanium Yellow, TSQ, umbrellaferone, violanthrone, yellow fluorescent protein, and YOYO-1.Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxylrhodamine 6G, carboxy-X-rhodamine (ROX), lysamine-rhodamine B, rhodamine 6G, rhodamine green, rhodamine red) Red), tetramethylrhodamine (TMR), etc., coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green dye (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.), Texas Red, Texas Red-X, Spectrum Red, Spectrum Green, cyanine dyes (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), Alexa Fluor dye (e.g., Alexa Fluor). 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor Examples include alpha (e.g., 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, etc.), IRD dyes (e.g., IRD40, IRD700, IRD800, etc.). Further preferred detectable agents are described in PCT / US14 / 56177. Non-limited examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioactive isotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioactive isotope is actinium-225 or lead-212.

[0137] Peptides may be conjugated with radiosensitizers or photosensitizers. Examples of radiosensitizers, but not limited to, include: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines such as 5-fluorodeoxyuridine). Examples of photosensitizers, but not limited to, include fluorescent molecules or beads that generate heat upon irradiation, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, and chalcogenapyrrillium dyes. Examples include dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphirins, texapphirins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, Nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrelins, and cercosporines), psoralens, quinones, retinoids, rhodamines, thiophenes, verzins, xanthene dyes (e.g., eosins, erythrosines, rose bengal), dimers and oligomers of porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, this method makes it possible to target cells of interest (e.g., immune cells) with great specificity by simultaneously using both therapeutic agents (e.g., drugs) and electromagnetic energy (e.g., irradiation or light). In some embodiments, the peptide is fused with the drug, for example, directly or via a linker, or covalently or non-covalently linked.

[0138] Peptides may be produced recombinantly or synthetically, for example, by solid-phase peptide synthesis or liquid-phase peptide synthesis. Peptide synthesis may be carried out by known synthetic methods, for example, using fluorenylmethyloxycarbonyl (Fmoc) chemistry or butyloxycarbonyl (Boc) chemistry. Peptide fragments may be linked together enzymatically or synthetically.

[0139] In some embodiments, this application provides a nucleic acid or fragment thereof encoding the SARS-CoV-2 immunogenic polypeptide described herein, for example, a DNA molecule encoding the SARS-CoV-2 immunogenic peptide. In some embodiments, the composition comprises an expression vector comprising an open reading frame or fragment thereof encoding the SARS-CoV-2 immunogenic peptide described herein. In some embodiments, the nucleic acid comprises regulatory elements necessary for the expression of the open reading frame. Such elements may include, for example, a promoter, a start codon, a stop codon, and a polyadenylation signal. Enhancers may also be included. These elements can be operably linked to the sequence or fragment thereof encoding the SARS-CoV-2 immunogenic polypeptide.

[0140] Examples of promoters, though not limited to them, include promoters derived from monkey virus 40 (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV), such as the HIV long terminal repeat (LTR) promoter, Moloney virus, cytomegalovirus (CMV), such as the CMV immediate early promoter, Epstein-Barr virus (EBV), and Rous sarcoma virus (RSV), as well as promoters derived from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein. Suitable polyadenylation signals include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal.

[0141] In addition to the regulatory elements necessary for expression, other elements may also be included in the nucleic acid molecule. Such additional elements include enhancers. Examples of enhancers include promoters as described herein. Preferred enhancers / promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as viral enhancers derived from CMV, RSV, and EBV.

[0142] In some embodiments, the nucleic acids may be used alone (for example, as naked nucleic acids) or operably incorporated into a carrier or delivery vector, as further described below. Useful delivery vectors include, but are not limited to, biodegradable microcapsules, immuno-stimulating complexes (ISCOMs) or liposomes, and genetically modified or attenuated biological carriers such as viruses or bacteria.

[0143] In some embodiments, the vector is a viral vector, such as a lentivirus, retrovirus, herpesvirus, adenovirus, adeno-associated virus, vaccinia virus, baculovirus, fowlpox, AV-pox, modified vaccinia ankara (MVA), and other recombinant viruses. For example, a lentiviral vector may be used to infect T cells.

[0144] III. Nucleic acids, vectors, and cells A further object of the present invention relates to nucleic acid sequences encoding described immunogenic polypeptides, SARS-CoV-2 immunogenic peptides and their fragments, MHC molecules, and TCRs and their fragments. In some embodiments, the present application discloses nucleic acid vector constructs that maximize the size of the vector for the efficacy of the T cell response by packing an optimal immunodominant epitope within it for antigen expression in cells. Generally, as further described below, the nucleic acids encompassed by the present invention may be direct vaccine constructs (or vectors used to produce them) [e.g., mRNA (or in vitro transcription expression vectors that produce mRNA), mammalian expression vectors for use as DNA vaccines, etc.], whether naked or incorporated into a vector. The nucleic acids encompassed by the present invention may have codons optimized for specific purposes, such as high expression in human subjects. Nucleic acids encompassed by the present invention may be manipulated to have high guanine and cytosine (GC) content, for example, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more, or any comprehensive range between these, for example, 60-70% GC content.

[0145] In certain embodiments, the present invention relates to a nucleic acid sequence encoding the SARS-CoV-2 immunogenic peptide described in this application. In certain embodiments, the present invention relates to a nucleic acid sequence encoding the immunogenic polypeptide described in this application.

[0146] Typically, the nucleic acids are DNA (e.g., cDNA) or RNA (e.g., mRNA) molecules, which may be basic or contained within a suitable vector (e.g., plasmid, cosmid, episome, artificial chromosome, phage, virus, or viral vector, etc.).

[0147] Such basic nucleic acids may be “primary constructs” (e.g., primary mRNA constructs, which refer to polynucleotide transcripts [which encode one or more polypeptides of interest and retain sufficient structural and / or chemical features to enable translation into the polypeptides of interest encoded therein]). If structurally or chemically modified, such primary constructs may be called modified nucleic acids, such as modified mRNA. Nucleic acid constructs may include sequences (e.g., capping sequences, tailing sequences, cyclization sequences, etc., as are well known in the art) in addition to the polypeptide-coding sequence. For example, tailing sequences may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). If the tailing region is a polyA tail, its length may be determined by the number of polyA-binding protein bindings, or by the number of units. In this embodiment, the poly(A) tail is long enough to bind to at least four monomers of the poly(A) binding protein. The poly(A) binding protein monomers bind to a width of about 38 nucleotides. Thus, poly(A) tails of about 80 nucleotides and 160 nucleotides have been observed to be functional. The capping region may include a single cap or a series of nucleotides forming the cap. In this embodiment, the capping region may be 1 to 10 nucleotides long (e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or less nucleotides). In some embodiments, the cap is absent.Accordingly, the nucleic acids encompassed by the present invention may include protein-coding regions from 2 to 40 or more, for example, 2-19, 2-28, etc., and may further include one or more additional elements described in this application, such as start and / or stop codons, translation sequences, internal ribosomal entry sequences, protein cleavage sequences, signal sequences, capping sequences, tail sequences, restriction sequences, self-replicating sequences, etc. In some embodiments, the nucleic acids encompassed by the present invention may be cyclized and / or concatemerized by chemical, enzymatic, and / or ribozyme-catalyzed methods well known in the art. The newly formed 5'- / 3'- bonds may be intramolecular or intermolecular.

[0148] In some embodiments, the nucleic acids encompassed by this application are self-replicating, for example, self-replicating RNA-like mRNA. RNA is cost-effective to produce in large quantities, and it can be generated from any given discovered sequence or from commercially synthesized DNA precursors in almost the same time, without endotoxin. RNA can be administered more safely and easily than DNA because it does not pose the risk of genome integration and only requires access to the cytoplasm of a cell to function. Furthermore, because self-replicating RNA (repRNA) based on the genome of an alphavirus or flavivirus can be utilized, maximum immunogenicity and antigen production levels can be achieved using very low doses. In some embodiments, repRNA is an attenuated viral genome lacking the viral structural proteins necessary to produce progeny virions, but retaining the ability to translate and replicate, and thus effectively increasing the translation half-life of the RNA. For example, delivery of a RepRNA encoding one or more exogenous genes to a cell can effectively increase the translation and expression of the exogenous genes in the cell compared to what would result from delivery of an equimolar amount of conventional mRNA encoding one or more exogenous genes to the cell. In some embodiments, the RepRNA is a non-cellular-pathogenic RepRNA. In some embodiments, the repRNA is an alphavirus self-amplifying repRNA [e.g., including a 5' cap; 5' untranslated region (5' UTR); non-structural genes encoded within a first open reading frame (e.g., NSP1-4); genome promoter region (e.g., 26S sub-genome promoter); second open reading frame; 3' untranslated region (3' UTR); and 3' polyadenylated tail, etc.]. The repRNA molecule is typically between 9,000 and 20,000 nucleotides in length, depending on the size of the gene sequence it encodes. Non-structural genes encode RNA-dependent RNA polymerase (RdRp).Typically, the RdRp does not tolerate classical nucleotide modifications used to protect conventional RNA from endonucleose and autocatalytic degradation. Therefore, nanoencapsulation (e.g., nanoparticles, lipids, lipid nanoparticles, cationic molecules, polymers, etc.) may be used to effectively deploy the expression platform. repRNA is a modulator and can manipulate the open reading frame to accommodate the exogenous sequence of interest. When the repRNA is placed in the cytoplasm of a host cell, RNA-dependent RNA polymerase (RdRp), encoded by the repRNA NS gene, is expressed in the cell. The RdRp can then replicate the entire repRNA or only the RdRp copy of the antigen encoded by the repRNA (i.e., by a sub-genomic promoter). Replicon RNA increases the overall effectiveness of RNA-mediated gene delivery because the repRNA can synthesize more copies of the full-length replicon, as well as more copies of the mRNA encoding the gene contained within the second open reading frame. Host cell ribosomes continue to translate full-length replicon copies or shorter antigen-only mRNAs, resulting in enhanced expression of the genes encoded by the repRNAs.

[0149] To further enhance protein production, the nucleic acids included in the present invention may be replaced with other polynucleotides, dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen and mitomycin C), porphyrins (TPPC4, texaphylline, and saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine and dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphates, amino acids, mercaptos, PEGs (e.g., PEG-40K), MPEGs, MPEG2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, and folic acid), synthetic ribonucleases, proteins (e.g., glycoproteins), or peptides (e.g., molecules having specific affinity for co-ligands), or antibodies (e.g., Nucleic acids may be designed to conjugate antibodies (which bind to specific cell types such as cancer cells, endothelial cells, or osteocytes), hormones and hormone receptors, and / or non-peptide species (e.g., lipids, lectins, carbohydrates, vitamins, cofactors, and drugs). Typical examples of nucleic acids encompassed by the present invention are described in this application, e.g., in the examples and drawings, and are well known in the art (see at least U.S. Patent Publication No. 2020 / 0354423, U.S. Patent Publication No. 2020 / 0254086, and U.S. Patent Publication No. 2020 / 0155660).

[0150] The terms “vector,” “cloning vector,” and “expression vector” refer to a medium that can introduce a DNA or RNA sequence (e.g., an exogenous gene) into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequence. Therefore, a further object encompassed by the present invention relates to a nucleic acid-containing vector as encompassed by the present invention.

[0151] Such vectors may contain regulatory elements, such as promoters, enhancers, and terminators, which may induce or instruct the expression of the polypeptide upon administration to a target. Examples of promoters and enhancers used in animal cell expression vectors include the initial promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney's mouse leukemia virus (Kuwana Y et al. 1987), and the promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chains.

[0152] Any expression vector for animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), and pSG1 beta d2-4- (Miyaji H et al. 1990). Other representative examples of plasmids include replication plasmids containing origins of replication, or integrative plasmids such as pUC, pcDNA, and pBR. Representative examples of viral vectors include vectors for adenoviruses, retroviruses, herpesviruses, and AAVs. Such recombinant viruses may be produced by techniques known in the art, such as transfection of packaging cells or transient transfection using helper plasmids or viruses. Typical examples of virus-packaging cells include PA317 cells, PsiCRIP cells, GPenv-positive cells, and 293 cells. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Patents 5,882,877, 6,013,516, 4,861,719, 5,278,056, and WO 94 / 19478. In some embodiments, a virus vector-based platform may be used.Representative, non-exclusive examples include vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus, and lentivirus, and, for example, but not limited to, second-generation, third-generation, or hybrid second / third-generation lentiviruses, and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see at least Hu et al. (2011) Immunol Rev. 239:45-61, Sakuma et al. (2012) Biochem J. 443:603-618, Cooper et al. (2015) Nucl. Acids Res. 43:682-690, Zufferey et al. (1998) J. Virol. 72:9873-9880, and U.S. Patent Application Publication 2020 / 0010849).

[0153] A further object of the present invention relates to cells that have been transfected, infected, or transformed by nucleic acids and / or vectors according to the present invention. The term “transformation” means the introduction of an “external” (i.e., extrinsic or extracellular) gene, DNA, or RNA sequence into a host cell, as a result, the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is “transformed.”

[0154] The nucleic acids encompassed by the present invention may be used to produce recombinant polypeptides encompassed by the present invention in a suitable expression system. The term “expression system” means, for example, a host cell and a compatible vector under suitable conditions for expressing a protein encoded by foreign DNA delivered by the vector and introduced into the host cell.

[0155] Common expression systems include E. coli (Escherichia coli) host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Other examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells lacking the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") (Urlaub G et al.; 1980), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL 1662, hereinafter referred to as "YB2 / 0 cells"). The aforementioned YB2 / 0 cells are preferred because the ADCC activity of the chimeric antibody or humanized antibody is enhanced when expressed in these cells.

[0156] The present invention also relates to a method for producing recombinant host cells expressing SARS-CoV-2 immunogenic peptides and their fragments, MHC molecules, and TCRs and their fragments, which are encompassed by the present invention, wherein the method comprises the steps of: (i) introducing such recombinant nucleic acids or vectors into competent host cells in vitro or ex vivo; (ii) culturing the obtained recombinant host cells in vitro or ex vivo; and (iii) optionally selecting cells that express the SARS-CoV-2 immunogenic peptides and their fragments, MHC molecules, and TCRs and their fragments. Such recombinant host cells can be used in diagnostic methods, prognostic methods, and / or therapeutic methods, which are encompassed by the present invention.

[0157] In another embodiment, the present invention provides isolated nucleic acids that hybridize to the polynucleotides disclosed herein under selective hybridization conditions. Accordingly, the polynucleotides of this embodiment may be used to isolate, detect, and / or quantify nucleic acids containing such polynucleotides. For example, polynucleotides encompassed by the present invention may be used to identify, isolate, or amplify partial or full-length clones in a storage library. In some embodiments, the polynucleotides are genomic sequences or cDNA sequences isolated from, or otherwise complementary to, cDNA derived from a human or mammalian nucleic acid library. Preferably, the cDNA library contains at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences. The cDNA library is normalized to increase the expression of rare sequences. Low-stringency or medium-stringency hybridization conditions are typically, but not exclusively, used for sequences with low sequence identity compared to complementary sequences. Medium-stringency and high-stringency conditions can be optionally used for sequences with higher identity. Low-stringency conditions allow for selective hybridization of sequences with approximately 70% sequence identity and may be used to identify orthologous or paralogous sequences. Optionally, the polynucleotides included by this invention encode at least a portion of the antibodies encoded by the polynucleotides described in this application. The polynucleotides included by this invention encompass nucleic acid sequences that can be used for selective hybridization to the polynucleotides encoding the antibodies included by this invention (see, for example, Ausubel and Colligan above, each in whole incorporated by reference in this application).

[0158] IV. MHC-Peptide Complexes In certain embodiments, the present application provides a composition comprising the SARS-CoV-2 immunogenic peptide and MHC molecules described herein. In some embodiments, the SARS-CoV-2 immunogenic peptide forms a stable complex with the MHC molecules.

[0159] The MHC proteins provided and used in the compositions and methods encompassed by the present invention may be any suitable MHC molecules known in the art. Generally, they are of the formula (α-β-P) n The matrix comprises, where n is at least 2, e.g., 2-10, e.g., 4. α is the α chain of a class I or class II MHC protein. β is the β chain, defined in this application as the β chain of a class II MHC protein, or the β chain of β2 microglobulin for a class I MHC protein. P is a peptide antigen.

[0160] In some embodiments, the MHC protein is an MHC class I complex, such as an HLA I complex.

[0161] The aforementioned MHC proteins may be derived from any mammal or bird species, such as primates, especially humans; rodents, such as mice, rats, and hamsters; rabbits; horses, cattle, dogs, cats, etc. For example, the aforementioned MHC proteins may be derived from human HLA proteins or mouse H-2 proteins. Examples of HLA proteins include class II subunits HLA-DPα, HLA-DPβ, HLA-DQα, HLA-DQβ, HLA-DRα, and HLA-DRβ, as well as class I proteins HLA-A, HLA-B, HLA-C, and β2-microglobulin. Examples of H-2 proteins include class I subunits H-2K, H-2D, and H-2L, as well as class II subunits I-Aα, I-Aβ, I-Eα, and I-Eβ, and β2-microglobulin. Sequences of several representative MHC proteins can be found in Kabat et al. Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, pp724-815. MHC protein subunits suitable for use according to the present invention are typically soluble forms of membrane-bound proteins, which are prepared as known in the art (e.g., by deletion of the transmembrane and cytoplasmic domains).

[0162] In the case of class I proteins, the soluble form may include α1, α2, and α3 domains. Soluble class II subunits include the α1 and α2 domains for the α subunit, and the β1 and β2 domains for the β subunit.

[0163] The α and β subunits can be generated separately and associated in vitro to form a stable heterodouble-stranded complex, or both subunits can be expressed in a single cell. Methods for generating MHC subunits are known in the art.

[0164] In a particular embodiment, the MHC-peptide complex is a peptide epitope selected from Table 1A, and the MHC (the alpha chain of this MHC is HLA-A*02 serotype, for example, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, In other embodiments, the MHC-peptide complex includes a peptide epitope selected from Table 1C, and an MHC (the alpha chain of this MHC has a serotype encoded by an HLA-A*03 serotype, e.g., HLA-A*0301, HLA-A*0302, HLA-A*0305, and / or an HLA-A*307 allele). In yet another embodiment, the MHC-peptide complex comprises a peptide epitope selected from Table 1B, and an MHC (the alpha chain of this MHC having a serotype encoded by an HLA-A*01 serotype, e.g., HLA-A*0101, HLA-A*0102, HLA-A*0103, and / or HLA-A*116 allele). In yet another embodiment, the MHC-peptide complex comprises a peptide epitope selected from Table 1D, and an MHC (the alpha chain of this MHC having a serotype encoded by an HLA-A*11 serotype, e.g., HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, and / or HLA-A*1119 allele).In other embodiments, the MHC-peptide complex comprises a peptide epitope selected from Table 1E, and an MHC (the alpha chain of this MHC having a serotype encoded by an HLA-A*24 serotype, such as HLA-A*2402, HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, and / or HLA-A*2458 allele). In yet another embodiment, the MHC-peptide complex comprises a peptide epitope selected from Table 1F, and an MHC (the alpha chain of this MHC having a serotype encoded by an HLA-A*07 serotype, such as HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and / or HLA-B*0721 allele).

[0165] To prepare the MHC-peptide complex, the subunits can be combined with an antigenic peptide and folded in vitro to form a stable heterodimer complex having an intrachain disulfide-bonding domain. The peptide may be included in the initial folding reaction or added to an empty heterodimer in a later step. In the compositions and methods encompassed by the present invention, this is a SARS-CoV-2 immunogenic peptide or a fragment thereof. The conditions that enable the folding and association of the subunits and peptides are known in the art. As an example, nearly equimolar amounts of solubilized α and β subunits may be mixed in a solution of urea. Refolding is initiated by dilution to a urea-free buffer solution or by dialysis. The peptide is loaded into an empty class II heterodimer at about pH 5 to 5.5 for about 1 to 3 days, followed by neutralization, concentration, and buffer exchange. However, the specific folding conditions are not important for carrying out the present invention.

[0166] The monomeric complex (α-β-P) (the monomer of this application) may be polymerized, for example, into an MHC tetramer. The resulting polymer is stable over long periods of time. Preferably, the polymer may be formed by conjugating the monomer to a polyvalent entity via a specific attachment site on the α or β subunit, as is known in the art (for example, as described in U.S. Patent No. 5,635,363). The MHC proteins may also be conjugated to beads or any other retainer, either with their monomers or polymers.

[0167] The multimer complex may be labeled, making it directly detectable when used in immunohistochemical staining or other methods known in the art, or it may be used in conjunction with a secondary labeling immunoreagent that specifically binds to the complex (e.g., to MHC protein subunits), as known in the art. For example, detectable labels include fluorescent dye molecules such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin (PE), allophycocyanin (APC), and Brilliant Violet. TM 421, Brilliant UV TM 395, Brilliant Violet TM 480, Brilliant Violet TM 421 (BV421), Brilliant Blue TM This may include 515, APC-R700, or APC-Fire750, etc. In some embodiments, the polymeric complex is labeled with a substructure that can specifically bind to another substructure. For example, the label may be biotin, streptavidin, oligonucleotide, or ligand. Other labels of interest include fluorescent dyes, pigments, enzymes, chemiluminescent materials, particles, radioisotopes, or other directly or indirectly detectable agents.

[0168] In some embodiments, cells that present immunogenic peptides within MHC molecules on their cell surface are generated by transfecting or transducing a vector (e.g., a viral vector) containing nucleic acids encoding recombinant or heterozygous antigens into the cells. In some embodiments, the vector is introduced into the cells under conditions (conditions in which one or more peptide antigens, if any, one or more peptide antigens of expressed heterozygous proteins, are expressed by the cells, undergo processing, and are presented on the surface of the cells within major histocompatibility complex (MHC) molecules).

[0169] Generally, the cells into which the vector is contacted are MHC-expressing cells, i.e., MHC-expressing cells. These cells may be cells that normally express MHC on their cell surface, i.e., cells that are induced to express and / or upregulated MHC expression on their cell surface, or cells that have been engineered to express MHC on their cell surface. In some embodiments, the MHC includes polymorphic peptide binding sites or grooves that can, in some cases, form complexes with polypeptide peptide antigens (e.g., peptide antigens processed by a cell machinery). In some cases, the MHC molecule may be presented or expressed on the cell surface, for example, as a complex with a peptide (i.e., an MHC-peptide complex) for antigen presentation in a conformation recognizable by the TCR on T cells or other peptide-binding molecules.

[0170] In some embodiments, the cells are nucleated cells. In some embodiments, the cells are antigen-presenting cells. In some embodiments, the cells are macrophages, dendritic cells, B cells, endothelial cells, or fibroblasts. In some embodiments, the cells are endothelial cells, such as an endothelial cell line or primary endothelial cells. In some embodiments, the cells are fibroblasts, such as a fibroblast cell line or primary fibroblast cells.

[0171] In some embodiments, the cells are artificial antigen-presenting cells (aAPCs). Typically, aAPCs include the characteristics of natural APCs (e.g., expression of MHC molecules, irritant molecules and co-irritant molecules, Fc receptors, adhesion molecules, and / or the ability to produce or secrete cytokines (e.g., IL-2), etc.). Typically, aAPC is a cell line lacking the expression of one or more of the above, and MHC molecules, low-affinity Fc receptors (CD32), high-affinity Fc receptors (CD64), and one or more co-stimulatory signals (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta-receptor, ILT3, ILT4, 3 / TR6, or ligands for B7-H3; or CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, It is produced by introducing one or more of the missing elements from among Toll ligand receptors or CD83 ligands (antibodies that specifically bind to these ligands / receptors or ligands of CD83), cell adhesion molecules (e.g., ICAM-1 or LFA-3), and / or cytokines (e.g., IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon-alpha (IFNalpha), interferon-beta (IFNbeta), interferon-gamma (IFNgamma), tumor necrosis factor-alpha (TNFalpha), tumor necrosis factor-beta (TNFbeta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (GCSF)), for example, through transfection or transduction.In some cases, aAPCs do not normally express MHC molecules but may be engineered to express them, or in some cases may be induced to express MHC molecules by stimuli such as cytokine stimulation. In some cases, aAPCs may also be loaded with stimulatory ligands (such as, for example, anti-CD3 antibodies, anti-CD28 antibodies or anti-CD2 antibodies). Exemplary cell lines that can be used as a backbone for generating aAPCs are the K562 cell line or fibroblast cell lines. Various aAPCs are known in the art (see, for example, U.S. Patent No. 8,722,400, Published Application No. US2014 / 0212446; Butler and Hirano (2014) Immunol Rev., 257(1):10. 1111 / imr.12129; Suhoshki et al. (2007) Mol. Ther., 15:981-988).

[0172] Determining or identifying the specific MHC or allele expressed by a cell is well within the level of one of ordinary skill in the art. In some embodiments, prior to contacting the cell with the vector, the expression of a specific MHC molecule may be evaluated or confirmed, for example, by using an antibody specific for said specific MHC molecule. Antibodies to MHC molecules are known in the art and are, for example, any of those described below.

[0173] In some embodiments, the cells may be selected to express MHC alleles of desired MHC restriction conditions. In some embodiments, cells of such MHC typing are well known in the art, such as cell lines. In some embodiments, cells of MHC typing (e.g., primary cells obtained from a subject) may be determined using procedures well known in the art, such as performing tissue typing using a molecular haplotype assay (BioTest ABC SSPtray, BioTest Diagnostics Corp., Denville, N.J.; SeCore Kits, Life Technologies, Grand Island, N.Y.). In some cases, standard typing of cells to determine the HLA genotype, for example, by using sequence-based typing (SBT) (Adams et al. (2004) J. Transl. Med., 2:30; Smith (2012) Methods Mol Biol., 882:67-86), is well within the level of those skilled in the art. In some cases, cells of such HLA typing are known, such as fibroblasts. For example, the human fetal lung fibroblast cell line MRC-5 is HLA-A*0201, A29, B13, B44 Cw7 (C*702); the human foreskin fibroblast cell line Hs68 is HLA-A1, A29, B8, B44, Cw7, Cw16; and the WI-38 cell line is A*6801, B*0801 (Solache et al. (1999) J Immunol, 163:5512-5518; Ameres et al. (2013) PloS Pathog. 9:e1003383). The human transfected fibroblast cell line M1DR1 / Ii / DM expresses HLA-DR and HLA-DM (Karakikes et al. (2012) FASEB J., 26:4886-96).

[0174] In some embodiments, the cells that come into contact with the vector, or into which the vector is introduced, are cells that are engineered or transfected to express MHC molecules. In some embodiments, the cell line may be prepared by genetically modifying a parent cell line. In some embodiments, the cells typically lack a specific MHC molecule and are engineered to express such a specific MHC molecule. In some embodiments, the cells are genetically engineered using recombinant DNA technology.

[0175] In some embodiments, the stable MHC-peptide complex described in this application is used to detect T cells that bind to the stable MHC-peptide complex. In some embodiments, the stable MHC-peptide complex described in this application is used to monitor the T cell response in a subject by detecting, for example, the quantity and / or proportion of T cells (e.g., CD8+ T cells) that specifically bind to the fluorescently labeled MHC-peptide complex. Methods for generating, labeling, and using MHC-peptide complexes (e.g., MHC-peptide tetramers) to detect MHC-peptide complex-specific T cells are well known in the art. Further descriptions can be found, for example, in U.S. Patent No. 7,776,562; U.S. Patent No. 8,268,964; and U.S. Patent Application Publication No. 2019 / 0085048 (the entirety of which is incorporated by reference in this application).

[0176] V. Immunogenic compositions In some embodiments, the present application provides a pharmaceutical composition (e.g., a vaccine composition) comprising a SARS-CoV-2 immunogenic peptide and / or a nucleic acid encoding the SARS-CoV-2 immunogenic peptide, and an adjuvant. In some embodiments, the present application provides a pharmaceutical composition (e.g., a vaccine composition) comprising an immunogenic peptide and / or a nucleic acid encoding the immunogenic peptide, and an adjuvant. In some embodiments, the present application provides a pharmaceutical composition (e.g., a vaccine composition) comprising a stable MHC-peptide complex containing the SARS-CoV-2 immunogenic peptide in an MHC molecule, and an adjuvant. In some embodiments, the composition comprises a combination of several (e.g., two or more) SARS-CoV-2 immunogenic peptides or nucleic acids, and an adjuvant. In some embodiments, the composition comprises several (e.g., two or more) stable MHC-peptide complexes containing the SARS-CoV-2 immunogenic peptide in an MHC molecule, and an adjuvant. In some embodiments, the above-described composition further comprises a pharmaceutically acceptable carrier.

[0177] The pharmaceutical compositions disclosed herein may be specially formulated for administration in solid or liquid form, wherein such administration includes, for example, administration adapted for the following purposes: (1) oral administration, e.g., drench (aqueous or non-aqueous solution or suspension), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, bolus, powder, granules, paste for application to the tongue; or (2) parenteral administration, e.g., as a sterile solution or suspension, or as a sustained-release formulation, e.g., by subcutaneous injection, intramuscular injection, intravenous injection, or epidural injection.

[0178] Methods for preparing these formulations or compositions include the step of associating the SARS-CoV-2 immunogenic peptide and / or nucleic acid described in this application with an adjuvant, a carrier, and optionally one or more accessory components. Generally, the formulations are prepared by uniformly and closely associating the agent described in this application with a liquid carrier, or a micronized solid carrier, or both, and then, if necessary, shaping the product.

[0179] Pharmaceutical compositions suitable for parenteral administration include, in combination with an adjuvant and one or more pharmaceutically acceptable, sterile, sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes to be isotonic with the blood of the recipient of the formulation, or suspending agents or thickeners.

[0180] Suitable aqueous and non-aqueous carriers that may be used in the aforementioned pharmaceutical composition include, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Appropriate fluidity may be maintained, for example, by using a coating substance such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant.

[0181] Regardless of the selected route of administration, the agents provided in this application, and / or the pharmaceutical compositions disclosed in this application, which may be used in a preferred hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0182] In some embodiments, the pharmaceutical compositions described may, when administered to a subject, induce an immune response against cells infected with SARS-CoV-2. Such pharmaceutical compositions may be useful as vaccine compositions for the prophylactic and / or therapeutic treatment of COVID-19.

[0183] In some embodiments, the pharmaceutical composition further comprises a physiologically acceptable adjuvant. In some embodiments, the adjuvant used increases the immunogenicity of the pharmaceutical composition. Such further immune response stimulating compounds or adjuvants may (i) be mixed with the pharmaceutical composition according to the present invention after the reconstitution of the peptide and optionally emulsified with an oil-based adjuvant as defined above, (ii) be part of a reconstitution composition encompassed by the present invention as defined above, (iii) be physically linked to the peptide to be reconstituted, or (iv) be administered separately to a subject to be treated, mammal or human. The adjuvant may be an adjuvant that provides sustained release of an antigen (for example, the adjuvant may be a liposome), or the adjuvant may be immunogenic itself and thereby function synergistically with the antigen (i.e., the antigen present in the SARS-CoV-2 immunogenic peptide). For example, the adjuvant may be a known adjuvant or another substance that promotes antigen uptake, recruits immune system cells to the administration site, or promotes immune activation of responsive lymphoid cells. Examples of adjuvants, but not limited to, include immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, glucans, dextran sulfate, iron oxides, sodium alginate, bacto-adjuvant, synthetic polymers (e.g., polyamino acids and amino acid copolymers), saponins, paraffin oils, and muramyl dipeptides. In some embodiments, the adjuvant is adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptide, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ, IL-17, lipid A, lipopolysaccharide, lipovant, montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, trehalose dimicolate, or zymosan.

[0184] In some embodiments, the adjuvant is an immunomodulatory molecule. For example, the immunomodulatory molecule may be a recombinant protein cytokine, chemokine, or immunostimulatory agent, or a nucleic acid encoding a cytokine, chemokine, or immunostimulatory agent, designed to enhance the immune response.

[0185] Examples of immunomodulatory cytokines include interferons (e.g., IFNα, IFNβ, and IFNγ), interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17, and IL-20), tumor necrosis factor (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1 alpha, MIP-1 β, Rantes, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as any of the functional fragments mentioned above.

[0186] In some embodiments, immunomodulatory chemokines that bind to chemokine receptors (i.e., CXC, CC, C, or CX3C chemokine receptors) may also be included in the compositions provided in this application. Examples of chemokines, but not limited to, include Mip1α, Mip-1β, Mip-3α (Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2, Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tarc, Elc, I309, IL-8, Gcp-2 Gro-α, Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, and Bca-1 (Blc), as well as any of the functional fragments mentioned above.

[0187] In some embodiments, the composition comprises a nucleic acid encoding the SARS-CoV-2 immunogenic polypeptide described in this application, for example, a DNA molecule encoding the SARS-CoV-2 immunogenic peptide. In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding the SARS-CoV-2 immunogenic peptide.

[0188] When taken up by cells (e.g., muscle cells, antigen-presenting cells (APCs) [e.g., dendritic cells, macrophages, etc.]), DNA molecules may exist in the cells as extrachromosomal molecules and / or be incorporated into the chromosomes. DNA may be introduced into cells in the form of plasmids, which are maintained as separate genetic material. Alternatively, linear DNA that can be incorporated into the chromosomes may be introduced into the cells. When DNA is introduced into cells selectively, reagents that promote the incorporation of DNA into the chromosomes may be added.

[0189] VI. Connected substructure In some embodiments, binding substructures are provided that bind to the peptides described in this application and / or to the stable MHC-peptide complexes described in this application. For example, binding proteins such as T cell receptors (TCRs), antibodies, etc. -7 M (for example, about 10 -7 , about 10 -8 , about 10 -9 , about 10 -10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 The following are provided: a binding protein that specifically binds to the peptide and / or the stable MHC-peptide complex, with a Kd of the following magnitude.

[0190] In some embodiments, the MHC molecule comprises an MHC alpha chain which is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and / or HLA-B*07. In some embodiments, the HLA alleles are HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, The HLA allele is selected from the group consisting of the HLA-A*0274 allele. In certain embodiments, the HLA allele is HLA-A*0201. In some embodiments, the binding protein is genetically engineered, isolated, and / or purified.

[0191] In some embodiments, the binding proteins provided herein include a constant region that is chimeric, humanized, human, primate, or rodent (e.g., rat or mouse). For example, the human variable region may be chimeric using the mouse constant region, or the mouse variable region may be humanized using the human constant region and / or human skeletal region. In some embodiments, the constant region may be mutated to modify its function (e.g., introducing non-native cysteine ​​substitutions at opposing residue positions in the TCR alpha and beta chains to provide disulfide bonds useful for increasing affinity between the TCR alpha and beta chains). Similarly, the transmembrane domain of the constant region may be mutated to modify its function (e.g., increasing hydrophobicity by introducing non-native substitutions of residues using hydrophobic amino acids).

[0192] In some embodiments, each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions, or combinations thereof compared to the reference CDR sequence.

[0193] In some embodiments, the binding protein disclosed herein may include a T cell receptor (TCR), an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). In some embodiments, the binding protein disclosed herein may include two polypeptide chains, each including a variable region comprising CDR3 of the TCR alpha chain and CDR3 of the TCR beta chain, or CDR1, CDR2, and CDR3 of both the TCR alpha chain and the TCR beta chain. In some embodiments, the binding protein may include a single-chain TCR (scTCR), which is a TCR V α and TCR V β It includes both domains, but there is only one TCR constant domain (C α or C β) and includes. The term "chimeric antigen receptor" (CAR) refers to a fusion protein engineered to include two or more naturally occurring amino acid sequences linked together in a non-naturally occurring manner or in a manner not naturally occurring in a host cell, where the fusion protein can function as a receptor when present on the surface of a cell. The CARs encompassed by the present invention include an extracellular portion comprising an antigen-binding domain (i.e., an antigen-binding domain obtained from or derived from an immunoglobulin or immunoglobulin-like molecule [e.g., an antibody or TCR], or an antigen-binding domain derived from or obtained from a killer immunoreceptor derived from a NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally including a co-stimulatory domain) (see, for example, Sadelain et al. (2013) Cancer Discov. 3:388, Harris and Kranz (2016) Trends Pharmacol. Sci. 37:220, and Stone et al. (2014) Cancer Immunol. Immunother. 63:1163).

[0194] In some embodiments, the binding proteins disclosed in the present application (e.g., a TCR, an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR)) are chimeric (e.g., comprising amino acid residues or motifs from two or more donors or species), humanized (e.g., comprising residues from non-human organisms that have been modified or substituted to reduce the risk of immunogenicity in humans), or human.

[0195] Methods for producing manipulated binding proteins (e.g., TCRs, CARs, and their antigen-binding fragments, etc.) are well known in the art (e.g., Bowerman et al. (2009) Mol. Immunol. 5:3000; U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Application Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Application Publication WO 2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al. (2007) Clin. Cancer Res. 73:5426).

[0196] In some embodiments, the binding protein described herein is a TCR or its antigen-binding fragment expressed on the cell surface, wherein the TCR expressed on the cell surface can associate with the CD3 protein more efficiently than endogenous TCRs. The binding proteins encompassed by the present invention (e.g., TCRs) exhibit higher surface expression on the surface of cells such as T cells compared to endogenous binding proteins (e.g., endogenous TCRs) when expressed on the surface of said cells. In some embodiments, a CAR is provided herein, wherein the binding domain of the CAR includes an antigen-specific TCR binding domain (see, for example, Walseng et al. (2017) Scientific Reports 7:10713).

[0197] Furthermore, the V disclosed in this application may be used as a starting material for manipulating modified binding proteins, whose properties may have changed from the starting binding protein. α and / or V β Modified binding proteins (e.g., TCR, antigen-binding fragments of TCR, or CARs) that may be prepared according to well-known methods using binding proteins having one or more of the sequences are also provided. α and / or V βThe binding protein may be manipulated by modifying one or more residues within, for example, one or more CDR regions and / or one or more skeletal regions. Additionally or alternatively, the binding protein may be manipulated by modifying residues within the constant region.

[0198] Another type of modification of the variable region is V α and / or V β The objective is to mutate amino acid residues within the CDR1, CDR2, and / or CDR3 regions of the protein by thereby improving one or more binding properties (e.g., affinity) of the binding protein of interest. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on protein binding, or other functional properties of interest, may be evaluated in vitro or in vivo assays as described in this application and provided in the examples. In some embodiments, conservative modifications (as described above) may be introduced. The mutations may be amino acid substitutions, additions, or deletions. In some embodiments, the mutations are substitutions. Furthermore, typically, one, two, three, four, or five or fewer residues within the CDR region are modified.

[0199] In some embodiments, the binding proteins described herein (e.g., TCR, antigen-binding fragment of TCR, or CAR) may have one or more amino acid substitutions, deletions, or additions compared to naturally occurring TCRs. In some embodiments, each CDR of the binding protein may have up to five amino acid substitutions, insertions, deletions, or combinations thereof compared to a reference CDR sequence. Conservative amino acid substitutions are well known and may be naturally occurring, or may be introduced when the binding protein is produced by recombination. Amino acid substitutions, deletions, and additions may be introduced into proteins using mutagenesis methods known in the art (e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, NY). Modified polynucleotides having specific codons modified according to desired substitutions, deletions, or insertions may be provided using oligonucleotide-targeted site-specific (or segment-specific) mutagenesis methods. Alternatively, immunogen polypeptide variants may be prepared using random mutagenesis techniques or saturated mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-targeted mutagenesis (see, for example, Sambrook et al. cited above).

[0200] Various criteria known to those skilled in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conserved (or analogous). For example, an analogous amino acid substitution or a conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having an analogous side chain. Analogous amino acids may be included in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline (which is considered more difficult to classify) shares properties with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some embodiments, substituting glutamine for glutamic acid or asparagine for aspartic acid can be considered similar substitutions, in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As is understood in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequence of the first polypeptide and the conserved amino acid substitutions therefor with the sequence of the second polypeptide (e.g., GENEWORKS). TM Determine using the Align, BLAST algorithm, or other algorithms described in this application and implemented in the art.

[0201] In any embodiment described herein, the encoded binding protein (e.g., TCR, antigen-binding fragment of TCR, or CAR) may include a “signal peptide” (also known as a leader sequence, leader peptide, or transit peptide). The signal peptide targets the newly synthesized polypeptide to an appropriate location inside or outside the cell. The signal peptide may be removed from the polypeptide during or after localization or secretion. The polypeptide having the signal peptide is referred to herein as a “pre-protein,” and the polypeptide from which the signal peptide has been removed is referred to herein as a “mature” protein or polypeptide. In some embodiments, the binding protein described herein (e.g., TCR, antigen-binding fragment of TCR, or CAR) may include a mature V α Domain, mature V β The domain, or both. In some embodiments, the binding protein described herein (e.g., TCR, antigen-binding fragment of TCR, or CAR) comprises a mature TCR β-chain, a mature TCR α-chain, or both.

[0202] In some embodiments, the binding protein is a fusion protein comprising: (a) an extracellular component comprising a TCR or its antigen-binding fragment; (b) an intracellular component comprising an effector domain or its functional portion; and (c) a transmembrane domain linking the extracellular and intracellular components. In some embodiments, the fusion protein can specifically bind to an MHC-peptide antigen complex containing the peptide epitope described herein within an MHC molecule (e.g., an MHC class I molecule).

[0203] As used in this application, “effector domain” or “immune effector domain” is an intracellular portion or domain of a fusion protein or receptor that may directly or indirectly promote an immune response in a cell when it receives an appropriate signal. In some embodiments, the effector domain is derived from an immune cell protein or portion thereof or immune cell protein complex that receives a signal when it binds (e.g., CD3ζ) or when the immune cell protein or portion thereof or immune cell protein complex directly binds to a target molecule, thereby inducing signal transduction from the effector domain in an immune cell.

[0204] An effector domain may directly promote a cellular response if it contains one or more signaling domains or motifs (e.g., intracellular tyrosine-based activation motifs (ITAMs) [e.g., motifs found in co-stimulatory molecules]). While we do not wish to be bound by theory, ITAMs are thought to be beneficial for T cell activation after ligand engagement by T cell receptors or by fusion proteins containing T cell effector domains. In some embodiments, the intracellular component or its functional site includes an ITAM. Examples of immunoeffector domains include, but are not limited to, immunoeffector domains derived from CD3ε, CD3δ, CD3ζ, CD25, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, Wnt, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof. In some embodiments, the effector domain includes a lymphocyte receptor signaling domain (e.g., CD3ζ, or a functional site or variant thereof).

[0205] In further embodiments, the intracellular components of the fusion protein include a co-stimulatory domain or a functional portion thereof, selected from ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD2, CD5, ICAM-l (CD54), LFA-l (CD11a / CD18), ICOS (CD278), GITR, CD30, CD40, BAFF-R, HVEM, LIGHT, MKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83, or functional variants thereof, or any combination thereof. In some embodiments, the intracellular components include the CD28 co-stimulatory domain, or a functional portion or variant thereof (these may optionally include the LL-GG mutation at position 186-187 of the native CD28 protein [e.g., Nguyen et al. (2003) Blood 702:4320]), the 4-1BB co-stimulatory domain, or a functional portion or variant thereof, or both.

[0206] In some embodiments, the effector domain includes the CD3ε end domain or its functional (e.g., signaling) portion, or its functional variant. In further embodiments, the effector domain includes the CD27 end domain or its functional (e.g., signaling) portion, or its functional variant. In further embodiments, the effector domain includes the CD28 end domain or its functional (e.g., signaling) portion, or its functional variant. In even further embodiments, the effector domain includes the 4-1BB end domain or its functional (e.g., signaling) portion, or its functional variant. In even further embodiments, the effector domain includes the OX40 end domain or its functional (e.g., signaling) portion, or its functional variant. In even further embodiments, the effector domain includes the CD2 end domain or its functional (e.g., signaling) portion, or its functional variant. In further embodiments, the effector domain includes the CD5 end domain or its functional (e.g., signaling) portion, or its functional variant. In further embodiments, the effector domain includes the ICAM-1 end domain or its functional (e.g., signaling) portion, or its functional variant. In further embodiments, the effector domain includes the LFA-1 end domain or its functional (e.g., signaling) portion, or its functional variant. In further embodiments, the effector domain includes the ICOS end domain or its functional (e.g., signaling) portion, or its functional variant.

[0207] The extracellular and intracellular components encompassed by the present invention are linked by transmembrane domains. A “transmembrane domain,” as used in this application, is a portion of a transmembrane protein that can be inserted into or penetrate the cell membrane. A transmembrane domain has a three-dimensional structure that is thermodynamically stable within the cell membrane and generally ranges in length from about 15 to about 30 amino acids. The structure of a transmembrane domain may include an alpha-helix, beta-barrel, beta-sheet, beta-helix, or any combination thereof. In some embodiments, the transmembrane domain includes or is derived from known transmembrane proteins (e.g., CD4 transmembrane domain, CD8 transmembrane domain, CD27 transmembrane domain, CD28 transmembrane domain, or any combination thereof).

[0208] In some embodiments, the extracellular components of the fusion protein further include a linker positioned between the binding domain and the transmembrane domain. When referring to the components of the fusion protein to which the binding domain and the transmembrane domain are linked, as used in this application, the “linker” may be an amino acid sequence having about 2 to about 500 amino acids, which may provide flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules linked by the linker. For example, a linker as encompassed by the present invention may position the binding domain away from the surface of a host cell expressing the fusion protein, thereby enabling proper contact, antigen binding, and activation between the host cell and the target cell (Patel et al. (1999) Gene Therapy 6:412-419). The linker length can be varied to maximize antigen recognition based on the selected target molecule, selected binding epitope, or the captureability and affinity of the antigen-binding domain (e.g., Guest et al. (2005) Immunother. 28:203-11 and PCT application publication WO 2014 / 031687). An example linker is Gly x Ser y Examples include linkers having a glycine-serine amino acid chain with 1 to approximately 10 repeats, where x and y are each independent integers from 0 to 10, except that both x and y are not 0 (e.g., (Gly4Ser)2, (Gly3Ser)2, Gly2Ser, or combinations thereof [e.g., ((Gly3Ser)2Gly2Ser) etc.]).

[0209] In some embodiments, the binding substructures encompassed by the present invention may be an engineered protein scaffold, an antibody or its antigen-binding fragment, a TCR-mimicking antibody, etc. Such binding substructures may be designed and / or fabricated for the peptides and / or MHC-peptide complexes described in this application using routine immunological methods, such as immunizing a host, obtaining antibody-producing cells and / or antibodies, and constructing hybridomas useful for producing monoclonal antibodies (e.g., Watt et al. (2006) Nat. Biotechnol. 24:177-183; Gebauer and Skerra (2009) Curr. Opin. Chem Biol. 13:245-255; Skerra et al. (2008) FEBS J. 275:2677-2683; Nygren et al. (2008) FEBS J. 275:2668-2676; Dana et al. (2012) Exp. Rev. Mol. Med. 14:e6; Sergeva et al. (2011) Blood 117:4262-4272; PCT Publications WO 2007 / 143104, PCT / US86 / 02269, and WO 86 / 01533; US Patent No. 4,816,567; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, SL(1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; US Patent No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060). If desired, the binding substructure may be isolated or purified using conventional methods such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, and high-performance liquid chromatography (HPLC) (e.g., *Current Protocols in Immunology*, or *Current Protocols in Protein Science*, John Wiley & Sons, NY, NY).

[0210] The terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric antibodies, and humanized antibodies, as well as multispecific antibodies, and fragments and derivatives relating to all of the above (these fragments and derivatives have at least an antigen-binding site). Antibody derivatives may include proteins or chemical substructures conjugated to antibodies.

[0211] Furthermore, intrabodies are well-known antigen-binding molecules that possess antibody properties but can be expressed intracellularly to bind to and / or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). For example, methods for adapting antibodies to target (e.g., inhibit) intracellular substructures are well known in the art, such as the use of single-chain antibodies (scFv), modification of the immunoglobulin VL domain for hyperstability, modification of antibodies to resist reductive intracellular environments, and the creation of fusion proteins that increase intracellular stability and / or regulate intracellular localization. For example, intracellular antibodies may also be introduced and expressed in one or more cells, multicellular organisms, tissues or organs for preventive and / or therapeutic purposes (e.g., as gene therapy) (at least PCT publications WO 08 / 020079, WO 94 / 02610, WO 95 / 22618, and WO 03 / 014960; U.S. Patent No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag publs.); Kontermann (2004) Methods 34:163-170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412; (See Shaki-Loewenstein et al. (2005) J. Immunol. Meth. 303:19-39).

[0212] As used in this application, the term “antibody” also includes the “antigen-binding portion” (or simply “antibody portion”) of an antibody. As used in this application, the term “antigen-binding portion” refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., a peptide and / or MHC-peptide complex as described in this application). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the antibody term "antigen-binding region" include: (i) a monovalent fragment consisting of the Fab fragment, VL, VH, CL, and CH1 domains; (ii) a bivalent fragment containing two Fab fragments linked by disulfide crosslinking in the hinge region, the F(ab')2 fragment; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of the VH domain; and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be linked by a synthetic linker that allows them to be produced as a single protein chain [in which the VL and VH regions pair up to form a monovalent polypeptide (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778)]. Such single-chain antibodies are also intended to be included within the aforementioned terminology of antibodies as "antigen-binding portion".To generate expression vectors encoding complete IgG polypeptides or other isotypes, any VH and VL sequences of a particular scFv may be ligated to the cDNA or genomic sequence of the constant region of a human immunoglobulin. VH and VL may also be used when generating Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single-chain antibodies, such as diabodies, are also included. Diabody is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used, thereby causing the domains to pair with complementary domains on another chain, thereby creating two antigen-binding sites (see, for example, Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0213] Furthermore, an antibody or its antigen-binding moiety may be part of a larger immunoadhesive polypeptide formed by the covalent or noncovalent association of the antibody or antibody moiety with one or more other proteins or peptides. Examples of such immunoadhesive polypeptides include the preparation of tetrameric scFv polypeptides using streptavidin core regions (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the preparation of divalent and biotinylated scFv polypeptides using cysteine ​​residues, protein subunit peptides, and C-terminal polyhistidine tags (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). The antibody moieties (e.g., Fab and F(ab')2 fragments) may be prepared from the whole antibody, for example, by conventional techniques such as papain digestion or pepsin digestion, respectively. Furthermore, antibodies, antibody portions, and immunoadhesive polypeptides may be obtained using standard recombinant DNA technology as described in this application.

[0214] Antibodies may be polyclonal or monoclonal; heterogeneous, homogeneous, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies may also be entirely human. Preferably, the antibodies encompassed by this application bind specifically or substantially specifically to the peptides and / or MHC-peptide complexes described herein. The terms “monoclonal antibody” and “monoclonal antibody composition” as used in this application refer to a group of antibody polypeptides containing only one type of antigen-binding site capable of eliciting an immune response with a specific epitope of an antigen, while the terms “polyclonal antibody” and “polyclonal antibody composition” refer to a group of antibody polypeptides containing multiple types of antigen-binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity to the specific antigen with which they elicit an immune response.

[0215] Similar to other binding substructures described in this application, antibodies may also be “humanized,” which is intended to include antibodies produced by non-human cells having variable and constant regions that have been modified to more closely resemble antibodies produced by human cells. For example, by modifying the amino acid sequence of a non-human antibody to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies as encompassed by this application may include amino acid residues not encoded by human germline immunoglobulin sequences (mutations introduced, for example, by random mutagenesis or site-directed mutagenesis in vitro, or by somatic mutation in vivo) in the CDR. As used in this application, the term “humanized antibody” also includes antibodies in which a CDR sequence derived from the germline of another mammalian species has been transferred and conjugated to a human skeletal sequence.

[0216] In some embodiments, the binding protein encompassed by the present invention may be covalently bound to a substructure. In some embodiments, the covalently bound substructure includes an affinity tag or label. The affinity tag may be selected from the group consisting of glutathione-S-transferase (GST), calmodulin-binding protein (CBP), protein C tag, Myc tag, HaloTag, HA tag, Flag tag, His tag, biotin tag, and V5 tag. The label may be a fluorescent protein. In some embodiments, the covalently bound substructure may be selected from the group consisting of inflammatory agents, anti-inflammatory agents, cytokines, toxins, cytotoxic molecules, radioisotopes, or antibodies such as single-chain Fv.

[0217] The binding protein may be conjugated to drugs used in imaging, research, therapy, theranostics, pharmaceuticals, chemotherapy, chelation therapy, targeted drug delivery, and radiotherapy. In some embodiments, the binding protein may be conjugated or fused with detectable drugs, such as fluorescent dye molecules, near-infrared dyes, contrast agents, nanoparticles, metal-containing nanoparticles, metal chelates, X-ray contrast agents, PET agents, metals, radioisotopes, dyes, radionuclide chelators, or other suitable substances that may be used for imaging. In some embodiments, detectable substructures 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more may be linked to the binding protein. Non-limiting examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212. In some embodiments, the near-infrared dye does not readily quench with biological tissues and fluids. In some embodiments, the fluorescent dye molecule is a fluorescent agent that emits electromagnetic waves at wavelengths between 650 nm and 4000 nm, and such electromagnetic waves are used to detect such an agent. Non-exclusive examples of fluorescent dyes that may be used as conjugate molecules include DyLight-680, DyLight-750, VivoTag-750, DyLight-800, IRDye-800, VivoTag-680, Cy5.5, ZQ800, or indocyanine green (ICG).In some embodiments, cyanine dyes (e.g., Cy7, Cy5.5, and Cy5) are often used as near-infrared dyes. Additional non-limiting examples of fluorescent dyes for use as conjugate molecules according to the present invention include acradine orange or yellow and Alexa Fluors. (登録商標) (For example, Alexa Fluor (登録商標) 790, 750, 700, 680, 660, and 647) and any derivative thereof, 7-actinomycin D, 8-anilinonaphthalene-1-sulfonic acid, ATTO (登録商標) Dynes and their derivatives, auramine-rhodamine stains and their derivatives, bensantrone, biman, 9-10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)nathacene, bisbenzimide, brainbow, calcein, carbodyfluorescein and their derivatives, 1-chloro-9,10-bis(phenylethynyl)anthracene and its derivatives, DAPI, DiOC6, DyLight (登録商標) Fluors (登録商標) and any derivative thereof, epicocconone, ethidium bromide, FlAsH-EDT2 (登録商標) Fluo dyes and any derivatives thereof, FluoProbe (登録商標) and any derivative thereof, fluorescein and any derivative thereof, Fura (登録商標) and any derivative thereof, GelGreen (登録商標) and any derivative thereof, GelRed (登録商標)and any derivatives thereof, fluorescent proteins and any derivatives thereof, for example, m-isoform proteins such as mCherry and any derivatives thereof, hetamethine dye and any derivatives thereof, Hoechst stain, iminocoumarin, Indian yellow, indo-1 and any derivatives thereof, laurdan, Lucifer yellow and any derivatives thereof, luciferin and any derivatives thereof, luciferase and any derivatives thereof, mercocyanin and any derivatives thereof, Nile dye and any derivatives thereof, perylene, phloxine, phyco dye (phyco Examples include dyes and any derivatives thereof, propium iodide, pyranine, rhodamine and any derivatives thereof, ribogreen, RoGFP, rubrene, stilbene and any derivatives thereof, sulforhodamine and any derivatives thereof, SYBR and any derivatives thereof, synapto-pHluorin, tetraphenyl butadiene, tetrasodium tris, Texas Red, Titanium Yellow, TSQ, umbrellaferone, violanthrone, yellow fluorescent protein, and YOYO-1.Other suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxyfluorescein, 6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxylrhodamine 6G, carboxy-X-rhodamine (ROX), lysamine-rhodamine B, rhodamine 6G, rhodamine green, rhodamine red) Red), tetramethylrhodamine (TMR), etc., coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green. TM ) Pigments (e.g., Oregon Green) TM 488, 500, 514, etc.), Texas Red 登録商標 ), Texas Red 登録商標 )-X, Spectrum Red 登録商標 ), Spectrum Green 登録商標 ), cyanine pigments (e.g., CY-3, Cy-5, CY-3.5, CY-5.5, etc.), Alexa Fluor 登録商標 Pigments (for example, Alexa Fluor 登録商標 350, 488, 532, 546, 568, 594, 633, 660, 680, etc.), BODIPY 登録商標 Pigments (for example, BODIPY 登録商標 FL, R6G, TMR, TR, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, etc.), IRD dyes (e.g., IRD40) TM IRD700 TM IRD800 TMExamples include, etc. Further suitable detectable agents are well known in the art (e.g., PCT publication number PCT / US14 / 56177). Non-limited examples of radioisotopes include alpha emitters, beta emitters, positron emitters, and gamma emitters. In some embodiments, the metal or radioisotope is selected from the group consisting of actinium, americium, bismuth, cadmium, cesium, cobalt, europium, gadolinium, iridium, lead, lutetium, manganese, palladium, polonium, radium, ruthenium, samarium, strontium, technetium, thallium, and yttrium. In some embodiments, the metal is actinium, bismuth, lead, radium, strontium, samarium, or yttrium. In some embodiments, the radioisotope is actinium-225 or lead-212.

[0218] The binding protein may be conjugated to a radiosensitizer or photosensitizer. Examples of radiosensitizers, but not limited to, include: ABT-263, ABT-199, WEHI-539, paclitaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, etanidazole, misonidazole, tirapazamine, and nucleic acid base derivatives (e.g., halogenated purines or pyrimidines such as 5-fluorodeoxyuridine). Examples of photosensitizers, but not limited to, include fluorescent molecules or beads that generate heat upon irradiation, nanoparticles, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines), metalloporphyrins, metallophthalocyanines, angelicins, and chalcogenapyrrillium dyes. Examples include dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphirins, texapphirins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives, naphthalimides, Nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrelins, and cercosporines), psoralens, quinones, retinoids, rhodamines, thiophenes, verzins, xanthene dyes (e.g., eosins, erythrosines, rose bengal), dimers and oligomers of porphyrins, and prodrugs such as 5-aminolevulinic acid. Advantageously, this method makes it possible to target cells of interest (e.g., immune cells) with great specificity by simultaneously using both therapeutic agents (e.g., drugs) and electromagnetic energy (e.g., irradiation or light). In some embodiments, the binding protein fuses with the drug, or links covalently or non-covalently, for example, directly or via a linker.

[0219] In some embodiments, the binding protein may be chemically modified. For example, mutations may be introduced into the binding protein to modify peptide properties such as detectability, stability, in vivo distribution, pharmacokinetics, half-life, surface charge, hydrophobicity, conjugation site, pH, and function. N-methylation is one example of methylation that may occur in the binding protein encompassed by the present invention. In some embodiments, the binding protein may be modified by methylation on a free amine, for example, by reductive methylation with formaldehyde and sodium cyanoborohydride.

[0220] Chemical modifications may include polymers, polyethers, polyethylene glycol, biopolymers, amphoteric polymers, polyamino acids, fatty acids, dendrimers, Fc regions, monosaturated carbon chains such as palmitic acid or myristoleic acid, or albumin. Chemical modifications of the binding protein by the Fc region may result in a fusion Fc-protein. Examples of polyamino acids include polyamino acid sequences in which single amino acids are repeated (e.g., polyglycine), polyamino acid sequences having mixed polyamino acid sequences that may or may not follow a pattern, or any combination of the above.

[0221] In some embodiments, the binding protein encompassed by the present invention may be modified. In some embodiments, the modification generates a functional variant that has substantial or significant sequence identity with the parent binding protein and maintains one or more biophysical and / or biological activities of the parent binding protein (e.g., maintains binding specificity). In some embodiments, the mutation is a conserved amino acid substitution.

[0222] In some embodiments, the binding proteins encompassed by the present invention may include synthetic amino acids instead of one or more natural amino acids. Such synthetic amino acids are well known in the art and, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1, 2, 3, Examples include 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane-carboxylic acid, OC-aminocyclohexane-carboxylic acid, α-aminocycloheptane-carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and OC-tert-butylglycine.

[0223] The binding proteins encompassed by the present invention may be modified, for example, by glycosylation, amidation, carboxylation, phosphorylation, esterification, N-acylation, cyclization (e.g., via disulfide crosslinking), conversion to an acid addition salt, and / or optionally dimerization, polymerization, or conjugation.

[0224] In some embodiments, the half-life of the peptides encompassed by the present invention may be extended by attaching hydrophobic substructures, for example, to the N-terminus, C-terminus, or internal amino acids. In other embodiments, the binding protein may include post-translational modifications (e.g., methylation and / or amidation) that may affect the serum half-life. In some embodiments, a simple carbon chain may be conjugated to the binding protein (e.g., by myristoylation and / or palmitylation). In some embodiments, the simple carbon chain may allow the binding protein to be easily separated from the unconjugated substance. For example, methods that may be used to separate the binding protein from the unconjugated substance include, but are not limited to, solvent extraction and reverse-phase chromatography. Lipid-soluble substructures may extend the half-life via reversible binding to serum albumin. The conjugated substructure may be a lipid-soluble substructure that extends the peptide's half-life via reversible binding to serum albumin. In some embodiments, the lipid-soluble substructure may be cholesterol or cholesterol derivatives, such as cholestanes, cholestanes, cholestadienes, and oxysterols. In some embodiments, the binding protein may be conjugated to myristic acid (tetradecanoic acid) or its derivatives. In other embodiments, the binding protein may be coupled (e.g., conjugated) to a half-life-modifying agent. Examples of half-life-modifying agents include, but are not limited to, polymers, polyethylene glycol (PEG), hydroxyethyl starch, polyvinyl alcohol, water-soluble polymers, amphoteric water-soluble polymers, water-soluble poly(amino acids), water-soluble polymers of proline, alanine, and serine, water-soluble polymers containing glycine, glutamic acid, and serine, Fc domains, fatty acids, palmitic acid, or molecules that bind to albumin.In some embodiments, the spacer or linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues, etc., which may couple to the binding protein and act as a spacer or linker to facilitate conjugate or fusion to another molecule, and to facilitate cleavage of the peptide from such conjugated or fused molecule. In some embodiments, the binding protein may be conjugated to, for example, another substructure that can alter or change the properties of the binding protein.

[0225] The binding protein may be generated recombinantly or synthetically, for example, by solid-phase peptide synthesis or liquid-phase peptide synthesis. Polypeptide synthesis may be carried out by known synthetic methods, for example, using fluorenylmethyloxycarbonyl (Fmoc) chemistry or butyloxycarbonyl (Boc) chemistry. Polypeptide fragments may be linked together enzymatically or synthetically.

[0226] In embodiments encompassed by the present invention, the present application provides a method for producing the binding protein described herein, the method comprising the steps of: (i) culturing transformed host cells, which have been transformed with a nucleic acid comprising a sequence encoding the binding protein described herein, under conditions suitable for enabling the expression of the binding protein; and (ii) recovering the expressed binding protein.

[0227] Useful methods for isolating and purifying recombinantly produced binding proteins may include, for example, obtaining a supernatant from a host cell / vector system suitable for secreting the binding protein into a culture medium, and then concentrating the medium using a commercially available filter. After concentration, the concentrate may be applied to a single suitable purification matrix or a series of suitable matrices, such as affinity matrices or ion exchange resins. Recombinant polypeptides may be further purified using one or more reverse-phase HPLC steps. These purification methods may also be used to isolate immunogens from their natural environment. Methods for producing one or more binding proteins on a large scale as described in this application may include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the binding proteins may be carried out according to methods described in this application and known in the art.

[0228] Various assays are known for evaluating binding affinity and / or determining whether a binding molecule specifically binds to a particular ligand (e.g., a peptide antigen-MHC complex). Determining the binding affinity of a binding protein to a target polypeptide, such as a T cell peptide epitope, is within the scope of the art, for example, by using any of the many binding assays known in the art. For example, in some embodiments, Biacore TM An instrument may be used to determine the binding constant of a complex between two proteins. The dissociation constant (K) of the complex. D) may be determined by monitoring the change in refractive index over time as the buffer passes over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), or measurement of binding by monitoring changes in the spectroscopic or optical properties of the protein by fluorescence, UV absorption, circular dichroism or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore TM ) analysis (see, for example, Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660, Wilson (2002) Science 295:2103, Wolff et al. (1993) Cancer Res. 53:2560, and U.S. Pat. Nos. 5,283,173 and 5,468,614), flow cytometry, sequencing and other methods for detecting expressed nucleic acids. In one example, the apparent affinity for a target is measured by flow cytometry using labeled multimers, for example, MHC-antigen peptide multimers, by evaluating binding to tetramers at various concentrations. In one representative example, the apparent K D of the binding protein is measured over a concentration range using serial dilutions of the labeled tetramer, followed by determination of the binding curve by non-linear regression. The apparent K D is determined as the concentration of ligand that produces half-maximal binding.

[0229] VII. USES AND METHODS a. Diagnostic methods In some embodiments, the present application provides a diagnostic method for determining whether a subject has been exposed to SARS-CoV-2 and / or has protection from SARS-CoV-2, including: (a) incubating a sample obtained from the subject (e.g., blood, isolated PBMCs, or isolated T cells) with cells encoding and / or presenting SARS-CoV-2 immunogenic peptides described in the present application (e.g., peptide epitopes selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F), MHC-peptide complexes described in the present application, or MHC-peptide complexes described in the present application derived from, for example, an immunogenic polypeptide construct described in the present application; and (b) detecting a level of responsiveness, where a higher level of responsiveness compared to a control level indicates that the subject has been exposed to SARS-CoV-2 and / or has protection from SARS-CoV-2.

[0230] In some embodiments, the level of responsiveness is indicated by T cell activation or effector function, such as, but not limited to, T cell proliferation, injury, or cytokine release. The control level may be a reference figure or level for healthy subjects who have not been exposed to SARS-CoV-2.

[0231] b. Treatment method In some embodiments, the present application provides methods for preventing COVID-19 (i.e., SARS-CoV-2 infection), and / or treating COVID-19, and / or inducing an immune response to the SARS-CoV-2 protein or fragments thereof. In certain embodiments, the method includes the step of administering an immunogenic composition described in the present application to a subject.

[0232] The methods described herein may be used to treat any subject in need thereof. As used herein, “subject in need thereof” includes any subject having COVID-19, any subject having had COVID-19 in the past, and / or any subject susceptible to COVID-19. For example, in some embodiments, the subject has COVID-19. In some embodiments, the subject is receiving treatment for COVID-19. In some embodiments, the subject has a deficiency in the immune system or other serious underlying condition that makes the subject susceptible to COVID-19 due to aging or makes the subject susceptible to COVID-19.

[0233] The pharmaceutical compositions disclosed in this application may be delivered by any preferred route of administration, such as orally and parenterally. In certain embodiments, the pharmaceutical compositions are delivered generally (for example, by oral or parenteral administration). In certain embodiments, the pharmaceutical compositions are administered by subcutaneous injection.

[0234] The dosage of the drug may be determined by referring to its plasma concentration. For example, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity (AUC(0-4)) may be used. The dosage includes the dosage that yields the above values ​​for Cmax and AUC(0-4), as well as other dosages that yield larger or smaller values ​​for those parameters.

[0235] The actual dosage level of the active ingredient in the pharmaceutical composition may be modified to obtain an amount of the active ingredient that is not toxic to the patient and is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration.

[0236] The selected dosage level depends on various factors, such as the activity of the specific agent used, the route of administration, the timing of administration, the rate of excretion or metabolism of the specific compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the specific compound used, the age, sex, weight, symptoms, overall health, and medical history of the patient being treated, and similar factors known in the medical field.

[0237] A physician or veterinarian with ordinary skill can easily determine and prescribe the effective amount of a required pharmaceutical composition. For example, such a physician or veterinarian may prescribe and / or administer a dose of the agent used in the pharmaceutical composition at a lower level than required to achieve the desired therapeutic effect, and may gradually increase the dosage until the desired effect is achieved.

[0238] Generally, the preferred daily dose of the agent described in this application is the minimum dose of the agent that is effective in producing a therapeutic effect. Such an effective dose generally depends on the factors mentioned above.

[0239] In some embodiments, the immunogenic composition contains a certain amount of SRS-CoV-2 immunogenic peptide in combination with an adjuvant constituting a drug dosing unit. A drug dosing unit is defined in this application as the amount of active ingredient (e.g., SRS-CoV-2 immunogenic peptide and / or adjuvant) applied to a subject at a given time. A drug dosing unit may be applied to a subject in a single volume (e.g., a single shot), or in two, three, four, five or more separate volumes or shots applied to different locations on the body (e.g., the right and left limbs). There may be multiple reasons for applying a single drug dosing unit in separate volumes, such as avoiding negative side effects, avoiding antigen competition and / or considerations for compositional analysis. It should be understood in this application that separate volumes of pharmaceutical dosages may have different compositions, i.e., they may contain different types or compositions of active ingredients and / or adjuvants.

[0240] A drug dosage unit may be an effective dose or a portion of an effective dose. In this application, “effective dose” should be understood as the amount or dosage of the active ingredient required to prevent and / or reduce the symptoms of a disease (e.g., COVID-19) compared to an untreated subject. The effective dose of the active compound used to carry out the present invention for the prophylactic and / or therapeutic treatment of COVID-19 will vary depending on the mode of administration, the age, weight, and overall health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate dose and dosage regimen. Such a dose is referred to as the “effective” dose. This effective dose may also be the amount that can induce an effective cellular T cell response, or more preferably an effective systemic cellular T cell response, in the subject to be treated.

[0241] In one aspect, the present application provides a method for inducing an immune response in a subject to cells infected with the SARS-CoV-2 virus. The method comprises the steps of: administering a pharmaceutical composition described in the present application to the subject, wherein the pharmaceutical composition, upon administration to the subject, induces an immune response to cells infected with the SARS-CoV-2 virus.

[0242] Generally, the aforementioned immune responses include humoral immune responses, cell-mediated immune responses, or both.

[0243] Humoral responses may be measured by standard immunoassays for antibody levels in serum samples from subjects administered the pharmaceutical composition. Cellular immune responses are T cell-involved responses and may be measured in vitro or in vivo. For example, a general cellular immune response may be measured as T cell proliferation activity in cells (e.g., peripheral blood lymphocytes (PBLs)) sampled from the subject at a suitable time after administration of the pharmaceutical composition. For example, after incubating PBMCs with a stimulant for an appropriate period of time, 3 [H]thymidine uptake may be measured. A subset of proliferating T cells can be determined using flow cytometry.

[0244] In certain embodiments, the method provided in this application includes a step of administering to both human and non-human mammals. Veterinary use is also intended. In some embodiments, the subject may be any organism in which an immune response can be induced. Examples of subjects, but not limited to, include humans, livestock, dogs, cats, mice, rats, and their genetically modified species.

[0245] In some embodiments, the pharmaceutical composition may be administered at any appropriate time. For example, the administration step may be performed before or during treatment of a subject having COVID-19, and may be continued after the SARS-CoV-2 infection is no longer clinically detectable. The administration step may also be continued in subjects showing signs of relapse.

[0246] In some embodiments, the pharmaceutical composition may be administered in a therapeutically or prophylactically effective amount. The step of administering the pharmaceutical composition to the subject may be carried out using known procedures and in a dosage and duration sufficient to achieve the desired effect.

[0247] In some embodiments, the pharmaceutical composition may be administered to the subject at any preferred site. The route of administration may be parenteral, intramuscular, subcutaneous, intradermal, intraperitoneal, intranasal, intravenous (e.g., via an indwelling catheter), via afferent lymphatic vessels, or any other appropriate route considering the condition of the subject. Preferably, the dose is administered in an amount and duration effective in producing the desired response, to induce the immune response or to induce prophylactic or therapeutic treatment of SARS-CoV-2 infection and / or related symptoms.

[0248] The pharmaceutical composition may be administered prior to, before, or concurrently with other therapies, such as therapies that also induce an immune response in the subject. For example, the subject may have been treated beforehand or concurrently with other forms of immunomodulators, the other therapies preferably provided in a manner that does not interfere with the immunogenicity of the composition described herein.

[0249] The administration step may be appropriately timed by the care provider (e.g., physician, veterinarian) and may depend on the clinical symptoms of the subject, the purpose of administration, and / or other therapies intended or administered. In some embodiments, an initial dose may be administered and the subject may be monitored for immunological and / or clinical responses. A preferred method of immunological monitoring includes the step of using the patient's peripheral blood lymphocytes (PBLs) as responders and the immunogenic peptides or MHC-peptide complexes described herein as stimulants. Immunological responses may also be measured by delayed inflammatory responses at the administration site. One or more doses following the initial dose may be administered appropriately, typically monthly, nearly monthly, or weekly, until the desired effect is achieved. Thereafter, booster doses or maintenance doses may be administered as needed, particularly if the immunological or clinical benefits appear to be diminishing.

[0250] c. Methods for identifying molecules that bind to peptides within MHC molecules In some embodiments, the present application provides a method for identifying peptide-binding molecules or antigen-binding fragments thereof that bind to peptide epitopes selected from Tables 1A, 1B, 1C, 1D, 1E, and / or 1F.

[0251] In some embodiments, the peptide-binding molecule, i.e., the MHC-peptide-binding molecule, is a molecule or portion thereof that retains the ability to bind (e.g., specifically bind) to a peptide epitope (MHC-peptide complex) that is presented or displayed, for example, within an MHC molecule on the surface of a cell. Examples of peptide-binding molecules include T cell receptors or antibodies that exhibit specific binding activity to MHC-peptide complexes, or their antigen-binding portions, such as their single-chain immunoglobulin variable regions (e.g., scTCR, scFv). In some embodiments, the peptide-binding molecule is a TCR or its antigen-binding fragment. In some embodiments, the peptide-binding molecule is an antibody, such as a TCR-like antibody, or its antigen-binding fragment. In some embodiments, the peptide-binding molecule is a TCR-like CAR, including an antibody or its antigen-binding fragment, such as a TCR-like antibody, for example, one that has been modified to bind to an MHC-peptide complex. In some embodiments, the peptide-bonded molecules may be derived from natural sources, or may be produced partially or entirely synthetically or recombinantly.

[0252] In some embodiments, binding molecules that bind to peptide epitopes may be identified by contacting one or more candidate peptide binding molecules (e.g., one or more candidate TCR molecules, antibodies, or their antigen-binding fragments) with an MHC-peptide complex and evaluating whether each of the candidate binding molecules binds to the MHC-peptide complex (e.g., whether it binds specifically). The method may be performed in vitro, ex vivo, or in vivo. The method is well known in the art for screening, for example, as described in U.S. Patent Application Publication 2020 / 0102553.

[0253] In some embodiments, the method includes contacting a plurality or library of binding molecules (e.g., a plurality or library of TCRs or antibodies) with an MHC-restricted epitope, and identifying or selecting molecules that specifically bind to such an epitope. In some embodiments, a library or collection comprising a plurality of diverse binding molecules (e.g., a plurality of diverse TCRs or a plurality of diverse antibodies, etc.) may be screened or evaluated for binding to an MHC-restricted epitope. In some embodiments (e.g., for selecting antibody molecules that specifically bind to an MHC-restricted peptide), the hybridoma method may be used.

[0254] In some embodiments, a screening method may be used to contact a plurality of candidate binding molecules (e.g., a library or collection of candidate binding molecules) individually with a peptide binding molecule, either simultaneously or sequentially. Library members that specifically bind to a particular MHC-peptide complex may be identified or selected. In some embodiments, a library or collection of candidate binding molecules may comprise at least 2, 5, 10, 100, 10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、or more diverse peptide binding molecules.

[0255] In some embodiments, the method may be used to identify peptide-binding molecules (e.g., TCRs or antibodies) that exhibit binding to two or more MHC haplotypes or two or more MHC alleles. In some embodiments, the peptide-binding molecules (e.g., TCRs or antibodies) specifically bind to or specifically recognize peptide epitopes presented in multiple MHC class I haplotypes or alleles. In some embodiments, the peptide-binding molecules (e.g., TCRs or antibodies) specifically bind to or specifically recognize peptide epitopes presented in multiple MHC class II haplotypes or alleles.

[0256] Various assays are known for evaluating binding affinity and / or determining whether a binding molecule binds to a specific ligand (e.g., an MHC-peptide complex). Measuring the binding affinity of a target polypeptide to a T cell epitope of a TCR, for example, by using any of the many binding assays known in the art, is within the scope of the art. For example, in some embodiments, a BIAcore instrument may be used to determine the binding constant of a complex between two proteins. The dissociation constant (K) of the complex D This can be determined by monitoring the change in refractive index over time as the buffer passes over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA), or the measurement of binding by monitoring changes in the spectroscopic or optical properties of the protein by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays, but not limited to, include Western blotting, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance (Biacore). 登録商標) Analysis (e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; Wilson (2002) Science 295:2103; Wolff et al. (1993) Cancer Res. 53:2560; and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalent), flow cytometry, sequencing, and other methods for detecting expressed nucleic acids. In one example, the apparent affinity for TCR is measured by evaluating the binding to tetramers at various concentrations, for example, by flow cytometry using labeled tetramers. In another example, the apparent K of TCR D The apparent K is measured using a 2-fold dilution of the labeled tetramer within a certain concentration range, followed by the determination of the bond curve by non-linear regression. D This is determined as the concentration of the ligand that produces half of the maximum binding.

[0257] In some embodiments, the method may be used to identify a binding molecule that binds only when a specific peptide is present in the complex, and does not bind when the specific peptide is absent, or when another, non-overlapping, or unrelated peptide is present. In some embodiments, the binding molecule does not substantially bind to the MHC in the absence of its binding peptide, and / or does not substantially bind to the peptide in the absence of the MHC. In some embodiments, the binding molecule is at least partially specific. In some embodiments, an exemplary identified binding molecule may bind to the MHC-peptide complex when a specific peptide is present, and may also bind to the MHC-peptide complex when a related peptide having one or two substitutions for the specific peptide is present.

[0258] In some embodiments, the identified antibody (e.g., a TCR-like antibody) may be used to produce or generate a chimeric antigen receptor (CAR) containing a non-TCR antibody that specifically binds to an MHC-peptide complex.

[0259] In some embodiments, cells expressing or containing peptide-binding molecules (e.g., TCRs or TCR-like antibodies or TCR-like CARs) may be manipulated using a method for identifying the peptide-binding molecules. In some embodiments, the cells or manipulated cells are T cells. In some embodiments, the T cells are CD4+ or CD8+ T cells. In some embodiments, the peptide-binding molecules recognize MHC class I peptide complexes, MHC class II peptide complexes, and / or MHC-E peptide complexes. In some embodiments, CD8+ T cells may be manipulated using peptide-binding molecules (e.g., TCRs, antibodies, or CARs) that specifically recognize peptides in MHC class I. In some embodiments, compositions of manipulated CD8+ T cells expressing or containing TCRs, antibodies, or CARs for recognizing peptides presented in MHC class I are also provided. In any of such embodiments, the cells may be used in adoptive cell therapy methods.

[0260] In some embodiments, the TCR library may be generated by amplifying the Vα and Vβ repertoire derived from T cells isolated from a subject, including cells present in PBMCs, the spleen, or other lymphoid organs. In some cases, T cells may be amplified from tumor-infiltrating lymphocytes (TILs). In some embodiments, the TCR library may be generated from CD4+ or CD8+ cells. In some embodiments, the TCR may be amplified from a normal, healthy subject T cell source, i.e., from a normal TCR library. In some embodiments, the TCR may be amplified from a diseased subject T cell source, i.e., from a diseased TCR library. In some embodiments, the Vα and VP gene repertoire is amplified using degenerate primers, for example, by RT-PCR in a sample obtained from a human (e.g., T cells). In some embodiments, the scTv library may be assembled from naive Vα and Vβ libraries, where the amplification products are cloned or assembled so as to be separated by a linker. Depending on the source and cell of the subject, the library may be HLA allele-specific.

[0261] Alternatively, in some embodiments, a TCR library may be generated by mutagenesis or diversification of parent TCR molecules or scaffold TCR molecules. For example, in some embodiments, a subject (e.g., a human or other mammal such as a rodent) may be vaccinated with a peptide, such as the peptide identified by this method. In some embodiments, a sample, such as a sample containing blood lymphocytes, may be obtained from the subject. In some cases, a binding molecule (e.g., TCR) may be amplified from a sample (e.g., T cells contained in the sample). In some embodiments, antigen-specific T cells may be selected, for example, by screening to evaluate CTL activity to the peptide. In some embodiments, TCRs present on antigen-specific T cells may be selected, for example, by binding activity (e.g., a specific affinity or avidity to the antigen). In some embodiments, the TCRs may be subjected to targeted evolution (e.g., mutagenesis) (e.g., of the α or β chain). In some embodiments, specific residues within the CDR of the TCR may be modified. In some embodiments, the selected TCR may be modified by affinity maturation. In some embodiments, the selected TCR may be used as the parent scaffold TCR for the antigen.

[0262] In some embodiments, the subject is a human, for example, a human with COVID-19. In some embodiments, the subject is a rodent, for example, a mouse. In some such embodiments, the mouse is a genetically modified mouse, for example, a mouse expressing a human MHC (i.e., HLA) molecule (e.g., HLA-A2). See Nicholson et al. Adv Hematol. 2012; 2012: 404081.

[0263] In some embodiments, the subject is a transgenic mouse expressing human TCR or an antigen-negative mouse. See Li et al. (2010) Nat Med. 161029-1034; Obenaus et al. (2015) Nat Biotechnol. 33:402-407. In some embodiments, the subject is a transgenic mouse expressing human HLA molecules and human TCR.

[0264] In some embodiments, for example, when the subject is a genetically modified HLA mouse, the identified TCR is modified, for example, to be chimeric or humanized. In some embodiments, the TCR scaffold is modified, such as to be similar to known antibody humanization methods.

[0265] In some embodiments, such scaffold molecules are used to generate a library of TCRs.

[0266] For example, in some embodiments, the library contains a modified or manipulated TCR or its antigen-binding moiety compared to a parent TCR molecule or a scaffold TCR molecule. In some embodiments, directed evolution methods may be used to generate TCRs with altered properties, such as higher affinity for a specific MHC-peptide complex. In some embodiments, the display approach includes the step of manipulating or modifying a known, parent, or reference TCR. For example, in some cases, a wild-type TCR may be used as a template to produce a mutation-induced TCR in which one or more residues in the CDR are mutated, and a mutant having a desired altered property, such as higher affinity for a desired target antigen, is selected. In some embodiments, directed evolution is achieved by display methods, including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol 4:55-62; Holler et al. (2000) Proc Natl Acad Sci USA 97:5387-5392), phage display (Li et al. (2005) Nat Biotechnol 23:349-354), or T cell display (Chervin et al. (2008) J Immunol Methods 339:175-184), etc.

[0267] In some embodiments, the library may be soluble. In some embodiments, the library is a display library in which the TCRs are displayed on the surface of phages or cells, or bound to particles or molecules, such as cells, ribosomes, or nucleic acids, such as RNA or DNA. Typically, the TCR library (e.g., normal and disease TCR libraries, or diversified libraries, etc.) may be generated in any form (e.g., as heterodimers or as single-stranded forms, etc.). In some embodiments, one or more members of the TCR may be double-stranded heterodimers. In some embodiments, pairing of Vα and Vβ strands may be facilitated by the introduction of disulfide bonds. In some embodiments, the members of the TCR library may be single-stranded TCRs (scTv or ScTCR), and in some cases may include Vα and Vβ strands separated by a linker. Furthermore, in some cases, when TCRs are screened and selected from the library, the selected members may be generated in any form (e.g., full-length TCR heterodimers, single-chain forms, or antigen-binding fragments thereof).

[0268] Another method for identifying molecules that bind to peptides within MHC molecules is described in U.S. Patent Application No. 2020 / 0182884, which is incorporated in its entirety by reference in this application.

[0269] d. Monitoring of efficacy in clinical trials Monitoring the effects of SARS-CoV-2 therapies (e.g., compounds, drugs, vaccines, or cell therapies) on T-cell responsiveness (e.g., presence of binding, and / or T-cell activation, and / or effector function) may be applied not only during basic candidate peptide-binding molecule screening but also in clinical trials. For example, the efficacy of the SARS-CoV-2 immunogenic peptides or compositions described in this application, nucleic acids, MHC-peptide complexes encoding such SARS-CoV-2 immunogenic peptides, or cells expressing nucleic acids, vectors, immunogenic peptides, or MHC-peptide complexes, for enhancing the immune response to SARS-CoV-2 infection (e.g., T-cell immune response) may be monitored in clinical trials involving subjects with COVID-19. In such clinical trials, the presence of binding, and / or T-cell activation, and / or effector function (e.g., T-cell proliferation, injury, or cytokine release) may be used as a "readout" or marker for the phenotype of a particular cell, tissue, or system. Similarly, the efficacy of adaptive T cell therapy using T cells engineered to express the TCR determined by the screening assay described in this application, or the efficacy of adaptive T cell therapy using T cells stimulated with immunogenic peptides, MHC-peptide complexes, or cells encoding and / or presenting MHC-peptide complexes as described in this application to enhance the immune response to SARS-CoV-2 infected cells, may be monitored in clinical trials involving subjects with COVID-19. In such clinical trials, the presence of binding, and / or T cell activation, and / or effector function (e.g., T cell proliferation, injury, or cytokine release) may be used as a “readout” or marker for the phenotype of a particular cell, tissue, or system.

[0270] In one embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with a SARS-CoV-2 therapy (e.g., a compound, drug, vaccine, or cell therapy), the method comprising the steps of: a) measuring the presence or level of responsiveness between T cells obtained from a subject and one or more immunogenic polypeptides or one or more stable MHC-peptide complexes described herein, in a first sample obtained from the subject before providing at least a portion of the SARS-CoV-2 therapy to the subject; and b) measuring the presence or level of responsiveness between one or more immunogenic polypeptides or one or more stable MHC-peptide complexes described herein and T cells obtained from the subject, in a second sample obtained from the subject after providing at least a portion of the SARS-CoV-2 therapy, wherein the presence or higher level of responsiveness in the second sample compared to the first sample indicates that the therapy is effective in treating SARS-CoV-2 in the subject.

[0271] For example, increasing the prescription of the SARS-CoV-2 therapy may be desirable to increase the presence or level of responsiveness between T cells obtained from the subject and one or more immunogenic peptides or one or more stable MHC-peptide complexes described in this application, i.e., to increase the efficacy of the SARS-CoV-2 therapy. According to such embodiments, even in the absence of an observable phenotypic response, the presence or level of responsiveness between T cells obtained from the subject and one or more immunogenic peptides or one or more stable MHC-peptide complexes described in this application may be used as an indicator of the efficacy of the SARS-CoV-2 therapy. Similarly, patients receiving SARS-CoV-2 therapy may be selected by analyzing the presence or level of responsiveness between T cells and one or more immunogenic peptides or one or more stable MHC-peptide complexes described in this application, for example, by direct binding assays, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunochemical assays, Western blotting, or intracellular flow assays.

[0272] For example, in a direct binding assay, an immunogenic peptide or MHC-peptide complex may be coupled with a radioisotope label or enzyme label, and the binding may be measured by detecting the resulting labeled immunogenic peptide or MHC-peptide complex. 125 I, 35 S, 14 C, or 3The immunogenic peptide or MHC-peptide complex may be labeled directly or indirectly with H, and the radioactive isotope may be detected by direct counting of radioactivity or by scintillation counting. Alternatively, the immunogenic peptide or MHC-peptide complex may be enzymatically labeled, for example, with horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic labeling may be detected by measuring the conversion from a suitable substrate to a product. The interaction between the immunogenic peptide or MHC-peptide complex and T cells may also be measured using standard binding assays or enzymatic analysis assays. In one or more embodiments of the above assay methods, it may be desirable to immobilize the immunogenic peptide or MHC-peptide complex to adapt to the automation of the assay.

[0273] The binding of immunogenic peptides or MHC-peptide complexes to T cells may be carried out in any container suitable for containing the reaction product. Non-limiting examples of such containers include microtiter plates, test tubes, and microcentrifuge tubes. Immobilization forms of immunogenic peptides or MHC-peptide complexes described in this application also include immunogenic peptides or MHC-peptide complexes bound to a solid phase (e.g., membranes, cellulose, nitrocellulose, or glass fibers, etc.; beads, e.g., beads made of agarose, polyacrylamide, or latex, etc.; or, e.g., the surface of a dish, plate, or well made of polystyrene, etc.) such as porous, microporous (average pore diameter less than about 1 micron) or macroporous (average pore diameter greater than about 10 microns) material.

[0274] In some embodiments, T cell responsiveness to one or more immunogenic peptides or one or more stable MHC-peptide complexes means, in this application, the presence of binding and / or T cell activation and / or effector function. The term "T cell activation" refers to one or more cellular responses of cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers.

[0275] The response of T cells to one or more immunogenic peptides or one or more stable MHC-peptide complexes can be measured according to any of the T cell functional parameters described in this application (e.g., proliferation, cytokine release, cytotoxicity, changes in cell surface marker phenotype, etc.).

[0276] Cytokine production and / or release are performed using methods well known in the art [e.g., ELISA, enzyme-linked immune absorbent spot (ELISPOT), Luminex]. (登録商標) It can be measured by assays, intracellular cytokine staining, and flow cytometry, as well as combinations thereof (e.g., intracellular cytokine staining and flow cytometry). It can be measured according to the method of implementation.

[0277] As used in this application, the term "cytokine" refers to molecules that mediate and / or regulate biological or cellular functions or processes (e.g., immunity, inflammation, and hematopoiesis). As used in this application, the term "cytokine" includes "lymphokine," "chemokine," "monokine," and "interleukin." Examples of useful cytokines include GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-15, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β.

[0278] The proliferation and clonal growth of T cells resulting from the induction or stimulation of an antigen-specific immune response can be measured, for example, through the uptake of non-radioactive assays such as tritiated thymidine assays or MTT assays.

[0279] Cytotoxicity assays for measuring CTL activity may be performed using one of several techniques and methods routinely practiced in the art (e.g., Henkart et al. (2003) Fundamental Immunology 1127-1150). Further descriptions of methods for measuring antigen-specific T cell responsiveness can be found, for example, in U.S. Patent No. 10,208,086 and U.S. Patent Application No. 2017 / 0209573 (the entirety of which is incorporated by reference in this application).

[0280] VIII. Cell therapy In a particular embodiment, the method comprises adoptive cell therapy, thereby administering to a subject genetically engineered cells (e.g., cells expressing a TCR or a TCR-like CAR) that express a molecule that targets a provided MHC-restricted epitope. Such administration promotes the activation of the cells (e.g., T cell activation) in an antigen-targeting manner, and as a result, cells infected with SARS-CoV-2 become targets for destruction.

[0281] Accordingly, the methods and uses provided include methods and uses for adoptive cell therapy. In some embodiments, the methods include administering the cells or a composition containing the cells to a subject, tissue, or cells, for example, a subject, tissue, or cell, that is at risk of having, or suspected of having, the disease, condition, or disorder. In some embodiments, the cells, population, and composition are administered to a subject having a specific disease or condition to be treated (e.g., via adoptive cell therapy, e.g., adoptive T cell therapy, etc.). In some embodiments, the cells or composition are administered to the subject (e.g., a subject having, or at risk of having, the disease or condition, etc.). In some embodiments, the methods thereby treat, for example, improve one or more symptoms of a disease or condition.

[0282] Methods for administering cells for adoptive cell therapy are known and may be used in connection with methods and compositions that provide methods for administering cells for adoptive cell therapy. For example, adoptive T cell therapy is described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; U.S. Patent No. 4,690,915 by Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8:577-585). See, for example, Themeli et al. (2013) Nat Biotechnol. 31: 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438: 84-89; Davila et al. (2013) PLoS ONE 8:e61338.

[0283] In some embodiments, the cell therapy (e.g., adoptive cell therapy, e.g., adoptive T cell therapy) is carried out by autologous transplantation (in which the cells are isolated and / or otherwise prepared from or derived from a subject to receive the cell therapy). Thus, in some embodiments, the cells are derived from a subject in need of treatment (e.g., a patient), and the cells are isolated, processed, and then administered to the same subject.

[0284] In some embodiments, the cell therapy (e.g., adoptive cell therapy, e.g., adoptive T cell therapy) is carried out by allogeneic transplantation (in which the cells are isolated and / or otherwise prepared from a subject other than the subject receiving the cell therapy, or a subject other than the ultimate recipient [e.g., a first subject]). In such embodiments, the cells are then administered to another subject of the same species [e.g., a second subject]. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0285] In some embodiments, the subjects to whom the cells, cell populations, or compositions are administered are primates such as humans. In some embodiments, the primates are monkeys or apes. The subjects may be male or female, and of any appropriate age (e.g., infants, young, adolescents, adults, and elderly subjects). In some embodiments, the subjects are non-primate mammals such as rodents. In some embodiments, the patients or subjects are effective animal models for disease, for adoptive cell therapy, and / or for evaluating toxic outcomes such as cytokine release syndrome (CRS).

[0286] The aforementioned binding molecules [e.g., TCR, TCR-like antibodies, and chimeric receptors containing the TCR-like antibodies (e.g., CAR)], as well as cells expressing these molecules, may be administered by any preferred method, for example, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreous injection, transseptal injection, subscleral injection, choroidal injection, anterior chamber injection, subconjectval injection, subconjunctival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they may be administered parenterally, intrapulmonaryly, intranasally, and, if desired for local treatment, intralesional administration. Parenteral administration methods include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Medication and administration may depend in part on whether the administration is short-term or chronic. Various administration schedules, though not limited to these, include single or multiple doses, bolus doses, and pulse doses at various points in time.

[0287] For the purpose of preventing or treating a disease, the appropriate dosage of the binding molecule or cells depends on the type of disease to be treated, the type of binding molecule, the severity and course of the disease, whether the binding molecule is administered for preventive or therapeutic purposes, past treatments, the patient's clinical history and response to the binding molecule, and the discretion of the attending physician. The composition, molecules, and cells are administered appropriately to the patient, in some embodiments, either in a single dose or over a series of treatments.

[0288] In certain embodiments, the cells, or individual populations of cell subtypes, are defined as a range of about 1 million to about 100 billion cells and / or the amount of cells per kilogram of body weight, for example, 1 million to about 50 billion cells (for example, about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the aforementioned values), for example, about 10 million to about 100 billion cells (for example, about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, Approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined by any two of the aforementioned values), and in some cases, approximately 100 million to approximately 50 billion cells (for example, approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells, approximately 45 billion cells), or any value within these ranges, and / or per kilogram of body weight, shall be administered to the subject. The dosage may vary depending on the attributes specific to the disease or disorder, and / or the patient, and / or other treatments.

[0289] In some embodiments, for example, when the subject is human, the total dose is approximately 1 × 10⁻⁶. 8 Cells including recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) less than 1 × 10⁻¹⁶ 6 From 1 x 10 8 Such cells in the range of, for example, 2 × 10 6 , 5×10 6 , 1 x 10 7 , 5×10 7 , or 1 x 10 8Or all such cells, or a range between any two of the aforementioned values.

[0290] In some embodiments, the cells or binding molecules (e.g., TCR or TCR-like antibodies) may be administered as part of a combination therapy, for example, simultaneously or sequentially in any order, with another therapeutic intervention, such as another antibody or manipulated cells or receptors or drugs, such as cytotoxic agents or therapeutic agents.

[0291] In some embodiments, the cells or binding molecules (e.g., TCR or TCR-like antibodies) may be co-administered simultaneously or sequentially in any order with one or more additional therapeutic agents or in connection with another therapeutic intervention. In some situations, the cells are co-administered with another therapy at a sufficiently close time so that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells or binding molecules (e.g., TCR or TCR-like antibodies) are administered before one or more additional therapeutic agents. In some embodiments, the cells or binding molecules (e.g., TCR or TCR-like antibodies) are administered after one or more additional therapeutic agents.

[0292] When the cells are administered to a mammal (e.g., a human), in some embodiments, the biological activity of the manipulated cell population and / or binding molecule (e.g., TCR or TCR-like antibody) is measured by one of many known methods. Parameters to be evaluated include the specific binding of the manipulated T cells, native T cells, or other immune cells to the antigen, either in vivo (e.g., by imaging) or ex vivo (e.g., by ELISA or flow cytometry). In certain embodiments, the ability of the manipulated cells to destroy target cells may be measured using preferred methods known in the art (e.g., cytotoxicity assays, such as those described in Kochenderfer et al. (2009) J. Immunotherapy 32:689-702 and Herman et al. (2004) J. Immunological Methods 285:25-40). In certain embodiments, the biological activity of the cells may be measured by assaying the expression and / or secretion of certain cytokines (e.g., CD107a, IFNγ, IL-2, and TNF). In some embodiments, the biological activity may be measured by evaluating clinical outcomes, such as a reduction in tumor burden or load.

[0293] In certain embodiments, the manipulated cells are modified in any number of ways to increase their therapeutic or prophylactic efficacy. For example, the manipulated CAR or TCR expressed by the population may be conjugated to a targeting substructure, either directly or indirectly via a linker. The practice of conjugating compounds (e.g., CARs or TCRs) to targeting substructures is known in the art. See, for example, Wadwa et al. (1995) J. Drug Targeting 3:111 and U.S. Patent No. 5,087,616.

[0294] In certain embodiments, compositions and methods for providing SARS-CoV-2-primed antigen-presenting cells and / or SARS-CoV-2-specific lymphocytes produced using such antigen-presenting cells may utilize the SARS-CoV-2 immunogenic peptide described in this application, or nucleic acids encoding such SARS-CoV-2 immunogenic peptide. In some embodiments, such antigen-presenting cells and / or lymphocytes are used in the treatment and / or prevention of COVID-19 (i.e., SARS-CoV-2 infection).

[0295] In some embodiments, this application provides a method for producing SARS-CoV-2-primed antigen-presenting cells by contacting antigen-presenting cells with the SARS-CoV-2 immunogenic polypeptide described in this application, or with nucleic acids encoding at least one SARS-CoV-2 immunogenic polypeptide (by contacting the antigen-presenting cells alone or in combination with an adjuvant, in vitro under conditions sufficient to allow the antigen-presenting cells to present at least one SARS-CoV-2 immunogenic polypeptide).

[0296] In some embodiments, the SARS-CoV-2 immunogenic polypeptide, or the nucleic acid encoding the SARS-CoV-2 immunogenic polypeptide, may be contacted alone or in combination with an adjuvant to a homogeneous, substantially homogeneous, or heterogeneous composition containing antigen-presenting cells. For example, the composition may include, but is not limited to, whole blood, fresh blood, or fractions thereof (e.g., peripheral blood mononuclear cells, pia mater fraction of whole blood, concentrated red blood cells, irradiated blood, dendritic cells, monocytes, macrophages, neutrophils, lymphocytes, natural killer cells, and natural killer T cells, etc.). Optionally, if antigen-presenting cell precursors are used, the precursors may be cultured under suitable culture conditions sufficient to differentiate them into antigen-presenting cells. In some embodiments, the antigen-presenting cells (or their precursors) may be selected from monocytes, macrophages, myeloid cells, B cells, dendritic cells, or Langerhans cells.

[0297] Those skilled in the art may determine, by routine experimentation, the amount of the SARS-CoV-2 immunogenic polypeptide, or the amount of nucleic acid encoding the SARS-CoV-2 immunogenic polypeptide, to be placed in contact with antigen-presenting cells, either alone or in combination with an adjuvant. Generally, antigen-presenting cells are brought into contact with the SARS-CoV-2 immunogenic polypeptide, or the nucleic acid encoding the SARS-CoV-2 immunogenic polypeptide, either alone or in combination with an adjuvant, for a period of time sufficient for the cells to present the antigen in a processed form to modulate T cells. In one embodiment, antigen-presenting cells are incubated in the presence of the SARS-CoV-2 immunogenic polypeptide, or in the presence of nucleic acids encoding the SARS-CoV-2 immunogenic polypeptide, either alone or in combination with an adjuvant, for less than one week, for example, about 1 minute to about 48 hours, about 2 minutes to about 36 hours, about 3 minutes to about 24 hours, about 4 minutes to about 12 hours, about 6 minutes to about 8 hours, about 8 minutes to about 6 hours, about 10 minutes to about 5 hours, about 15 minutes to about 4 hours, about 20 minutes to about 3 hours, about 30 minutes to about 2 hours, and about 40 minutes to about 1 hour. The amount of SARS-CoV-2 immunogenic polypeptide, or the amount of nucleic acid encoding the SARS-CoV-2 immunogenic polypeptide, required by antigen-presenting cells to process and present the antigen, either alone or in combination with an adjuvant, may be determined, for example, using a pulse-chase method, which includes a washout period after contact and exposure to a read-out system, such as antigen-responsive T cells.

[0298] In certain embodiments, any suitable method for delivering an antigen to the endogenous processing pathway of an antigen-presenting cell may be used. Such methods include, but are not limited to, pH-sensitive liposomes, coupling the antigen to an adjuvant, apoptotic cell delivery, pulsing cells into dendritic cells, and delivering recombinant chimeric virus-like particles (VLPs) containing the antigen to the MHC class I processing pathway of a dendritic cell line.

[0299] In one embodiment, a solubilized SARS-CoV-2 immunogenic polypeptide is incubated with antigen-presenting cells. In some embodiments, the SARS-CoV-2 immunogenic polypeptide may be coupled to a cytolysin to deliver it via the MHC class I pathway, thereby enhancing the transfer of the antigen into the cytosol of the antigen-presenting cells. Exemplary cytolysins include saponin compounds, e.g., saponin-containing immunostimulating complexes (ISCOM5), pore-forming toxins (e.g., alpha-toxins), and innate cytolysins of Gram-positive bacteria, e.g., listeriolysin O (LLO), streptolysin O (SLO), and perfringolysin O (PFO)).

[0300] In some embodiments, antigen-presenting cells (e.g., dendritic cells and macrophages) may be isolated according to methods known in the art, and transfected with polynucleotides by methods known in the art for introducing nucleic acids encoding SARS-CoV-2 immunogenic polypeptides into the antigen-presenting cells. Transfection reagents and methods are known in the art and commercially available. For example, RNA encoding SARS-CoV-2 immunogenic polypeptide may be provided in a suitable medium and combined with lipids (e.g., cationic lipids) before contact with antigen-presenting cells. Non-limiting examples of such lipids include LIPOFECTIN. TM and LIPOFECTAMINE TM These are examples. The obtained polynucleotide-lipid complex may then be brought into contact with antigen-presenting cells. Alternatively, the polynucleotide may be introduced into antigen-presenting cells using techniques such as electroporation or calcium phosphate transfection. The polynucleotide-loaded antigen-presenting cells may then be used to stimulate the proliferation of T lymphocytes (e.g., cytotoxic T lymphocytes) in vivo or ex vivo. In one embodiment, the T lymphocytes proliferated ex vivo are administered to the subject by adoptive immunotherapy.

[0301] In certain embodiments, the present application provides a composition comprising antigen-presenting cells (antigen-presenting cells that have been contacted in vitro, either alone or in combination with an adjuvant, with a SARS-CoV-2 immunogenic polypeptide or a nucleic acid encoding a SARS-CoV-2 immunogenic polypeptide, under conditions sufficient to present a SARS-CoV-2 immunogenic epitope).

[0302] In some embodiments, this application provides a method for preparing lymphocytes specific to the SARS-CoV-2 protein. The method includes the step of contacting lymphocytes with the antigen-presenting cells under conditions sufficient to produce SARS-CoV-2 protein-specific lymphocytes that can induce an immune response against cells infected with the SARS-CoV-2 virus. Thus, the antigen-presenting cells may also be used to provide lymphocytes (e.g., T lymphocytes and B lymphocytes, etc.) for inducing an immune response against cells infected with the SARS-CoV-2 virus.

[0303] In some embodiments, a preparation of T lymphocytes is brought into contact with the antigen-presenting cells for a period of time (e.g., at least about 24 hours) to prime the T lymphocytes against the SARS-CoV-2 immunogenic epitope presented by the antigen-presenting cells.

[0304] In some embodiments, a population of antigen-presenting cells may be co-cultured with a heterogeneous population of peripheral blood T lymphocytes, either alone or in combination with a SARS-CoV-2 immunogenic polypeptide, or with a nucleic acid encoding the SARS-CoV-2 immunogenic polypeptide. The cells may be co-cultured for a period of time and under conditions sufficient for the SARS-CoV-2 epitope contained in the SARS-CoV-2 polypeptide to be presented by the antigen-presenting cells, and for the antigen-presenting cells to prime the population of T lymphocytes to respond to cells infected with SARS-CoV-2. In certain embodiments, the present application provides T lymphocytes and B lymphocytes primed to respond to cells infected with the SARS-CoV-2 virus.

[0305] T lymphocytes may be obtained from any suitable source (e.g., peripheral blood, spleen, and lymph nodes). The T lymphocytes may be used as a crude preparation or as a partially or substantially purified preparation, and these may be obtained by standard techniques, such as, but not limited to, antibody-based immunomagnetic techniques or flow cytometry techniques.

[0306] In certain embodiments, the present application provides a composition (e.g., a pharmaceutical composition) comprising the above-mentioned antigen-presenting cells or lymphocytes, and a pharmaceutically acceptable carrier and / or diluent. In some embodiments, the composition further comprises an adjuvant as described above.

[0307] In certain embodiments, the present application provides a method for inducing an immune response against cells infected with the SARS-CoV-2 virus, wherein the method comprises the step of administering the antigen-presenting cells or lymphocytes to a subject in an effective amount sufficient to induce the immune response. In some embodiments, a method for treating or preventing COVID-19, wherein the method comprises the step of administering an effective amount of the antigen-presenting cells or lymphocytes to a subject. In one embodiment, the antigen-presenting cells or lymphocytes are administered systemically, preferably by injection. Alternatively, they may be administered locally rather than systemically, for example, by direct injection into tissue, preferably as a depot or sustained-release formulation.

[0308] In certain embodiments, antigen-primed antigen-presenting cells described in this application, and antigen-specific T lymphocytes produced using these antigen-presenting cells, may be used as active compounds in an immunomodulatory composition for the prophylactic or therapeutic treatment of COVID-19. In some embodiments, SARS-CoV-2-primed antigen-presenting cells described in this application may be used for adoptive transfer to the subject, as described in CD8 + T lymphocytes, CD4+ They may be used to generate T lymphocytes and / or B lymphocytes. For example, SARS-CoV-2-specific lymphocytes may be adopted and transferred to subjects infected with COVID-19 for therapeutic purposes.

[0309] In certain embodiments, antigen-presenting cells and / or lymphocytes described herein may be administered to a subject, either by themselves or in combination, to induce an immune response, particularly an immune response against cells infected with the SARS-CoV-2 virus. In some embodiments, the antigen-presenting cells and / or lymphocytes may originate from the subject (i.e., autologous cells) or from another subject that is MHC-matched or mismatched (e.g., allogeneic) with the subject.

[0310] The antigen-presenting cells and lymphocytes may be administered as a single or multiple doses, in a number of cells and treatment selected by the care provider (e.g., a physician). In some embodiments, the antigen-presenting cells and / or lymphocytes are administered in a pharmaceutically acceptable carrier. A suitable carrier may be the growth medium in which the cells were grown, or any suitable buffer medium such as phosphate-buffered saline. The cells may be administered alone or as adjunctive therapy in combination with other therapeutic agents.

[0311] IX. Kit The present invention also encompasses kits. For example, such kits may include, packaged in suitable containers, immunogenic peptides, vectors containing sequences encoding immunogenic peptides, stable MHC-peptide complexes described in this application, adjuvants, and combinations thereof, and may further include instructions for using such reagents. Such kits may also include other components (e.g., administration tools packaged in separate containers).

[0312] The present disclosure is further illustrated by the following embodiments, which should not be construed as limiting. The contents of all references, patents, and published patent applications, as well as drawings, cited throughout this application are incorporated by reference. [Examples]

[0313] Example 1: SARS-CoV-2 vaccine construct We investigated how to construct a vaccine that would produce the most potent CD8 T cell response against SARS-CoV-2. This involved maximizing the magnitude of the response to each individual epitope, as well as the overall width of the targeted epitopes. At the same time, vaccine design faced several constraints. One important consideration was the limited size of the overall vaccine construct; that is, excessive size would pose a challenge to the vaccine delivery system (e.g., mRNA or viral vector) and could hinder the efficient expression of the complete construct.

[0314] There is considerable doubt in this field about whether polyepitope vaccines truly have the ability to produce robust T-cell responses. This doubt focuses on two main challenges. The first challenge concerns the limited set of epitopes included in the vaccine. Polyepitope vaccines primarily contain only separately encoded peptide epitopes. In contrast, vaccines containing whole proteins are processed into thousands of different epitopes—different epitopes presented on different MHC alleles. A protein the size of the SARS-CoV-2 S protein contains so many potential epitopes that for every conceivable MHC allele, there are numerous predicted high-affinity conjugates. The challenge for polyepitope vaccines is whether they can induce a stronger response to a limited, MHC-restricted set of epitopes than would naturally occur for the whole protein. The second challenge concerns the processing and presentation of epitopes in the context of polyepitope vaccines. In the context of SARS-CoV-2 infection, the efficient presentation of discovered epitopes does not necessarily mean that they will be presented in the context of polyepitope vaccines. This concern is particularly serious because of the limited number of epitopes contained in polyepitope vaccines; that is, even the failure to present a very small fraction of the epitopes in the vaccine could dramatically affect the vaccine's ability to induce a response to a specific MHC allele.

[0315] These challenges are widely understood in the field and have hindered the successful development of polyepitope vaccines against other viruses. Indeed, even considering recent SARS-CoV-2 epitope mapping studies and the identification of a limited number of highly immunodominant epitopes recognized on each MHC allele, there has been considerable skepticism about whether these biological insights can be translated into effective vaccines that overcome these fundamental challenges.

[0316] In designing a polyepitope vaccine, two sets of open-end decisions are made, including 1) the contents of the vaccine, and 2) the conditions under which the contents are expressed.

[0317] We explored a wide range of options for both of these decisions. Using our previously developed proprietary reagents (highly active memory CD8 T cells derived from a convalescent group of COVID-19 patients), we designed and experimentally tested various vaccine sets.

[0318] In particular, the DNA sequence was back-translated from the sequence of the designed protein vaccine, and then GenSmart TM The sequence was optimized using a codon optimization algorithm (GenScript, Inc.; see also PCT publication WO 2020 / 0024917). The resulting DNA was ordered as gBlocks (Integrated DNA Technologies Inc.). NEBuilder (登録商標) HiFi DNA Assembly Master Mix The assembly was assembled into the EcoRI-linear pHAGE-CMV-IRES-Puro vector (Kula et al. (2019) Cell 178:1016-1028) using (New England Biolabs; Cat. #E2621S). The assembly was then mixed using Mix & Go! Chemically competent cells The cells were transformed into (Zymo Research; catalog number T3007), and individual colonies were selected and subjected to Sanger sequencing.

[0319] A*02:01-expressing HEK293 cells were transduced using each vaccine construct at a single multiplicity of infection (MOI), and selected using puromycin (1.5 ug / mL, Gibco). 5x10 4The cells were seeded in a 96-well plate and allowed to stand for 16 hours. Memory CD8 T cells isolated from convalescent COVID-19 patients (available at Ferretti et al. (2020) Immunity S1074-7613(20)30447-7; doi.org / 10.1016 / j.immuni.2020.10.006) were added in an effector-to-target ratio of 2:1 and incubated for 16-18 hours. After incubation, the cells were mixed by pipetting, transferred to a V-bottom 96-well plate, and pelletized by centrifugation at 300xg for 5 minutes. The supernatant was collected, and IFN gamma was measured using Ella (Protein Simple). The cell pellet was stained with BV421-conjugated anti-CD8 (BioLegend), AF647-conjugated anti-CD69 (Biolegend), and PE-conjugated anti-CD137 (Miltenyi) antibodies, and analyzed using Cytoflex S (Beckman Coulter).

[0320] These results demonstrate that both the vaccine's context and composition have a significant, unpredictable impact on its performance. Furthermore, the designed vaccine construct was observed to be highly promising in that it generates a more robust CD8 T-cell response than the current vaccine candidates in clinical trials.

[0321] Six immunodominant epitopes presented on the most common MHC alleles were identified: HLA-A*02:01, HLA-A*01:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, and HLA-B*07:02. Polyepitope vaccines were designed encoding either all 29 identified immunodominant epitopes (6 A2, 8 A1, 4 A3, 5 A11, 3 A24, and 3 B7) or 19 immunodominant epitopes (3-4 from each MHC). In selecting the 19 epitopes, epitopes whose presentation on multiple MHC alleles was predicted or verified were included. These 19 epitopes include a subset derived from Table L1, as specified below.

[0322] Table L below, consisting of Tables L1 and L2, identifies 29 peptide epitopes that may be used in the disclosed constructs (e.g., sequences in Table L1 and further data in Table L2). Note that Table L clearly indicates that orf3a, M, and N, as well as orf1ab, proteins are hotspots for containing T cell epitopes.

[0323] Table L1 [Table 8-1] [Table 8-2]

[0324] Table L2 [Table 9]

[0325] Regarding the association with the vaccine, one important factor is the linkers between epitopes. These linkers are a critical determinant of whether the epitopes are likely to be efficiently processed and presented. The constructs tested had either no linkers at all (direct linking), 3aa upstream and downstream of each epitope (a total of 6aa spacing between adjacent epitopes), or optimized proteasome cleavage sequences "KAA" between each pair of epitopes. Further research also included optimizing the order of epitopes within the polyepitope vaccine. In this application, the objective was to minimize the generation of junctional epitopes that bind to MHC with high affinity but are not found in the SARS-CoV-2 genome; such epitopes compete with the desired epitopes for presentation. An algorithm was developed to iteratively rearrange the order of epitopes within polyepitope constructs to remove the epitope predicted to have the highest affinity. This algorithm was applied to 10,000 starting constructs, and the variant with the best final performance was selected. In particular, it was determined that the variable contributing to the differences was the choice of which HLA alleles to consider when evaluating junctional neoepitopes. For the purposes of the algorithm, six HLA alleles presenting the epitopes in Table L were selected. Next, a comprehensive set of all possible conjugates between the selected epitopes was generated. The MHC-binding epitopes on the six MHC alleles examined were determined using the NetMHC4.0 algorithm (Jurtz et al. (2017) J. Immunol. 199:3360-3368). For each conjugate, the conjugate with the highest predicted affinity that did not originate from the native viral sequence (i.e., resulting from the conjugate itself) was identified. Next, a random starting order was assigned to the epitopes. For a given order, the junctional epitope with the highest predicted affinity was identified. All possible ways in which two epitopes could be swapped within the structure were evaluated, and the epitope swap that resulted in the junctional epitope with the lowest affinity was selected. This process was repeated until the epitope swap could no longer further improve the structure.The entire algorithm was run with 10,000 random starting orders, and the best-performing final order (i.e., the order in which the highest-affinity junction epitope has the lowest-probability affinity) was selected.

[0326] During the design process of various constructs, we also altered the positions of individual epitopes within the polyepitope vaccines. Finally, we constructed the designed polyepitope vaccines as standalone constructs to be expressed in the cytoplasm or in a polycistronic expression context following the natural SARS-CoV-2 S protein (using the P2A sequence to enable cytoplasmic expression of our polyepitope vaccine immediately after transmembrane expression using the natural signaling sequence of the S protein).

[0327] To evaluate these constructs, each construct was expressed in HEK293 T cells expressing only HLA-A*02:01 using lentiviral transduction (at a low MOI). These modified HEK293 T cells were then co-cultured with memory CD8 T cells derived from a panel of convalescent COVID-19 patients (e.g., A*02:01-positive COVID-19 patients). The intensity of the T cell response to any presented epitope was assessed using IFN-gamma (IFNg) secretion. This provided a readout regarding how efficiently the epitope was presented in each vaccine context. Based on previous studies, it was known that most convalescent COVID-19 patients generate memory CD8 T cells for the tested epitopes; therefore, the intensity of the response provided a direct readout regarding how efficiently the epitope was presented in each vaccine construct.

[0328] Several important controls were included. First, HEK293 T cells alone (without any viral sequences) were tested, serving as a negative control ("A2 Screps"). Second, peptide pulses with a set of three immunodominant HLA-A*02:01 SARS-CoV-2 epitopes were used as a positive control (because peptide pulses load directly onto the MHC with high efficiency) ("A2 Screps + KLW / YLQ / LLY"). Third, the S protein alone was used. This is a lead construct currently being tested in clinical trials, and it allows for comparison of the magnitude of the T cell response induced by the aforementioned polyepitope constructs with this baseline. Finally, large fragments of ~500 aa spanning each of the three immunodominant HLA-A*02:01 epitopes were also included in the pulse experiments.

[0329] The results of this experiment are shown in Figures 3 and 4, and several important findings were determined.

[0330] Firstly, significant differences in the efficiency of epitope presentation were observed depending on the context of each vaccine. In particular, it was identified that using the three amino acids upstream and downstream of each epitope was superior to the absence of a linker or to the use of a KAA linker. Secondly, it was observed that the expression of the construct in the cytoplasm was important. In fact, despite the fact that the S protein contained one of the immunodominant HLA-A*02:01 epitopes, a very weak response to the full-length S protein was observed in all patients. Indeed, the expression of a 500 aa fragment derived from the S protein spanning this epitope ("ORF S-1") resulted in a stronger response, thereby highlighting that the full-length S protein resulted in particularly poor presentation. Thirdly, it was observed that direct expression of the polyepitope vaccine resulted in stronger expression than using the P2A sequence. Generally, expression from 27 epitopes yielded a stronger response than expression from 19 epitopes.

[0331] However, the most striking conclusion was the overall efficiency of the vaccine constructs. All of the polyepitope constructs tested resulted in dramatically more robust T-cell activity than the S protein alone, and some of the optimal constructs performed better than pulsing with the positive control peptide. This result is quite surprising and highlights the potential of polyepitope vaccines when using the appropriate epitope, linker, and protein context.

[0332] These results are in stark contrast to the level of technology (which was skeptical of the usefulness of polyepitope constructs). For example, Korber et al. (2009) J. Virol. (2009) 83: 8300-8314 provides a comprehensive overview of T-cell-based vaccine approaches against HIV, and its section on polyepitope vaccines highlights the lack of immunogenicity observed with this theoretical approach.

[0333] Based on the research conducted, two points were found to be important. First, the identity of the epitopes themselves. The included epitopes have the highest level of functional validation – they are immunodominant in the context of actual SARS-CoV-2 infection. Other epitopes have been identified as either predicted, detected on infected cells by mass spectrometry, or recognized by convalescent patients after proliferation specifically of susceptible antigens; that is, this does not indicate that they are immunodominant (and therefore likely to be the most immunogenic and efficiently recognized by the immune system in the context of the vaccine). Second, the number of included epitopes. In some embodiments, epitopes are selected to cover a wide diversity of HLA types (e.g., at least one immunodominant epitope for each of the six most common HLA types). It should be noted that there are several epitopes presented by multiple HLA alleles, and that it is possible to obtain a single immunodominant epitope for each of the six alleles using only four or five epitopes. Therefore, for example, minimal coverage for at least one immunodominant epitope for each of the six most common HLAs can be achieved using, for example, four, five, six, or more epitopes, thereby reasonably covering the HLA diversity in the population. Vaccines with less coverage of such HLA diversity have significant blind spots and miss patients with suboptimal HLA alleles. More broadly, the value of a vaccine is considered to increase significantly by adding more epitopes. In some embodiments, the construct has at least two immunodominant epitopes for each of the six most common HLAs. The tested constructs possess at least 18 epitopes (approximately 3 for each of the 6 HLA alleles), which is thought to increase the generation of robust and broad responses.

[0334] Having multiple epitopes per HLA allele is considered important because: 1) it is not always known which epitope (or protein) is the most protective; 2) it increases the likelihood of a robust T cell response against at least one of the included epitopes; and 3) T cell responses to multiple epitopes are important for efficacy and preventing antigen escape variants.

[0335] The following factors are measurable, but were judged to be of relatively low importance.

[0336] One factor is the identity of the linker between epitopes. Various linkers showed some degree of effectiveness. While the ~2x improvement in epitope presentation observed with 3-amino acid linkers is significant, longer linkers are equally suitable.

[0337] Another factor is the order of epitopes. While optimizing the order of epitopes restricted junctional epitopes, constructs with various epitope orders appeared to yield similar results. There is no definitive cutoff value for which junctional epitopes are problematic. Generally, high-affinity junctional epitopes are thought to compete with desired epitopes, thereby reducing the magnitude of the observed immune response. For the vaccine constructs, all conjugate epitopes with a predicted binding affinity < 77 nM (e.g., ≤ 75 nM, ≤ 70 nM, ≤ 65 nM, ≤ 60 nM, ≤ 55 nM, ≤ 50 nM, ≤ 45 nM, ≤ 40 nM, ≤ 35 nM, ≤ 30 nM, ≤ 25 nM, ≤ 20 nM, ≤ 15 nM, ≤ 10 nM, ≤ 5 nM, or any comprehensive range between these, e.g., 50-75 nM) were excluded. This means that, in the case of the 29 epitope vaccines disclosed, the 22 conjugates with the highest predicted affinity across the entire construct are particularly desirable epitopes. Even a single epitope predicted to have high affinity binding may not have a dramatic effect, and generally, it is considered difficult to measure the benefit of removing junctional epitopes.

[0338] Furthermore, studies in related contexts suggest that orientation has little effect on presentation efficiency. Theoretically, epitopes located at the N-terminus of a fragment might be presented more efficiently, because the N-terminus is always synthesized, while some defective protein products may lack a C-terminus. However, experimental data suggest that the presentation of epitopes at various orientations is generally equivalent.

[0339] Due to size constraints in the overall vaccine construct, there is a trade-off between adding epitope repeats and including additional novel epitopes. To cover a broader range of HLA and to increase the number of epitopes (covering more viral proteins), we prioritized adding additional novel epitopes.

[0340] Preliminary analysis suggested that the 3aa linker was relatively the most effective. Longer linkers are likely to be as effective as 3aa, but this would require a larger vaccine construct overall. Even vaccines without linkers showed some efficacy. Therefore, overall, having a linker is beneficial.

[0341] In some embodiments, ribosome arrest / restart sites are preferred because they are the most robust and minimal. IRESs or posttranslational cleavage sequences may also be used in certain embodiments. In some embodiments, the S protein may be co-expressed to enable an antibody response with the polyepitope vaccine. The two proteins can be expressed in either order. Typically, the first protein is expressed at a higher level, and the higher expression of the S protein is likely to be more important (if only for a direct comparison with a vaccine containing only the S protein). The vaccine is expected to function in either direction.

[0342] An alternative to polycistronic expression is generally to deliver two vaccines simultaneously (which can be easily done using delivery systems such as mRNA).

[0343] These studies may utilize a variety of detection methods. For example, tetramer staining may be used to quantify T cells against individual epitopes; T cell activation assays (e.g., CD137 staining, intracellular IFNg staining, etc.) may be used to quantify responsive T cells; and TCR sequencing may be used to demonstrate that vaccines reproduce the TCR repertoire that occurred in natural infection.

[0344] Example 2: Further SARS-CoV-2 vaccine constructs and validation results To further verify the above results, we designed additional SARS-CoV-2 vaccine constructs, such as those shown in Figures 9C-9E.

[0345] Further analyses were conducted to further verify the above results. For example, various in vitro analyses were performed using vaccine constructs formulated as lipid nanoparticles (LNPs). Figure 10 shows that memory T cells isolated from SARS-CoV-2 patients respond to cells treated with representative LNP formulations of the vaccine constructs described in Figure 9C, thereby demonstrating that cells effectively process and present epitopes so that they can be recognized in a manner similar to cells infected with SARS-CoV-2. Briefly, memory T cells (Tmem pool) were isolated from recently recovered SARS-CoV-2 patients using a CD8+ memory T cell isolation kit (Miltenyi, catalog number 130-094-412) according to the manufacturer's instructions. The Tmem pool was co-cultured with HLA class I-deficient HEK293T cells expressing either HLA-A*02:01 or HLA-B*07:02, treated with 1 ug / mL mRNA-LNP complexes from the construct in Figure 9C. After 24 hours, interferon-gamma was measured using human IFN-γ third-generation Simple Plex Ella Assay (Protein Simple, catalog number SPCKB-PS-002574) according to the manufacturer's instructions.

[0346] Furthermore, individual TCR clones can be used as reagents to evaluate the processing and presentation of specific epitopes (e.g., the representative clones and data epitopes shown in Figure 11). For example, Figure 11 shows that four individual epitopes included in the 27-epitope construct described in Figure 6A are processed and presented in a manner that can be recognized by TCRs specific to SARS-CoV-2 epitopes. Briefly speaking, monocytes are obtained from healthy donors who are positive for HLA-A*02:01 and HLA-B*07:02 using EasySep. TM Human CD14-positive selection kit II (StemCell Technologies, 17858) was used to isolate monocyte-derived dendritic cells (moDCs). These cells were cultured for 48 hours in the presence of interleukin-4 and granulocyte-macrophage colony-stimulating factor to differentiate them into monocyte-derived dendritic cells (moDCs). The moDCs were treated for 4 hours with LNPs containing 1 ug / mL mRNA from the construct described in Figure 6A, and then treated with TNF-alpha, IL-1 beta, IL-6, and PGE2. After 48 hours, the moDCs were co-cultured with T cells transduced to individual TCRs recognizing one of the LLY, SPR, KLW, or YLQ epitopes described in Tables 1A and 1F, respectively. TCR activation was measured by staining with AF647-conjugate anti-CD69 antibody (Biolegend, catalog number 310918) and PE-conjugate anti-CD137 antibody (Biolegend, catalog number 309804), and then detecting the staining by flow cytometry (C Cytoflex S, Beckman Coulter).

[0347] Interestingly, it was also determined that by formulating the vaccine construct shown in Figure 9C into an LNP formulation, a SARS-CoV-2 epitope-specific TCR utilizing a common TRAV gene was generated (Figures 13-16).

[0348] In response to natural infection with SARS-CoV-2, TCRs that recognize the same immunodominant epitope of SARS-CoV-2 share a common TRAV gene, and this TRAV gene is the dominant TRAV gene utilized by memory T cells (Tmem) of SARS-CoV-2 patients immediately after recovery from infection (Figure 12). To verify that the vaccine construct proliferates naive repertory T cells that use the same TCR gene as Tmem cells derived from COVID patients, an in vitro vaccine model was developed (Figure 13). Briefly, moDCs treated with LNPs containing 1 ug / mL of mRNA of the construct described in Figure 9C were subjected to EasySep TMHuman Naive CD8+ T Cell Isolation Kit II (StemCell, 17968) was used to co-culture naive CD8 T cells isolated from healthy donor blood collected in 2019, prior to the SARS-CoV-2 outbreak, with human naive CD8 T cells. The co-culture was divided into multiple wells of a 96-well plate, and the proliferation of specific clones derived from the naive repertoire was detected. After 10 days of proliferation, each well was divided into four copies, and each copy in each well was stained with fluorescently labeled tetramers for the indicated peptides (Tetramer Shop, catalog numbers HA02-070 and HB07-017) and measured by flow cytometry (Cytoflex S, Beckman Coulter). The remaining copies were pooled and restimulated with HEK293 T cells that were mono-allelemic, either HLA-A*02:01 or HLA-B*07:02, pulsed with 1 ug / mL of the respective peptides from Table 1A and Table 1F (Figure 13). Re-activated T cells, identified by CD69 and CD137 staining, were sorted and sequenced by flow cytometry (MoFlo Astrios EQ) to determine the TRAV gene utilized by the proliferated T cells. These results demonstrate that pulsed SARS-CoV-2 immunodominant peptides, such as the YLQ peptide, induce peptide-specific T cell proliferation in this assay (Figure 14), and that the vaccine construct described in Figure 9C induces SARS-CoV-specific T cell proliferation in this assay (Figure 15). Interestingly, it was also determined that by formulating the vaccine construct shown in Figure 9C into an LNP formulation, a SARS-CoV-2 epitope-specific TCR utilizing a common TRAV gene was generated (Figure 16).

[0349] Further verification experiments may be conducted.

[0350] In one representative embodiment, a processing and presentation assay is performed. For example, a construct (e.g., formulated as lipid nanoparticles encapsulating mRNA (mRNA-LNP)) is introduced into dendritic cells (DCs), which are the target cells for the construct. The treated DCs are co-cultured with epitope-specific T cells, and the responsiveness of the T cells is measured, for example, by analyzing surface activation markers and IFNg. If the T cells respond, the epitope is likely to be processed and properly presented. To compare the processing and presentation of epitopes of constructs, EasySep TM Monocytes are isolated from healthy donors with HLA types matching the epitope of interest using the Human CD14 Positive Selection Kit II (StemCell Technologies, 17858). The monocytes are cultured for 72 hours in the presence of granulocyte colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to differentiate into moDCs. After 72 hours, the moDCs are treated for 4 hours with LNPs containing 1 ug / mL of mRNA of each construct, and then treated with TNF-alpha, IL-1 beta, IL-6, and PGE2. After 48 hours, T cells transduced with epitope-specific TCRs in a 1:1 ratio of moDCs to T cells are added to the culture and co-cultured for 24 hours. T cell activation was measured by flow cytometry staining with AIM CD69 (Biolegend, catalog number 309804) and CD137 (Biolegend, catalog number 309804), and by flow cytometry (Cytoflex S, Beckman Coulter). Constructs that can induce epitope-specific T cell activation indicate that the epitope recognized by the TCR is processed and presented sufficiently to induce T cell activation. Furthermore, when comparing the processing and presentation of a single epitope between constructs, if a particular construct induces a higher level of T cell activation, it is interpreted that the processing and presentation of the measured epitope are at a higher level.

[0351] In another representative embodiment, an in vitro vaccine assay is performed. For example, a construct (e.g., an mRNA-LNP preparation) is reintroduced into DCs, and the mRNA-LNP DCs and the DCs are co-cultured with naive T cells derived from donor blood collected in 2019 or earlier (i.e., non-COVID exposed). The co-cultured cells are divided into several hundred wells of a 96-well plate, and antigen-specific proliferation is measured by peptide-conjugated MHC tetramer staining. The magnitude of the response is measured by the number of wells identified with antigen-specific clones (Figure 13). After 10 days of proliferation, the T cells are pooled and re-activated with HEK cells expressing a single HLA of interest, which have been treated again with LNP together with mRNA 1 ug / mL of the construct used to induce the initial response. The cells are stained with AIM CD69 (Biolegend, catalog number 309804) and CD137 (Biolegend, catalog number 309804), and AIM-double positive cells are selected, and rearranged TCR genes are sequenced. This assay may compare the T cells that respond to the co-culture with memory T cells derived from COVID patients, and analyzes the repertoire of proliferated T cells.

[0352] In yet another representative embodiment, an MHC proliferation assay is performed. For example, to determine whether a construct (e.g., an mRNA-LNP preparation) is introduced into cells such as human embryonic kidney (HEK) cells expressing a single HLA matching the HLA of COVID patient memory T cells, in order to determine whether it contains an epitope in which the immunogenic regions of the SARS-CoV-2 protein (e.g., N, Orf3a, and M) are presented on additional HLAs, and as a result, vaccination would be effective in individuals with HLAs other than those mentioned above. To assess the theoretical proportion of individuals that respond to the construct, CD8 Tmem cells are isolated and banked from patients who have recently recovered from SARS-CoV-2 infection and express an HLA other than one of the known epitopes listed in Table 1, using a CD8+ memory T cell isolation kit (Miltenyi, catalog no. 130-094-412). The Tmem cells are co-cultured with mono-allelegenic HEK cells expressing matching HLA, and T cell activation is measured by measuring IFN-gamma release using the human IFN-γ third-generation Simple Plex Ella Assay (Protein Simple, catalog number SPCKB-PS-002574). Since HEK cells treated with the construct contain a single HLA, Tmem cell activation indicates that the epitope on the construct is processed and presented on the tested HLA. Furthermore, recognition by Tmem cells from COVID patients indicates that the undefined epitope is sufficient to generate specific T cells in the memory repertoire of patients exposed to SARS-CoV-2, and that patients possessing the tested HLA are likely to generate a T cell response to the epitope delivered by the construct.

[0353] In yet another representative embodiment, an in vivo vaccine assay is performed. For example, an animal model (e.g., a humanized mouse model engineered to express the human TCR repertoire and human MHC, e.g., VELOCI-T) is used. (登録商標)Anti-SARS-CoV-2 immunity may be determined by immunizing mouse models (Regeneron, Inc.) and human subjects with constructs, and epitope-specific T cell responses may be measured using peptide-conjugated MHC tetramer staining, ELISPOT assay, or co-culture assay (in this co-culture assay, Tmem cells derived from vaccinated mice or patients are co-cultured with mono-allelic HEKs treated with LNPs containing the mRNA of the corresponding vaccine construct).

[0354] Import by reference All publications, patents, and patent applications referenced herein are incorporated by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually identified and incorporated by reference. In case of any conflict, this application (including any definitions thereof) shall prevail.

[0355] Any polynucleotide and polypeptide sequences that reference accession numbers corresponding to entries in public databases (for example, sequences maintained by the JC Venter Institute for Genomic Research (TIGR) at tigr.org on the World Wide Web, and / or the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov on the World Wide Web) are also incorporated in their entirety by reference in this application.

[0356] Equal portions Those skilled in the art can recognize or confirm, through routine experimentation, many equivalents to the particular embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. (a) Peptide epitopes described in SEQ ID NO: 1-4, 7-9, 15-17, 19-20, and 22-28; (b) Peptide epitopes described in SEQ ID NO: 1-9 and 11-28; (c) At least two peptide epitopes selected from the group consisting of SEQ ID NO: 1-4, 7-9, 15-17, 19-20, and 22-28; or (d) At least two peptide epitopes selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F, An immunogenic polypeptide comprising, optionally, wherein said immunogenic peptide is 1) Comprising said at least two peptide epitopes in the linked order, optionally, wherein at least one or more immunodominant epitopes are present as two or more copies; 2) Comprising at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more of said peptide epitopes, optionally, wherein said immunogenic polypeptide comprises at least one, two, and / or three immunodominant epitopes for each of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and HLA-B*07; 3) Comprising three peptide epitopes each derived from Table 1A, 1B, 1C, 1D, 1E, and 1F; 4) Further comprising a linker between said peptide epitopes, optionally, wherein a) Said linker comprises at least three amino acids for each of said peptide epitopes, optionally, wherein said at least three amino acids are contiguous with each of their respective peptide epitopes, or b) Said linker is a proteasome cleavage motif; 5) One or more full-length SARS-CoV-2 proteins selected from the group consisting of Orf1ab, M, N, Orf3a, and S, or one or more protein fragments thereof, optionally, wherein said fragments of said one or more full-length SARS-CoV-2 proteins include SARS-CoV-2 proteins that do not encode functional SARS-CoV-2 proteins; 6) further comprising a ribosome stop / restart segment, an IRES segment, and / or a post-translational cleavage segment, optionally, wherein the post-translational cleavage segment is a P2A segment; 7) comprising any one of the amino acid sequences provided in Table 1G or Table 1I; 8) comprising at least two peptide fragments, wherein each of the peptide fragments comprises at least two of the peptide epitopes, wherein the at least two peptide epitopes in each peptide fragment are derived from the same protein of SARS-CoV-2, optionally, wherein a) the at least two peptide fragments are derived from the N protein, M protein, ORF1a / b protein, or ORF3a protein of SARS-CoV-2; b) the immunogenic polypeptide comprises a maximum of six of the peptide fragments; c) the immunogenic polypeptide further comprises, optionally, one or more full-length SARS-CoV-2 proteins or one or more protein fragments thereof selected from the group consisting of Orf1ab, M, N, Orf3a, and S, wherein the fragments of the one or more full-length SARS-CoV-2 proteins include SARS-CoV-2 proteins that do not encode functional SARS-CoV-2 proteins; d) the immunogenic polypeptide further comprises, optionally, a ribosome stop / restart segment, an IRES segment, and / or a post-translational cleavage segment, wherein the post-translational cleavage segment is a P2A segment; and / or e) the immunogenic polypeptide comprises any one of the amino acid sequences provided in Table 1H or Table 1J; and / or 9) capable of inducing a T cell response in vitro and / or in vivo, optionally, wherein the T cell response is measured by a tetramer staining assay, a T cell activation assay, a CD137 staining assay, an intracellular IFNg staining assay, a cytokine release assay, and / or a T cell proliferation assay. **Claim 2** An immunogenic composition comprising at least one immunogenic polypeptide according to claim 1, optionally, wherein the immunogenic composition: 1) a) One or more full-length SARS-CoV-2 proteins, or one or more protein fragments thereof, selected from the group consisting of Orf1ab, M, N, Orf3a, and S, optionally, wherein the fragments of the one or more full-length SARS-CoV-2 proteins include SARS-CoV-2 proteins that do not encode functional SARS-CoV-2 proteins, and / or b) An adjuvant, further comprising; 2) Capable of inducing a T cell response in vitro and / or in vivo, optionally, wherein the T cell response is measured by a tetramer staining assay, a T cell activation assay, a CD137 staining assay, an intracellular IFNg staining assay, a cytokine release assay, and / or a T cell proliferation assay; 3) Capable of inducing a T cell response in a subject; and / or 4) Comprising MHC molecules, optionally, wherein the MHC molecules are a) MHC multimers, optionally, wherein the MHC multimers are tetramers; b) MHC class I molecules; and / or c) An MHC alpha chain comprising an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and / or HLA-B*07, wherein the HLA alleles are HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 alleles, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 alleles, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 alleles, HLA-A*2402, HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 alleles, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles, and are selected from the group consisting of.

3. A stable MHC-peptide complex comprising a peptide epitope of the at least one immunogenic polypeptide according to claim 1 in an MHC molecule, or an immunogenic composition comprising the stable MHC-peptide complex and an adjuvant, optionally, wherein 1) The MHC molecule is a) An MHC multimer, optionally, wherein the MHC multimer is a tetramer; b) An MHC class I molecule; and / or c) Optionally comprising an MHC alpha chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and / or HLA-B*07, wherein the HLA alleles are HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 alleles, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 alleles, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 alleles, HLA-A*2402, HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 alleles, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles, and are selected from the group consisting of; 2) The peptide epitope and the MHC molecule are covalently bound, and / or the alpha and beta chains of the MHC molecule are covalently bound; and / or 3) The stable MHC-peptide complex optionally comprises a detectable label, wherein the detectable label is a fluorescent dye molecule. **Claim 4** An isolated nucleic acid encoding the immunogenic polypeptide according to claim 1, or its complement, or a vector comprising the isolated nucleic acid, optionally, wherein (1) the isolated nucleic acid is DNA, RNA, chemically modified RNA, mRNA, cDNA, self-replicating, circularized, concatenated, wherein the isolated nucleic acid comprises a 5'untranslated region (5'UTR) and / or a 3'UTR, comprises an expression promoter, comprises an internal ribosome entry site (IRES), and / or comprises a self-cleaving 2A peptide such as P2A or T2A; and / or (2) the vector is an expression vector. **Claim 5** A cell that is: a) comprising the isolated nucleic acid or vector according to claim 4, and / or b) producing one or more of the immunogenic polypeptides according to claim 1 and / or presenting one or more of the stable MHC-peptide complexes according to claim 3 on its cell surface, optionally, wherein the cell is genetically engineered. **Claim 6** A binding moiety that specifically binds to the immunogenic polypeptide according to claim 1 and / or to the stable MHC-peptide complex according to claim 3, optionally, wherein the binding moiety is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain. **Claim 7** a) one or more of the immunogenic polypeptides according to claim 1, and / or b) one or more of the stable MHC-peptide complexes according to claim 3, comprised in a device or kit, wherein the device or kit optionally comprises reagents for detecting that a) and / or b) bind to a T cell receptor; optionally, wherein the immunogenic peptide is expressed by a cell and the cell is grown and / or isolated in one or more steps. **Claim 8** A method for detecting T cells that bind to a stable MHC-peptide complex, comprising: a) contacting a sample comprising T cells with the stable MHC-peptide complex according to claim 3; and, b) detecting binding of T cells to the stable MHC-peptide complex, optionally further measuring the proportion of stable MHC-peptide-specific T cells that bind to the stable MHC-peptide complex, optionally, wherein the sample comprises peripheral blood mononuclear cells (PBMCs), optionally, wherein 1) the T cells are CD8+ T cells; 2) performing the detecting step and / or the measuring step using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blot, or intracellular flow assay; 3) the sample comprises T cells that have been contacted with or are suspected of having been contacted with one or more SARS-CoV-2 proteins or fragments thereof; 4) placing the agent in contact with the stable MHC-peptide complex and T cells under conditions and for a time suitable for the formation of at least one immune complex; and / or 5) growing and / or isolating the stable MHC-peptide complex that is expressed by the cells and the cells in one or more steps.

9. A method for determining whether a subject has been exposed to and / or is protected from SARS-CoV-2, comprising: a) incubating a cell population comprising T cells obtained from the subject with the immunogenic polypeptide according to claim 1 or the stable MHC-peptide complex according to claim 3; and, b) detecting the presence or level of responsiveness, wherein the presence of responsiveness or a level higher than a control level indicates that the subject has been exposed to and / or is protected from SARS-CoV-2; optionally, wherein 1) the level of responsiveness is a) indicated by the presence of binding, and / or b) T cell activation and / or effector function, optionally, wherein the T cell activation or effector function is T cell proliferation, cytotoxicity, or cytokine release; 2) Optionally, the method further includes repeating steps a) and b) at a later time point, where the subject is undergoing treatment for improving SARS-CoV-2 infection between the first time point and the later time point; 3) Detect the T cell binding, activation, and / or effector function using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemistry, Western blot, or intracellular flow assay; 4) The control level is a) a reference number; and / or b) the level of a subject not exposed to SARS-CoV-2; 5) Place the agent in contact with the immunogenic peptide and the T cell under conditions and for a time suitable for the formation of at least one immune complex therebetween; 6) Express the immunogenic peptide by a cell and grow and / or isolate the cell in one or more steps; and / or 7) Optionally, the subject is a mammal, where the mammal is a human, a primate, or a rodent.

10. A method for predicting the clinical outcome of a subject suffering from SARS-CoV-2 infection, comprising: a) measuring the presence or level of responsiveness between T cells obtained from the subject and one or more immunogenic polypeptides according to claim 1 or one or more stable MHC-peptide complexes according to claim 3; and, b) comparing the presence or level of responsiveness with the presence or level of responsiveness of a control, where the control is obtained from a subject having a good clinical outcome; where the presence of responsiveness in the subject or a level higher compared to the control indicates that the subject has a good clinical outcome; Optionally, where 1) The level of responsiveness is a) the presence of binding, and / or b) T cell activation and / or effector function, optionally indicated by, where the T cell activation or effector function is T cell proliferation, cytotoxicity, or cytokine release; 2) The method further optionally includes repeating steps a) and b) at a later time point, where the subject is undergoing treatment for improving SARS-CoV-2 infection between the first time point and the later time point; 3) Detect the T cell binding, activation, and / or effector function using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blot, or intracellular flow assay; and / or 4) The control level is a) A reference number; and / or b) The level of a subject not exposed to SARS-CoV-2; 5) Place the agent in contact with the immunogenic peptide and the T cells under conditions and for a time suitable for forming at least one immune complex therebetween; 6) The immunogenic peptide is expressed by cells, and the cells are grown and / or isolated in one or more steps; and / or 7) The subject is optionally a mammal, where the mammal is a human, a primate, or a rodent.

11. A method for evaluating the effectiveness of a SARS-CoV-2 therapy, comprising: a) Measuring the presence or level of responsiveness between T cells obtained from the subject and one or more immunogenic polypeptides according to claim 1 or one or more stable MHC-peptide complexes according to claim 3 in a first sample obtained from the subject before providing at least a portion of the SARS-CoV-2 therapy to the subject, and b) Measuring the presence or level of responsiveness between the one or more immunogenic polypeptides according to claim 1 or one or more stable MHC-peptide complexes according to claim 3 and T cells obtained from the subject present in a second sample obtained from the subject after providing at least a portion of the SARS-CoV-2 therapy, where the presence or higher level of responsiveness in the second sample relative to the first sample indicates that the therapy is effective in treating SARS-CoV-2 in the subject; Optionally, where 1) The level of responsiveness is a) The presence of binding, and / or b) T cell activation and / or effector function, optionally, as indicated by, wherein said T cell activation or effector function is T cell proliferation, cytotoxicity, or cytokine release; 2) The method further optionally includes repeating steps a) and b) at a later time point, wherein the subject is undergoing treatment for improving SARS-CoV-2 infection between the first time point and the later time point; 3) Detecting said T cell binding, activation, and / or effector function using fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunochemical, Western blot, or intracellular flow assay; 4) The control level is a) a reference number; and / or b) the level of a subject not exposed to SARS-CoV-2; 5) Placing the agent in contact under conditions and for a time suitable for forming at least one immune complex between the immunogenic peptide and the T cell; 6) Expressing the immunogenic peptide by the cell and growing and / or isolating the cell in one or more steps; and / or 7) Optionally, the subject is a mammal, wherein the mammal is a human, primate, or rodent.

12. An immunogenic composition comprising and / or encoding the at least one immunogenic polypeptide according to claim 1 or the cell according to claim 5 for use in a method of preventing and / or treating SARS-CoV-2 infection in a subject, optionally, wherein 1) The immunogenic composition optionally comprises a nucleic acid encoding the immunogenic polypeptide according to claim 1, wherein the nucleic acid is DNA, RNA, mRNA, cDNA, self-replicating, circularized, and / or concatemerized; 2) The SARS-CoV-2 protein is selected from the group consisting of orf1a / b, S protein, N protein, M protein, orf3a, and orf7a; 3) The immunogenic polypeptide is capable of inducing a T cell response in a subject; 4) The immunogenic composition a) comprises two or more immunogenic polypeptides; b) comprising an adjuvant; and / or c) capable of inducing a T cell response in a subject; 5) The administered immunogenic composition a) induces an immune response against SARS-CoV-2 in the subject; and / or b) induces a T cell immune response against SARS-CoV-2 in the subject, Optionally, wherein the T cell immune response is a CD8+ T cell immune response; 6) placing the agent in contact, under conditions and for a time suitable for the formation of at least one immune complex between the peptide epitope, immunogenic peptide, stable MHC-peptide complex, T cell receptor, and / or T cell; 7) growing and / or isolating the peptide epitope, immunogenic peptide, stable MHC-peptide complex, and / or T cell receptor-expressing cells in one or more steps; and / or 8) The subject is a mammal, optionally, wherein the mammal is a human, primate, or rodent.

13. A method for identifying a peptide-binding molecule or an antigen-binding fragment thereof that binds to a peptide epitope of the at least one immunogenic polypeptide according to claim 1, comprising: a) providing a cell that presents a peptide epitope of the at least one immunogenic polypeptide according to claim 1 among MHC molecules on the surface of the cell, optionally, wherein the cell contains a nucleic acid encoding and expressing the at least one immunogenic polypeptide; b) measuring the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to the peptide epitope among the MHC molecules on the cell; and, c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to the peptide epitope among the MHC molecules; Optionally, wherein 1) step a) comprises contacting the MHC molecules on the surface of the cell with a peptide epitope selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F; 2) step a) comprises transfecting the cell with a basic nucleic acid and / or a vector containing the basic nucleic acid; 3) The binding part structure is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein containing a TCR and an effector domain; 4) The plurality of candidate peptide-binding molecules are a) one or more T cell receptors (TCRs), or one or more antigen-binding fragments of a TCR; b) at least 2, 5, 10, 100, 10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、or more than that, various candidate peptide binding molecules; and / or c) one or more candidate peptide-binding molecules obtained from a sample from a subject or a population of subjects, or the plurality of candidate peptide-binding molecules optionally include one or more candidate peptide-binding molecules containing mutations in a parental scaffold peptide-binding molecule obtained from a sample from a subject, where i) the subject or population of subjects is a) not infected with SARS-CoV-2 and / or has recovered from COVID-19, or b) infected with SARS-CoV-2 and / or has COVID-19; ii) the subject or population of subjects has been vaccinated with one or more immunogenic peptides, where the immunogenic peptides include peptide epitopes selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F; iii) the subject is a mammal, optionally, where the mammal is a human, a primate, or a rodent; iv) the subject is an HLA-transgenic mouse and / or a human TCR transgenic mouse; and / or v) the sample optionally includes T cells, where the sample includes peripheral blood mononuclear cells (PBMCs) or CD8+ memory T cells; 5) Place the agent in contact under conditions and for a time suitable for forming at least one immune complex between the peptide epitope, immunogenic peptide, stable MHC-peptide complex, T cell receptor, and / or T cells; 6) Express the peptide epitope, immunogenic peptide, stable MHC-peptide complex, and / or T cell receptor in cells and grow and / or isolate the cells in one or more steps; and / or 7) The subject is a mammal, optionally, where the mammal is a human, a primate, or a rodent.

14. A method of identifying a peptide-binding molecule or an antigen-binding fragment thereof that binds to a peptide epitope of said at least one immunogenic polypeptide according to claim 1, comprising: a) providing a stable MHC-peptide complex that contains a peptide epitope of said at least one immunogenic polypeptide according to claim 1 within an MHC molecule; b) determining the binding of a plurality of candidate peptide-binding molecules or antigen-binding fragments thereof to said stable MHC-peptide complex; and c) identifying one or more peptide-binding molecules or antigen-binding fragments thereof that bind to said stable MHC-peptide complex; Optionally, wherein 1) said MHC molecule is a) an MHC multimer, optionally, wherein said MHC multimer is a tetramer; b) an MHC class I molecule; and / or c) An MHC alpha chain comprising an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and / or HLA-B*07, wherein the HLA alleles are HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 alleles, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 alleles, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 alleles, HLA-A*2402, HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 alleles, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 alleles, selected from the group consisting of; 2) The peptide epitope and the MHC molecule are covalently bound, and / or the alpha chain and the beta chain of the MHC molecule are covalently bound; 3) The stable MHC-peptide complex optionally comprises a detectable label, wherein the detectable label is a fluorescent dye molecule; 4) The plurality of candidate peptide binding molecules are a) one or more T cell receptors (TCRs), or one or more antigen-binding fragments of a TCR; b) at least 2, 5, 10, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or more than that, various candidate peptide binding molecules; and / or c) one or more candidate peptide binding molecules obtained from a sample from a subject or a population of subjects; or, the plurality of candidate peptide binding molecules comprises one or more candidate peptide binding molecules that contain mutations within a parental scaffold peptide binding molecule obtained from a sample from a subject, optionally, where i) the subject or population of subjects is A) not infected with SARS-CoV-2 and / or has recovered from COVID-19, or B) infected with SARS-CoV-2 and / or has COVID-19; ii) the subject or population of subjects has been vaccinated with one or more immunogenic polypeptides, where the immunogenic polypeptides comprise peptide epitopes selected from Table 1A, 1B, 1C, 1D, 1E, and / or 1F; iii) the subject is a mammal, optionally, where the mammal is a human, primate, or rodent; iv) the subject is an HLA-transgenic mouse and / or a human TCR transgenic mouse; and / or v) the sample optionally comprises T cells, where the sample comprises peripheral blood mononuclear cells (PBMCs) or CD8+ memory T cells; 5) the binding moiety is an antibody, an antigen-binding fragment of an antibody, a TCR, an antigen-binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain; 6) placing the agent in contact under conditions and for a time suitable for forming at least one immune complex between the peptide epitope, immunogenic peptide, stable MHC-peptide complex, T cell receptor, and / or T cells; 7) expressing the peptide epitope, immunogenic peptide, stable MHC-peptide complex, and / or T cell receptor by cells and growing and / or isolating the cells in one or more steps; and / or 8) the subject is a mammal, optionally, where the mammal is a human, primate, or rodent. **Claim 15** Genetically engineered T cells expressing a TCR for use in the treatment of SARS-CoV-2 infection in a subject, wherein the genetically engineered T cells are 1) a TCR identified by the method according to claim 14; 2) a TCR that binds to a peptide epitope of the at least one immunogenic polypeptide according to claim 1; 3) a TCR that binds to a stable MHC-peptide complex comprising a peptide epitope of the at least one immunogenic polypeptide according to claim 1 within an MHC molecule, expressing, optionally, wherein the MHC molecule is an MHC multimer, and further optionally, wherein the MHC multimer is a tetramer; Optionally, wherein a) the MHC molecule is an MHC class I molecule; b) The MHC molecule comprises an MHC alpha chain that is an HLA serotype selected from the group consisting of HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, and / or HLA-B*07, optionally, wherein the HLA allele is HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 allele, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 allele, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 allele, HLA-A*2402, HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 allele, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, and HLA-B*0721 allele, and is selected from the group consisting of; c) The peptide epitope and the MHC molecule are covalently bound, and / or the alpha chain and beta chain of the MHC molecule are covalently bound; d) The stable MHC-peptide complex optionally comprises a detectable label, wherein the detectable label is a fluorescent dye molecule; e) The T cell is i) the subject, ii) a donor not infected with SARS-CoV-2, or iii) a donor recovered from COVID-19, and isolating from; f) placing the agent in contact, under conditions and for a time suitable for the formation of at least one immune complex between the peptide epitope, immunogenic peptide, stable MHC-peptide complex, T cell receptor, and / or T cells; g) growing and / or isolating the peptide epitope, immunogenic peptide, stable MHC-peptide complex, and / or T cell receptor-expressing cells, and / or the cells, in one or more steps; and / or h) optionally, the subject is a mammal, wherein the mammal is a human, primate, or rodent. **Claim 16** An antigen-specific T cell for use in a method of preventing or treating SARS-CoV-2 infection in a subject, wherein the antigen-specific T cell is generated by: a) stimulating PBMCs or T cells derived from the subject with an immunogenic polypeptide according to claim 1, a nucleic acid encoding the immunogenic polypeptide according to claim 1, a stable MHC-peptide complex comprising a peptide epitope of the at least one immunogenic polypeptide according to claim 1 in an MHC molecule, or a cell encoding and / or presenting the peptide epitope of the at least one immunogenic polypeptide according to claim 1 in an MHC molecule on the cell surface; and b) optionally, isolating the PBMCs or T cells from the subject prior to stimulating the PBMCs or T cells, and growing the antigen-specific T cells in vitro, optionally, wherein 1) the T cells are naive T cells, central memory T cells, or effector memory T cells, optionally, wherein the T cells are CD8+ memory T cells; 2) Place the agent in contact, under conditions and for a time suitable for the formation of at least one immune complex between the peptide epitope, immunogenic polypeptide, stable MHC-peptide complex, T cell receptor, and / or T cell; 3) Express the peptide epitope, immunogenic polypeptide, stable MHC-peptide complex, and / or T cell receptor by cells, and grow and / or isolate the cells in one or more steps; and / or 4) Optionally, the subject is a mammal, where the mammal is a human, primate, or rodent.

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