SARS-COV-2 ANTIGEN-TARGETING PEPTIDES AND ENGINEERED T-CELL RECEPTORS AND METHODS OF USE

Abstract

The present disclosure provides peptides, engineered T cell receptors (TCRs), cells containing the peptides and TCRs, and methods of making and using the peptides and TCRs, useful for vaccination and other applications. The present disclosure relates to TCRs that specifically recognize the SARS-Cov-2 HLA-A2 restricted peptide FVLAAVYRI (SEQ ID NO:22) from the membrane glycol-protein (MGP), MGP-65.

Description

[Technical field]

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 167,433, filed March 29, 2021; U.S. Provisional Patent Application No. 63 / 210,198, filed June 14, 2021; and U.S. Provisional Patent Application No. 63 / 314,870, filed February 28, 2022, which are incorporated by reference in their entireties.

[0002] II. FIELD OF THEINVENTION The present invention relates to the fields of immunotherapy and the treatment of viral infections. [Background technology]

[0003] III. Background Since the emergence of SARS-CoV-2 in December 2019, the World Health Organization reports that the infection has spread to more than 200 countries, with more than 170 million infections and over 3.5 million deaths worldwide. Despite this hardship, the quest to identify effective vaccines, therapeutics, and protective biomarkers continues.

[0004] SARS-CoV-2 manifests as a severe atypical pneumonia with high morbidity and mortality in humans. Dysregulated / excessive innate responses associated with T cell lymphopenia are the major drivers of lung pathology in this syndrome. Decreased T cell numbers correlate with the severity of the acute phase of SARS disease in humans and delayed viral clearance. Several lines of evidence from other respiratory viral infections, such as influenza A and parainfluenza, have demonstrated that virus-specific CD4 and CD8 T cells generated during primary and memory responses can clear the virus and protect the host from severe infection. Indeed, studies from patients and animals infected with SARS and MERS have also pointed to the important role of T cell-mediated adaptive immune responses in defense against and clearance of respiratory coronavirus infections. In patients who have recovered from SARS, neutralizing antibody titers and memory B cell responses are short-lived and last only a few months, whereas SARS-CoV-specific memory T cells persist for up to six years after infection.

[0005] Direct evidence for the role of virus-specific CD4 and CD8 T cells in SARS-CoV clearance and host defense has come from adoptive transfer studies. Adoptive transfer of SARS-Cov-specific effector CD4 and CD8 T cells into SARS-susceptible mice rapidly cleared the virus and improved clinical disease and survival of the animals. There is a need in the art for effective coronavirus treatments and prevention. Summary of the Invention

[0006] The present disclosure provides peptides, engineered T cell receptors (TCRs), cells containing the peptides and TCRs, and methods of making and using the peptides and TCRs, useful for vaccination and other applications. The present disclosure relates to TCRs that specifically recognize coronavirus peptides, such as those represented by SEQ ID NOs:22-81.

[0007] Thus, an aspect of the present disclosure relates to a polypeptide comprising an antigen-binding variable region comprising an amino acid sequence of a CDR3 of the present disclosure or an amino acid sequence having at least 80% sequence identity to a CDR3 of the present disclosure. A further aspect relates to an engineered TCR comprising a T cell receptor (TCR)-b polypeptide and a TCR-a polypeptide, wherein the TCR-b polypeptide comprises an amino acid sequence of a CDR3 of the present disclosure or an amino acid sequence having at least 80% sequence identity to a CDR3 of the present disclosure, and the TCR-a polypeptide comprises an amino acid sequence of a CDR3 of the present disclosure or an amino acid sequence having at least 80% sequence identity to a CDR3 of the present disclosure. A further aspect relates to a nucleic acid encoding a TCR-b polypeptide comprising an amino acid sequence of a CDR3 of the present disclosure or an amino acid sequence having at least 80% sequence identity to a CDR3 of the present disclosure and / or a TCR-a polypeptide comprising an amino acid sequence of a CDR3 of the present disclosure or an amino acid sequence having at least 80% sequence identity to a CDR3 of the present disclosure. The CDR3 can be the CDR3 of SEQ ID NO:7, 14, 21, 92, 99, 106, 113, 120, 127, 134 and 141. Aspects of TCR-a CDR3 include CDRs having the amino acid sequences of SEQ ID NO:7, 14, 92, 106, 120 and 134. Aspects of TCR-b CDR3 include CDRs having the amino acid sequences of SEQ ID NO:21, 99, 113, 127 and 141.

[0008] Further aspects relate to nucleic acids encoding the peptides, polypeptides, fusion proteins, and engineered TCRs. The disclosure also describes nucleic acid vectors comprising one or more nucleic acids of the disclosure, and cells comprising the peptides, fusion proteins, polypeptides, engineered TCRs, and / or nucleic acids of the disclosure. Compositions comprising the peptides, polypeptides, cells, nucleic acids, or engineered TCRs of the disclosure are also provided. Further aspects relate to methods of making engineered cells, comprising introducing the nucleic acid or vector of the disclosure into a cell. Further aspects relate to methods for treating or preventing coronavirus infection in a subject, comprising administering a polypeptide, composition, cell, nucleic acid, or engineered TCR to the subject. In some aspects, the method is for treating a sequela of a novel coronavirus infection, comprising administering a polypeptide, composition, cell, nucleic acid, or engineered TCR to the subject. Also provided are methods of stimulating an immune response in a subject, comprising administering a composition or cell of the disclosure to the subject. Methods also include a method of reducing tumor burden; a method of lysing cancer cells; a method of killing tumors / cancer cells; a method of increasing overall survival; a method of reducing the risk of developing cancer or a tumor; a method of increasing recurrence-free survival; a method of preventing cancer; and / or a method of reducing, eliminating or decreasing the spread or metastasis of cancer, comprising administering a polypeptide, composition, cell, nucleic acid or engineered TCR to a subject in need thereof.

[0009] Further aspects relate to fusion proteins comprising the TCR of the present disclosure, or its antigen-binding fragment, and a CD3-binding region. The CD3-binding region can comprise a CD3-specific fragment antigen-binding (Fab), a single-chain variable fragment (scFv), a single-domain antibody, or a single-chain antibody. Exemplary CD3-specific fragment antigen-binding (Fab) are known in the art. For example, US20180222981, which is incorporated herein by reference, discloses a variable region that specifically binds to CD3 and can be used in aspects of the present disclosure. Anti-CD3 antibodies and variable regions are disclosed in US20180117152, which is also incorporated by reference.

[0010] Aspects of the disclosure relate to peptides that comprise at least 60% sequence identity to one of the peptides of SEQ ID NOs:22-81. Also provided are peptides that comprise at least 60% sequence identity to one of the peptides of SEQ ID NOs:22-38. The peptide or polypeptide may have at least 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 or 100% sequence identity to one of SEQ ID NOs:22-81. (or any range derivable therein), or may have at least 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 or 100% (or any range derivable therein) sequence identity. The peptide may be selected from the group consisting of SEQ ID NOs: 22-38 and 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 or 100%. (or any range derivable therein) of sequence identity, or may have at least 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 or 100% (or any range derivable therein) of sequence identity. Polypeptides comprising the peptides of the disclosure are also provided. A further aspect relates to a molecular complex comprising a peptide or polypeptide of the disclosure and an MHC polypeptide.Another aspect of the present disclosure relates to a method for producing peptide / coronavirus-specific immune effector cells, comprising the steps of: (a) obtaining a starting population of immune effector cells; and (b) contacting the starting population of immune effector cells with a peptide of the present disclosure, thereby generating peptide / coronavirus-specific immune effector cells. The present disclosure also describes engineered T cells and TCRs specific for peptide / coronavirus produced by the method of the present disclosure. A further aspect relates to in vitro isolated dendritic cells comprising the peptide, polypeptide, nucleic acid or expression vector of the present disclosure.

[0011] Also provided is a method for prognosing a patient or detecting a T cell response in a patient, comprising contacting a biological sample from the patient with a composition, peptide or polypeptide of the present disclosure. A further aspect relates to a peptide / coronavirus-specific binding molecule that binds to the peptide of the present disclosure or binds to a peptide-MHC complex. Exemplary binding molecules include antibodies, TCR mimetic antibodies, scFvs, nanobodies, camelids, aptamers and DARPINs. A related method provides a method comprising contacting a composition comprising at least one MHC polypeptide and a peptide or polypeptide of the present disclosure with a composition comprising T cells, and detecting the T cells with bound peptide and / or MHC polypeptide by detecting the detection tag. A further aspect relates to a kit comprising the peptide, polypeptide, nucleic acid, expression vector or composition of the present disclosure. Further method aspects relate to a method of cloning a coronavirus T cell receptor (TCR), comprising: (a) obtaining a starting population of immune effector cells; (b) contacting the starting population of immune effector cells with a coronavirus peptide of the present disclosure, thereby generating coronavirus-specific immune effector cells; (c) purifying the coronavirus peptide-specific immune effector cells; and (d) isolating the TCR sequence from the purified immune effector cells. Further aspects relate to a method of generating cells, comprising transferring a nucleic acid or expression vector of the present disclosure into the cells. Further aspects relate to an in vitro method for generating a therapeutic T cell vaccine, comprising co-culturing T cells with a peptide of the present disclosure. In some aspects, the method is for treating or preventing SARS. In some aspects, the method is for treating or preventing COVID-19. In some aspects, COVID-19 is further defined as a sequela of a novel coronavirus infection.

[0012] Nucleic acids of the present disclosure include those encoding the CDR regions, variable regions, engineered TCRs, polypeptides, TCR-a polypeptides, TCR-b polypeptides, peptides, polypeptides and fusion proteins described herein. The nucleic acid may be RNA. The nucleic acid may be cDNA or DNA encoding a peptide or polypeptide, or the complement of a peptide or polypeptide. The nucleic acid may comprise one of SEQ ID NOs: 1, 8, 15, 86, 93, 100, 107, 114, 121, 128 or 135, or a fragment thereof. In some aspects, the nucleic acid has 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs: 1, 8, 15, 86, 93, 100, 107, 114, 121, 128 or 135, or a fragment thereof. (or any range derivable therein).

[0013] The polypeptide or TCR-a polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:7, or having at least 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 or 100% (or any range derivable therein) sequence identity. In some aspects, the polypeptide or TCR-a polypeptide comprises a CDR3 having the amino acid sequence of SEQ ID NO:7. The polypeptide or TCR-a polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:14, or at least 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 or 100% (or any range derivable therein) sequence identity. In some aspects, the polypeptide or TCR-a polypeptide comprises a CDR3 having the amino acid sequence of SEQ ID NO:14. The polypeptide may comprise a CDR3 comprising the amino acid sequence of one of SEQ ID NOs:7, 14 or 21.The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:21, or having at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO:21. The engineered TCR may comprise: (i) a TCR-a polypeptide comprising a CDR3 having an amino acid sequence of SEQ ID NO:7 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:7; or (ii) a TCR-a polypeptide comprising a CDR3 having an amino acid sequence of SEQ ID NO:14 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:14; and a TCR-b polypeptide comprising a CDR3 having an amino acid sequence of SEQ ID NO:21 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:21.The engineered TCR may comprise (i) a CDR3 having an amino acid sequence of SEQ ID NO:7 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:7; or (ii) a CDR3 having an amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:7; a TCR-a polypeptide comprising a CDR3 having an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:14, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:14; and The TCR-b polypeptide may comprise a TCR-b polypeptide comprising a CDR3 having an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to NO:21, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0014] The polypeptide or TCR may comprise a variable region comprising CDR1, CDR2, and / or CDR3 from a TCR-a polypeptide and / or a TCR-b polypeptide. The variable region may comprise a CDR1 having an amino acid sequence of one of SEQ ID NO:5, 12, or 19, or having at least 80% sequence identity to one of SEQ ID NO:5, 12, or 19. The variable region may comprise a CDR1 having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:5, 12 or 19, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0015] The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:5 and 12, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising the amino acid sequence of one of SEQ ID NOs:5 and 12. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:19, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:19.

[0016] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:5 and 12, or at least 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:5 and 12, The TCR-b polypeptide may comprise a TCR-b polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to NO:19, or at least 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 or 100% (or any range derivable therein) sequence identity. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:5 or 12 and a TCR-b polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:19.

[0017] The polypeptide, TCR-a or TCR-b, may comprise a variable region. The variable region may comprise a CDR2 having an amino acid sequence of one of SEQ ID NOs:6, 13 and 20, or having at least 80% sequence identity to one of SEQ ID NOs:6, 13 and 20. The variable region may comprise a CDR2 having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:6, 13 and 20, or having at least 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 or 100% (or any range derivable therein) sequence identity.

[0018] The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:6 and 13, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising the amino acid sequence of one of SEQ ID NOs:6 and 13. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:20, or having at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:20.

[0019] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:6 and 13, or at least 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:6 and 13, and 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:20, or may comprise a TCR-b polypeptide comprising a CDR2 having an amino acid sequence with at least 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 or 100% (or any range derivable therein) sequence identity to NO:20. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having the amino acid sequence of one of SEQ ID NOs:6 and 13, and a TCR-b polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:20.

[0020] The variable region may comprise an amino acid sequence having at least 70% sequence identity to one of SEQ ID NOs:3, 10, and 17. The variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:3, 10 and 17, or has at least 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 or 100% (or any range derivable therein) sequence identity. The variable region may comprise the amino acid sequence of one of SEQ ID NOs:3, 10 and 17. The TCR-a variable region may comprise an amino acid sequence having at least 70% sequence identity to one of SEQ ID NOs:3 and 10. The TCR-a variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity, or has at least 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:3 and 10. The TCR-a variable region may comprise an amino acid sequence of one of SEQ ID NOs:3 and 10. The TCR-b variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:17.The TCR-b variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:17, or has at least 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 or 100% (or any range derivable therein) sequence identity. The TCR-b variable region may comprise the amino acid sequence of SEQ ID NO:17.

[0021] The polypeptide may comprise a T cell receptor alpha (TCR-a) variable region. The variable region may comprise CDR1, CDR2, and / or CDR3. The polypeptide may comprise a TCR-a variable and constant region. The polypeptide may comprise or further comprise a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to one of SEQ ID NOs:4, 11, and 18. The signal peptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity, or having at least 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:4, 11 and 18. The signal peptide may comprise an amino acid sequence of one of SEQ ID NOs:4, 11 and 18.

[0022] The TCR aspects of the disclosure relate to TCRs comprising TCR-a and TCR-b polypeptides, wherein the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise (i) the amino acid sequences of SEQ ID NOs:5, 6, and 7, respectively; or (ii) the amino acid sequences of SEQ ID NOs:12, 13, and 14, respectively; and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs:19, 20, and 21, respectively.

[0023] The TCR-a polypeptide may comprise the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:2, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:2, and the TCR-b polypeptide may comprise the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: The polypeptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to NO:16, or at least 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 or 100% (or any range derivable therein) sequence identity to NO:16.The TCR-a polypeptide may comprise the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:9, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:9, and the TCR-b polypeptide may comprise the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:16, or an amino acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity to NO:16.

[0024] The TCR-a polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:1, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:1, and the TCR-b polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:15 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:1. The nucleic acid sequence may have 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 or 100% (or any range derivable therein) sequence identity to NO:15, or may be encoded by a nucleic acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity.The TCR-a polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:8 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:8, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:8, and the TCR-b polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:15 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: The nucleic acid sequence may have 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 or 100% (or any range derivable therein) sequence identity to NO:15, or may be encoded by a nucleic acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity.

[0025] The polypeptide or TCR-a polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:92, or at least 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 or 100% (or any range derivable therein) sequence identity. In some aspects, the polypeptide or TCR-a polypeptide comprises a CDR3 having the amino acid sequence of SEQ ID NO:92. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:99, or having at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO:99. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having an amino acid sequence of SEQ ID NO:92 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:92, and a TCR-b polypeptide comprising a CDR3 having an amino acid sequence of SEQ ID NO:99 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:99.The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO:92 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:92, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:92; 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:99, or may comprise a TCR-b polypeptide comprising a CDR3 having an amino acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity to NO:99.

[0026] The polypeptide or TCR may comprise a variable region comprising CDR1, CDR2, and / or CDR3 from a TCR-a polypeptide and / or a TCR-b polypeptide. The variable region may comprise a CDR1 having the amino acid sequence of SEQ ID NO:90 or 97, or having at least 80% sequence identity to SEQ ID NO:90 or 97. The variable region may comprise a CDR1 having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:90 or 97, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0027] The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:90, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:90. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:97, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:97.

[0028] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:90, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:90, The TCR-b polypeptide may comprise a TCR-b polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to NO:97, or at least 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 or 100% (or any range derivable therein) sequence identity. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:90 and a TCR-b polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:97.

[0029] The polypeptide, TCR-a or TCR-b, may comprise a variable region. The variable region may comprise a CDR2 having an amino acid sequence of one of SEQ ID NOs:91 or 98, or having at least 80% sequence identity to one of SEQ ID NOs:91 or 98. The variable region may comprise a CDR2 having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:91 or 98, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0030] The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:91, or having at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:91. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:98, or having at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:98.

[0031] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:91, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:91, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:98, or may comprise a TCR-b polypeptide comprising a CDR2 having an amino acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity to NO:98. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:91 and a TCR-b polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:98.

[0032] The variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:88 or 95. The variable region may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:88 or 95, or at least 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 or 100% (or any range derivable therein) sequence identity. The variable region may comprise the amino acid sequence of SEQ ID NO:88 or 95. The TCR-a variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:88. The TCR-a variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:88, or has at least 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 or 100% (or any range derivable therein) sequence identity. The TCR-a variable region may comprise the amino acid sequence of SEQ ID NO:88. The TCR-b variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:95.The TCR-b variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:95, or has at least 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 or 100% (or any range derivable therein) sequence identity. The TCR-b variable region may comprise the amino acid sequence of SEQ ID NO:95.

[0033] The polypeptide may comprise a T cell receptor alpha (TCR-a) variable region. The variable region may comprise CDR1, CDR2, and / or CDR3. The polypeptide may comprise a TCR-a variable and constant region. The polypeptide may comprise or further comprise a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to one of SEQ ID NOs:89 and 96. The signal peptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity, or having at least 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs:89 and 96. The signal peptide may comprise the amino acid sequence of one of SEQ ID NOs:89 and 96.

[0034] The TCR aspects of the disclosure relate to TCRs comprising TCR-a and TCR-b polypeptides, wherein the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs:90, 91, and 92, respectively; and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs:97, 98, and 99, respectively.

[0035] The TCR-a polypeptide may comprise the amino acid sequence of SEQ ID NO:87 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:87, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:87, and the TCR-b polypeptide may comprise the amino acid sequence of SEQ ID NO:94 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: The polypeptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to NO:94, or at least 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 or 100% (or any range derivable therein) sequence identity to NO:94.

[0036] The TCR-a polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:86 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:86, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:86, and the TCR-b polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:93 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: The nucleic acid sequence may have 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 or 100% (or any range derivable therein) sequence identity to NO:93, or may be encoded by a nucleic acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity.

[0037] The polypeptide or TCR-a polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:106, or at least 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 or 100% (or any range derivable therein) sequence identity. In some aspects, the polypeptide or TCR-a polypeptide comprises a CDR3 having the amino acid sequence of SEQ ID NO:106. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:113, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO: 113. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 106 and a TCR-b polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO: 113 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 113.The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having an amino acid sequence of SEQ ID NO:106 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:106, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:106, and 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:113, or may comprise a TCR-b polypeptide comprising a CDR3 having an amino acid sequence with at least 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 or 100% (or any range derivable therein) sequence identity.

[0038] The polypeptide or TCR may comprise a variable region comprising CDR1, CDR2, and / or CDR3 from a TCR-a polypeptide and / or a TCR-b polypeptide. The variable region may comprise a CDR1 having the amino acid sequence of SEQ ID NO:104 or 111, or having at least 80% sequence identity to SEQ ID NO:104 or 111. The variable region may comprise a CDR1 having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs: 104 or 111, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0039] The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:104, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:104. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:111, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:111.

[0040] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:104, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:104. 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:111, or may comprise a TCR-b polypeptide comprising a CDR1 having an amino acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity to NO:111. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:104 and a TCR-b polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:111.

[0041] The polypeptide, TCR-a or TCR-b, may comprise a variable region. The variable region may comprise a CDR2 having an amino acid sequence of SEQ ID NO:105 or 112, or having at least 80% sequence identity to SEQ ID NO:105 or 112. The variable region may have 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:105 or 112, or may comprise a CDR2 having at least 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 or 100% (or any range derivable therein) sequence identity.

[0042] The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:105, or having at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:105. The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:105. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:112, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:112.

[0043] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:105, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:105. 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:112, or may comprise a TCR-b polypeptide comprising a CDR2 having an amino acid sequence with at least 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 or 100% (or any range derivable therein) sequence identity to NO:112. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:105 and a TCR-b polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:112.

[0044] The variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:102 or 109. The variable region may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:102 or 109, or at least 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 or 100% (or any range derivable therein) sequence identity. The variable region may comprise the amino acid sequence of SEQ ID NO:102 or 109. The TCR-a variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:102. The TCR-a variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:102, or has at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:102. The TCR-a variable region may comprise the amino acid sequence of SEQ ID NO:102. The TCR-b variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:109.The TCR-b variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: 109, or has at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: 109. The TCR-b variable region may comprise the amino acid sequence of SEQ ID NO: 109.

[0045] The polypeptide may comprise a T cell receptor alpha (TCR-a) variable region. The variable region may comprise CDR1, CDR2, and / or CDR3. The polypeptide may comprise a TCR-a variable and constant region. The polypeptide may comprise or further comprise a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to one of SEQ ID NOs: 103 and 110. The signal peptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity, or having at least 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs: 103 and 110. The signal peptide may comprise an amino acid sequence of one of SEQ ID NOs: 103 and 110.

[0046] The TCR aspects of the disclosure relate to TCRs comprising TCR-a and TCR-b polypeptides, wherein the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs:104, 105, and 106, respectively; and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs:111, 112, and 113, respectively.

[0047] The TCR-a polypeptide may comprise the amino acid sequence of SEQ ID NO:101 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:101, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:101, and the TCR-b polypeptide may comprise the amino acid sequence of SEQ ID NO:108 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: The polypeptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to NO:108, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0048] The TCR-a polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:100 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:100, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:100, and the TCR-b polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:107 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:107. The nucleic acid sequence may have 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 or 100% (or any range derivable therein) sequence identity to ID NO:107, or may be encoded by a nucleic acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity.

[0049] The polypeptide or TCR-a polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:120, or at least 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 or 100% (or any range derivable therein) sequence identity. In some aspects, the polypeptide or TCR-a polypeptide comprises a CDR3 having the amino acid sequence of SEQ ID NO:120. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:127, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO: 127. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO: 120 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 120 and a TCR-b polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO: 127 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 127.The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having an amino acid sequence of SEQ ID NO:120 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:120, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:120; The TCR-b polypeptide may comprise a TCR-b polypeptide comprising a CDR3 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to NO:127, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0050] The polypeptide or TCR may comprise a variable region comprising CDR1, CDR2, and / or CDR3 from a TCR-a polypeptide and / or a TCR-b polypeptide. The variable region may comprise a CDR1 having the amino acid sequence of SEQ ID NO:118 or 125, or having at least 80% sequence identity to SEQ ID NO:118 or 125. The variable region may comprise a CDR1 having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs: 118 or 125, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0051] The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:118, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:118. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:125, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:125.

[0052] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:118, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:118. The TCR-b polypeptide may comprise a TCR-b polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to NO:125, or at least 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 or 100% (or any range derivable therein) sequence identity. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:118 and a TCR-b polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:125.

[0053] The polypeptide, TCR-a or TCR-b, may comprise a variable region. The variable region may comprise a CDR2 having an amino acid sequence of SEQ ID NO:119 or 126, or having at least 80% sequence identity to SEQ ID NO:119 or 126. The variable region may have 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:119 or 126, or may comprise a CDR2 having at least 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 or 100% (or any range derivable therein) sequence identity.

[0054] The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:119, or having at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:119. The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:119. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:126, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:126.

[0055] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:119, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:119, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:126, or may comprise a TCR-b polypeptide comprising a CDR2 having an amino acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity to NO:126. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:119 and a TCR-b polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:126.

[0056] The variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:116 or 123. The variable region may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:116 or 123, or at least 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 or 100% (or any range derivable therein) sequence identity. The variable region may comprise the amino acid sequence of SEQ ID NO:116 or 123. The TCR-a variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:116. The TCR-a variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:116, or has at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:116. The TCR-a variable region may comprise the amino acid sequence of SEQ ID NO:116. The TCR-b variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:123.The TCR-b variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: 123, or has at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: 123. The TCR-b variable region may comprise the amino acid sequence of SEQ ID NO: 123.

[0057] The polypeptide may comprise a T cell receptor alpha (TCR-a) variable region. The variable region may comprise CDR1, CDR2, and / or CDR3. The polypeptide may comprise a TCR-a variable and constant region. The polypeptide may comprise or further comprise a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to one of SEQ ID NOs:117 and 124. The signal peptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity, or having at least 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs: 117 and 124. The signal peptide may comprise an amino acid sequence of one of SEQ ID NOs: 117 and 124.

[0058] The TCR aspects of the disclosure relate to TCRs comprising TCR-a and TCR-b polypeptides, wherein the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs:118, 119, and 120, respectively; and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs:125, 126, and 127, respectively.

[0059] The TCR-a polypeptide may comprise the amino acid sequence of SEQ ID NO:115 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:115, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:115, and the TCR-b polypeptide may comprise the amino acid sequence of SEQ ID NO:122 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: The polypeptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to NO:122, or at least 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 or 100% (or any range derivable therein) sequence identity to NO:122.

[0060] The TCR-a polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:114 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:114, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:114, and the TCR-b polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:121 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:121. The nucleic acid sequence may have 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 or 100% (or any range derivable therein) sequence identity to ID NO:121, or may be encoded by a nucleic acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity to ID NO:121.

[0061] The polypeptide or TCR-a polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:134, or at least 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 or 100% (or any range derivable therein) sequence identity. In some aspects, the polypeptide or TCR-a polypeptide comprises a CDR3 having the amino acid sequence of SEQ ID NO:134. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:141, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR3 comprising the amino acid sequence of SEQ ID NO:141. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO:134 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:134 and a TCR-b polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO:141 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:141.The engineered TCR may comprise a TCR-a polypeptide comprising a CDR3 having the amino acid sequence of SEQ ID NO:134 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:134, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:134, and 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:141, or may comprise a TCR-b polypeptide comprising a CDR3 having an amino acid sequence with at least 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 or 100% (or any range derivable therein) sequence identity to NO:141.

[0062] The polypeptide or TCR may comprise a variable region comprising CDR1, CDR2, and / or CDR3 from a TCR-a polypeptide and / or a TCR-b polypeptide. The variable region may comprise a CDR1 having the amino acid sequence of SEQ ID NO:132 or 139, or having at least 80% sequence identity to SEQ ID NO:132 or 139. The variable region may comprise a CDR1 having 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs: 132 or 139, or at least 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 or 100% (or any range derivable therein) sequence identity.

[0063] The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:132, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-a polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:132. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:139, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide, TCR or TCR-b polypeptide may comprise a CDR1 comprising the amino acid sequence of SEQ ID NO:139.

[0064] The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:132, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:132, The TCR-b polypeptide may comprise a TCR-b polypeptide comprising a CDR1 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to NO:139, or at least 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 or 100% (or any range derivable therein) sequence identity. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:132 and a TCR-b polypeptide comprising a CDR1 having the amino acid sequence of SEQ ID NO:139.

[0065] The polypeptide, TCR-a or TCR-b, may comprise a variable region. The variable region may comprise a CDR2 having the amino acid sequence of SEQ ID NO:133 or 140, or having at least 80% sequence identity to SEQ ID NO:133 or 140. The variable region may have 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:133 or 140, or may comprise a CDR2 having at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:133 or 140.

[0066] The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:133, or having at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:133. The polypeptide or TCR-a polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:133. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:140, or at least 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 or 100% (or any range derivable therein) sequence identity. The polypeptide or TCR-b polypeptide may comprise a CDR2 comprising the amino acid sequence of SEQ ID NO:140.

[0067] The engineered TCR comprises a TCR-a polypeptide comprising a CDR2 having an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:133, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:133, 95, 96, 97, 98, 99 or 100% (or any range derivable therein) sequence identity to NO:140, or may comprise a TCR-b polypeptide comprising a CDR2 having an amino acid sequence with at least 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 or 100% (or any range derivable therein) sequence identity to NO:140. The engineered TCR may comprise a TCR-a polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:133 and a TCR-b polypeptide comprising a CDR2 having the amino acid sequence of SEQ ID NO:140.

[0068] The variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:130 or 137. The variable region may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:130 or 137, or at least 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 or 100% (or any range derivable therein) sequence identity. The variable region may comprise the amino acid sequence of SEQ ID NO:130 or 137. The TCR-a variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:130. The TCR-a variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:130, or has at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:130. The TCR-a variable region may comprise the amino acid sequence of SEQ ID NO:130. The TCR-b variable region may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:137.The TCR-b variable region may comprise an amino acid sequence that has 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO: 137, or has at least 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 or 100% (or any range derivable therein) sequence identity. The TCR-b variable region may comprise the amino acid sequence of SEQ ID NO: 137.

[0069] The polypeptide may comprise a T cell receptor alpha (TCR-a) variable region. The variable region may comprise CDR1, CDR2, and / or CDR3. The polypeptide may comprise a TCR-a variable and constant region. The polypeptide may comprise or further comprise a signal peptide. The signal peptide may comprise an amino acid sequence having at least 80% identity to one of SEQ ID NOs:131 and 138. The signal peptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity, or having at least 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 or 100% (or any range derivable therein) sequence identity to one of SEQ ID NOs: 131 and 138. The signal peptide may comprise an amino acid sequence of one of SEQ ID NOs: 131 and 138.

[0070] The TCR aspects of the disclosure relate to TCRs comprising TCR-a and TCR-b polypeptides, wherein the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs:132, 133, and 134, respectively; and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs:1139, 140, and 141, respectively.

[0071] The TCR-a polypeptide may comprise the amino acid sequence of SEQ ID NO:129 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:129, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:129, and the TCR-b polypeptide may comprise the amino acid sequence of SEQ ID NO:136 or an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:136. The polypeptide may comprise an amino acid sequence having 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 or 100% (or any range derivable therein) sequence identity to NO:136, or at least 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 or 100% (or any range derivable therein) sequence identity to NO:136.

[0072] The TCR-a polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:128 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:128, or at least 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:128, and the TCR-b polypeptide may be encoded by the nucleic acid sequence of SEQ ID NO:135 or a nucleic acid sequence having 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 or 100% (or any range derivable therein) sequence identity to SEQ ID NO:135. The nucleic acid sequence may have 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 or 100% (or any range derivable therein) sequence identity to ID NO:135, or may be encoded by a nucleic acid sequence having at least 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 or 100% (or any range derivable therein) sequence identity to ID NO:135.

[0073] The TCR may contain modifications or may be chimeric. The variable region of the TCR may be fused to a TCR constant region that is different from the constant region of the cloned TCR that specifically binds the peptide of the present disclosure.

[0074] The TCR-a polypeptide and the TCR-b polypeptide may be operably linked. The term "operably linked" may refer to a covalent bond, such as a peptide bond (e.g., two elements are polypeptides and are on the same polypeptide), or a non-covalent bond, such as van der Waals forces (e.g., two polypeptides that have some degree of specific binding affinity for each other). The TCR-a polypeptide and the TCR-b polypeptide may be operably linked by a peptide bond. The TCR-a polypeptide and the TCR-b polypeptide may be on the same polypeptide, with the TCR-b being amino-proximal to the TCR-a. The TCR-a polypeptide and the TCR-b polypeptide may be on the same polypeptide, with the TCR-a being amino-proximal to the TCR-b. Thus, the TCR may be a single chain TCR. The single chain TCR may include or further include a linker between the TCR-a polypeptide and the TCR-b polypeptide. The linker may be a linker described herein or known in the art. The linker may also comprise or consist of glycine and serine residues. In some aspects, the linker is composed of only glycine and serine residues (glycine-serine linker). The linker may be a flexible linker. Exemplary flexible linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n, (G4S)n and (GGGS)n, where n is an integer of at least 1). In some aspects, n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or any range derivable therein), at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or any range derivable therein), or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or any range derivable therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art may be used as linkers in the polypeptides of the present disclosure. Exemplary linkers may include or consist of GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, and the like.When a first region is attached to the carboxy terminus of a second region, the first region is carboxy-proximal to the second region. There may be additional intervening amino acid residues between the first and second regions. Thus, unless specifically specified as having no intervening amino acid residues, the regions need not be directly adjacent. The term "amino-proximal" is similarly defined as when a first region is attached to the amino terminus of a second region, the first region is amino-proximal to the second region. Similarly, there may be additional intervening amino acid residues between the first and second regions, unless specifically specified.

[0075] CDRs may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 18, 19, 20, 21, 22, 23 or more (or any range derivable therein) contiguous amino acid residues flanking either or both sides of the particular CDR sequence in the context of the variable region of the TCR-a or TCR-b polypeptide; thus, there may be one or more additional amino acids at the N-terminus or C-terminus of the particular CDR sequence, such as those shown in SEQ ID NOs: 3, 10 and 17. Alternatively, or in combination, the CDRs may also be fragments of the CDRs described herein, lacking at least 1, 2, 3, 4 or 5 amino acids from the C-terminus or N-terminus of the particular CDR sequence.

[0076] The TCR or fusion protein may be conjugated to a detection or therapeutic agent. The agent may include a fluorescent molecule, a radioactive molecule, or a toxin. The TCR or fusion protein may be conjugated to an agent described herein.

[0077] The peptide may comprise at least 6 consecutive amino acids of one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 consecutive amino acids of one of the peptides of SEQ ID NOs:22-81. The peptide may comprise or consist of one of SEQ ID NOs:22-81. The peptide may comprise or consist of one of SEQ ID NOs:22-38. In some aspects, the peptide is 13 amino acids in length or less. A peptide may have at least 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (or any range derivable therein), at most 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (or any range derivable therein), exactly 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (or any range derivable therein), or may consist of 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids (or any range derivable therein). In certain aspects, the peptide consists of 9 amino acids. The peptide may consist of 10 amino acids. The peptide may consist of 11 amino acids. The peptide may consist of 12 amino acids. The peptide may consist of 13 amino acids. In some aspects, the peptide is immunogenic. The term immunogenic may refer to the generation of an immune response, such as a protective immune response. The peptide may be modified. The modification may include conjugation with a molecule. The molecule may be an antibody, a lipid, an adjuvant, or a detection moiety (tag). The peptide may include 100% sequence identity to one of the peptides of SEQ ID NO:22-81.The peptides of the disclosure share 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 or 100% sequence identity to one of the peptides of SEQ ID NOs:22-81. or having at least 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 or 100% sequence identity. The peptide may comprise at least 63% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 66% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 70% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 72% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 77% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 80% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 80% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 81% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 88% identity to one of the peptides of SEQ ID NOs:22-81. The peptide may comprise at least 90% identity to one of the peptides of SEQ ID NOs:22-81. The peptides of the present disclosure may have one, two or three substitutions to one of the peptides of SEQ ID NOs:18-21.The peptides may have at least 1, 2, 3, 4 or 5 substitutions, or at most 1, 2, 3, 4 or 5 substitutions, relative to one of SEQ ID NOs:22-81.

[0078] The pharmaceutical composition may be formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection. The peptide may be contained in a liposome, lipid-containing nanoparticle, or lipid-based carrier. The pharmaceutical preparation may be formulated for injection or for inhalation as a nasal spray. In some aspects, the composition of the present disclosure is formulated as a vaccine. The composition may further comprise an adjuvant.

[0079] In some aspects of the present disclosure relating to dendritic cells, the dendritic cells include mature dendritic cells. The cells can be cells with HLA-A type. The HLA can be HLA-A, HLA-B, or HLA-C. In some aspects, the cells are HLA-A24 type. In some aspects, the cells are HLA-A11 type. In some aspects, the cells are HLA-A01, HLA-A02, HLA-A11, HLA-A24, HLA-B07, HLA-B08, HLA-B15, or HLA-B40.

[0080] The method may further comprise isolating the expressed peptide or polypeptide. In some aspects, the T cell comprises a CD8+ T cell. In some aspects, the T cell is a CD4+ T cell, a Th1, Th2, Th17, Th9 or Tfh T cell, a cytotoxic T cell, a memory T cell, a central memory T cell, or an effector memory T cell.

[0081] In aspects of the methods of the present disclosure, the contacting step is further defined as co-culturing the starting population of immune effector cells with an antigen-presenting cell (APC), an artificial antigen-presenting cell (aAPC), or an artificial antigen-presenting surface (aAPS); wherein the APC, aAPC, or aAPS presents the peptide on its surface. In some aspects, the APC is a dendritic cell.

[0082] In an aspect of the present disclosure, the immune effector cells are T cells, peripheral blood lymphocytes, natural killer (NK) cells, invariant NK cells, or NKT cells. The immune effector cells may be differentiated from mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells. The T cell aspect is CD8 + T cells, CD4 + The T cells further include T cells defined as T cells or γδ T cells. In certain aspects, the T cells are cytotoxic T lymphocytes (CTLs). In some aspects, the obtaining step includes isolating the starting population of immune effector cells from peripheral blood mononuclear cells (PBMCs).

[0083] The nucleic acid may comprise TCR-a (TRA) and TCR-b (TRB) genes. The nucleic acid may be polycistronic. The nucleic acid may comprise a 2A cleavable linker, such as an internal ribosome entry site (IRES) or a P2A linker. The nucleic acid may comprise a cDNA encoding the TCR-a and / or TCR-b genes. The nucleic acid may further encode a polypeptide comprising a CD3 binding region. The CD3 binding region may comprise a CD3 specific fragment antigen binding (Fab), a single chain variable fragment (scFv), a single domain antibody, or a single chain antibody.

[0084] The vector may include both TCR-a and TCR-b genes. The vector may include a promoter that directs expression of the nucleic acid. The promoter may include a murine stem cell virus (MSCV) promoter. The cells may include stem cells, progenitor cells, immune cells, or natural killer (NK) cells. The cells may include hematopoietic stem or progenitor cells, T cells, cells differentiated from mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs). The cells may be isolated or derived from peripheral blood mononuclear cells (PBMCs). The T cells may include cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, invariant NK T (iNKT) cells, gamma-delta T cells, NKT cells, or regulatory T cells. The cells may be isolated from a patient with a current or past coronavirus infection. In some aspects, the cells are isolated from a healthy subject without a current or past coronavirus infection. In some aspects, the cells are isolated from a healthy patient. The cells may be frozen or not. The cells may be in cell culture. In some aspects, the cells lack endogenous expression of TCR genes. The cells may further comprise a chimeric antigen receptor (CAR).

[0085] The composition may be certified serum-free, mycoplasma-free, endotoxin-free, and / or sterile. The method may further include culturing the cells in a medium, incubating the cells under conditions that permit the cells to divide, screening the cells, and / or freezing the cells.

[0086] In aspects of the disclosure, the coronavirus may refer to a coronavirus isolated from a bat. In some aspects of the disclosure, the coronavirus is SARS-CoV, the virus that causes SARS in humans. In some aspects, the coronavirus is SARS-CoV-2, the virus that causes COVID-19 in humans. In some aspects, the method is for treating or preventing SARS. In some aspects, the method is for treating or preventing COVID-19. In some aspects, the coronavirus is a coronavirus expressing a polypeptide comprising one of SEQ ID NOs:22-81. The subject may include a laboratory test animal, such as a mouse, rat, rabbit, dog, cat, horse, or pig. In some aspects, the subject is a human. The subject may have one or more symptoms of a coronavirus infection. In some aspects, the subject does not have any symptoms of a coronavirus infection. The subject may have and / or have been diagnosed with a coronavirus infection. In some aspects, the subject does not have and / or has not been diagnosed with a coronavirus infection. In some aspects, the subject has been previously treated for coronavirus infection. The subject may have been determined to be resistant or non-responsive to previous treatment. In some aspects, the subject is administered an additional therapeutic agent. In some aspects, the additional therapeutic agent comprises a steroid or an antiviral therapeutic agent. In some aspects, the additional therapeutic agent comprises dexamethasone, a monoclonal antibody, remdesivir, paxlovid, molnupiravir, convalescent plasma, or a combination thereof.

[0087] In some aspects, the subject has and / or has been diagnosed with SARS. In some aspects, the subject has been diagnosed with complications associated with coronavirus infection. In some aspects, the subject has been diagnosed with complications associated with COVID-19 or SARS. The complications may include pneumonia, dyspnea or shortness of breath, chest pain or pressure, acute respiratory failure, acute respiratory distress syndrome, acute cardiac injury, secondary infection, acute kidney injury, septic shock, blood clots, multisystem inflammatory syndrome, chronic fatigue, rhabdomyolysis, disseminated intravascular coagulation, or acute liver injury. In some aspects, the subject is vaccinated against coronavirus. In some aspects, the subject is vaccinated against SARS-CoV-2 or SARS-CoV. The subject may not have been vaccinated against coronavirus. In some aspects, the subject is diagnosed with a sequela of a new coronavirus infection. In some aspects, the patient is immunocompromised or immunosuppressed.

[0088] The composition of the present disclosure can be formulated as a vaccine. The composition and method of the present disclosure provide prophylactic therapy to prevent COVID-19 or SARS-Cov-2 infection. The composition can further include an adjuvant. Adjuvants are known in the art and include, for example, TLR agonists and aluminum salts.

[0089] The methods of the disclosure may further include screening the cells for one or more cellular properties, such as for immunogenicity, such as TCR expression, incorporation of a nucleic acid encoding a TCR gene, or binding of the TCR to an antigen.

[0090] The method can include administering cells, including autologous cells, or a composition including the cells. In some aspects, the cells include non-autologous cells. The cells can also be defined as allogeneic or xenogeneic cells.

[0091] The biological sample in the disclosed method may include a blood sample or a fraction thereof. In some aspects, the biological sample includes lymphocytes. In some aspects, the biological sample includes a fraction sample including lymphocytes. The biological sample may be as described herein.

[0092] The composition may comprise the MHC polypeptides and peptides of the present disclosure, where the MHC polypeptides and / or peptides are conjugated to a detection tag. Thus, suitable detection tags include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The tag may be simply detected or quantified. A simply detected response generally includes a response that simply confirms its presence, while a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other properties. In luminescence or fluorescence assays, the detectable response may occur directly using a luminophore or fluorophore associated with the assay component that actually participates in the binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component. Examples of luminescence tags that generate signals include, but are not limited to, bioluminescence and chemiluminescence. Examples of suitable fluorescent tags include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th). Detection tags also include streptavidin or its binding partner, biotin.

[0093] The MHC polypeptide and the peptide may be operably linked. The term "operably linked" refers to a situation where two components are combined or can be combined to form a complex. For example, the components may be covalently linked and / or on the same polypeptide, such as a fusion protein, or the components may have some degree of binding affinity for each other, such as binding affinity caused by van der Waals forces. Thus, aspects of the present disclosure relate to the MHC polypeptide and the peptide being operably linked by a peptide bond. Further aspects relate to the MHC polypeptide and the peptide being operably linked by van der Waals forces. The peptide-MHC may be operably linked to form a pMHC complex. In some aspects, at least two pMHC complexes are operably linked together. Other aspects include 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes operably linked to each other, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes operably linked to each other, or at most 2, 3, 4, 5, 6, 7, 8, 9, or 10 pMHC complexes operably linked to each other. In some aspects, at least two MHC polypeptides are linked to one peptide. In other aspects, the average ratio of MHC polypeptide to peptide is 1:1 to 4:1. In some aspects, the ratio or average ratio is at least 1, 2, 3, 4, 5 or 6 to about 1, 2, 3, 4, 5 or 6 (or any range derivable therein), at most 1, 2, 3, 4, 5 or 6 to about 1, 2, 3, 4, 5 or 6 (or any range derivable therein), or about 1, 2, 3, 4, 5 or 6 to about 1, 2, 3, 4, 5 or 6 (or any range derivable therein).

[0094] In some aspects of the present disclosure, the peptide is complexed with MHC. In some aspects, the MHC comprises HLA-A type. The MHC can be further defined as HLA-A3 type or HLA-A11 type. The peptide can be loaded onto dendritic cells, lymphoblastoid cells, peripheral blood mononuclear cells (PBMCs), artificial antigen presenting cells (aAPCs), or artificial antigen presenting surfaces. In some aspects, the artificial antigen presenting surface comprises MHC polypeptides conjugated or linked to the surface. Exemplary surfaces include beads, microplates, slides, or cell culture plates.

[0095] The method of the present disclosure may further include counting the number of T cells that bind to the peptide and / or MHC. The composition comprising T cells may be isolated from a subject. The subject may be as defined herein, such as a human subject. The method may further include sorting the number of T cells that bind to the peptide and / or MHC. The method of the present disclosure may also include or further include sequencing one or more TCR genes from the T cells that bind to the peptide and / or MHC. In some aspects, the method includes or further includes sequencing TCR alpha and / or beta genes from a TCR, such as a TCR that binds to a peptide of the present disclosure. The method may also include or further include grouping lymphocyte interactions by paratope hotspot (GLIPH) analysis, which is further described in Glanville et al., Nature. 2017 Jul 6; 547(7661): 94-98, which is incorporated herein by reference.

[0096] The composition of the present disclosure can be serum-free, mycoplasma-free, endotoxin-free, and / or sterile.The method can further comprise culturing the cell of the present disclosure in a medium, incubating the cell under conditions that allow the cell to divide, screening the cell, and / or freezing the cell.The method can also further comprise isolating the peptide or polypeptide expressed from the cell of the present disclosure.

[0097] The disclosed method may include or may further include screening the dendritic cells for one or more cell characteristics. In some aspects, the method further includes contacting the cells with one or more cytokines or growth factors. The one or more cytokines or growth factors may include GM-CSF. In some aspects, the cell characteristics include cell surface expression of one or more of CD86, HLA, and CD14. The dendritic cells may be derived from CD34+ hematopoietic stem or progenitor cells.

[0098] The contacting step in the disclosed method may be further defined as co-culturing the starting population of immune effector cells with antigen-presenting cells (APCs) that present the peptide on their surface. In certain aspects, the APCs are dendritic cells. In some aspects, the dendritic cells are derived from peripheral blood monocytes (PBMCs). In some aspects, the dendritic cells are isolated from PBMCs. The cells from which the dendritic cells or DCs are derived are isolated by leukapheresis.

[0099] A peptide-MHC (pMHC) complex in an aspect of the present disclosure may be generated by contacting a peptide of the present disclosure with an MHC complex. In some aspects, the peptide is expressed in a cell and binds to an endogenous MHC complex to form pMHC. In some aspects, a pMHC complex is generated using peptide exchange. For example, a cleavable peptide may be designed, such as a photocleavable peptide, that binds to and stabilizes MHC. Cleavage of the peptide (e.g., by irradiation in the case of a photocleavable peptide) dissociates the peptide from the HLA complex, resulting in an empty HLA complex that rapidly disintegrates unless UV irradiation is performed in the presence of a "rescue peptide". Thus, a peptide of the present disclosure may be used as a "rescue peptide" in a peptide exchange procedure. A further aspect of the present disclosure relates to a pMHC complex comprising a peptide of the present disclosure. The pMHC complex may be operably linked to a solid support or may be attached to a detectable moiety, such as a fluorescent molecule, a radioisotope, or an antibody. Further aspects of the disclosure relate to peptide-MHC multimeric complexes comprising at least 1, 2, 3, 4, 5 or 6 peptide-MHC molecules or at most 1, 2, 3, 4, 5 or 6 peptide-MHC molecules operably linked together. Linkage may be covalent, such as by a peptide bond, or non-covalent. In some aspects, the pMHC molecules may be linked to a biotin molecule. Such pMHC molecules may be multimerized by binding with a streptavidin molecule. The pMHC multimers may be used to detect antigen-specific T cells or TCR molecules present in a composition or tissue. The multimers may be used to detect peptide-specific T cells or coronavirus-specific T cells in situ or in a biopsy sample. The multimers may be bound to a solid support or deposited on a solid support, such as an array or slide. Cells may then be added to the slide and detection of binding between the pMHC multimers and the cells may be performed.Thus, the pMHC molecules and multimers of the present disclosure may be used to detect and diagnose SARS-Cov-2 infection in a subject or to determine the immune response in an individual with COVID-19.

[0100] As defined in the methods described herein, the obtaining step may include or may further include isolating a starting population of immune effector cells from peripheral blood mononuclear cells (PBMCs). In some aspects, the starting population of immune effector cells is obtained from a subject. The method of the present disclosure may include or may further include a step of introducing a peptide or a nucleic acid encoding a peptide into dendritic cells prior to co-culture. The introduction of the peptide may be performed by transfecting or infecting dendritic cells with a nucleic acid encoding the peptide, or by incubating the peptide with dendritic cells. The peptide or the nucleic acid encoding the peptide may be introduced by electroporation. Other methods of nucleic acid transfer, such as lipofection, calcium phosphate transfection, transfection with DEAE-dextran, microinjection, and virus-mediated transduction, are known in the art and are useful in the method of the present disclosure for transferring the nucleic acid of the present disclosure into cells. In some aspects, the peptide or the nucleic acid encoding the peptide is introduced by adding the peptide or the nucleic acid encoding the peptide to dendritic cell culture medium. In some aspects, the immune effector cells are co-cultured with a second population of dendritic cells transfected with a peptide or a nucleic acid encoding the peptide. In some aspects, after co-culture, a population of CD4-positive or CD8-positive and peptide-MHC tetramer-positive T cells is purified from the immune effector cells. In some aspects, the population of CD4-positive or CD8-positive and peptide-MHC tetramer-positive T cells is purified by fluorescence-activated cell sorting (FACS). In some aspects, a clonal population of coronavirus-specific immune effector cells is generated by limiting or serial dilution followed by expansion of individual clones by a rapid expansion protocol.

[0101] In some aspects, the purifying step further comprises generating a clonal population of coronavirus-specific immune effector cells by limiting or serial dilution of the sorted cells followed by expansion of individual clones by a rapid expansion protocol. The disclosed method may include or further comprise cloning of a T cell receptor (TCR) from a clonal population of coronavirus-specific immune effector cells. The term isolation in the disclosed method may be defined or further defined as cloning of a T cell receptor (TCR) from a clonal population of coronavirus-specific immune effector cells. In some aspects, the cloning of the TCR is cloning of the TCR α and β chains. In some aspects, the TCR is cloned using the 5'-Rapid Amplification of cDNA Ends (RACE) method. In some aspects, the TCR α and β chains are cloned using the 5'-Rapid Amplification of cDNA Ends (RACE) method. In some aspects, the cloned TCR is subcloned into an expression vector. In some aspects, the expression vector comprises a linker domain between the TCR α and TCR β sequences. The expression vector can be a retroviral vector or a lentiviral vector. The vector can be an expression vector as described herein. The linker domain can include a sequence that encodes one or more peptide cleavage sites. The one or more cleavage sites can be furin cleavage sites and / or P2A cleavage sites. In some aspects, the TCR alpha and TCR beta sequences are linked by an IRES sequence.

[0102] The host cells of the present disclosure may be transduced with an expression vector to generate engineered cells expressing the TCR α and / or β chains. In some aspects, the host cells are immune cells. The immune cells may be T cells, and the engineered cells may be referred to as engineered T cells. The T cells are CD8 +The T cells may be of the type of T cells described herein, such as T cells, CD4+ T cells, or γδ T cells. The starting population of immune effector cells may be obtained from a subject with SARS-Cov-2 infection, and the host cells are allogeneic or autologous to the subject. The coronavirus-specific T cells may be autologous or allogeneic. A population of CD4-positive or CD8-positive and peptide-MHC tetramer-positive engineered T cells may be purified from the transduced host cells. In some aspects, a clonal population of coronavirus-specific engineered T cells is generated by limiting or serial dilution, followed by expansion of individual clones by a rapid expansion protocol. In some aspects, the purifying step in the method of the present disclosure is defined as purifying a population of CD4-positive or CD8-positive and peptide-MHC tetramer-positive T cells from the immune effector cells after co-culture.

[0103] The peptide can be linked to a solid support. In some aspects, the peptide is conjugated to a solid support or bound to an antibody that is conjugated to a solid support. The solid support can include a microplate, a bead, a glass surface, a slide, or a cell culture dish. In some aspects, the solid support includes a nanofluidic chip. Detecting the T cell response can include or further include detecting the binding of the peptide to a T cell or TCR. In some aspects, detecting the T cell response includes ELISA, ELISPOT, or tetramer assay.

[0104] The methods of the present disclosure may also be used to determine the effectiveness of a vaccine, such as a coronavirus, SARS or SARS-CoV2 vaccine.

[0105] Aspects of the kit of the present disclosure may include a peptide of the present disclosure in a container. The peptide may be included in a pharmaceutical preparation. The pharmaceutical preparation may be formulated for parenteral administration or inhalation. In some aspects, the peptide is included in a cell culture medium.

[0106] The terms "subject" and "patient" may be used interchangeably and may refer to a human subject. A subject may be defined as a mammalian subject. A subject may be a mouse, rat, pig, horse, non-human primate, cat, dog, cow, etc.

[0107] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning in the field of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being utilized to determine the value.

[0108] When used in conjunction with the term "comprising," the use of the words "a" or "an" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0109] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" only, "y" only, "z" only, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z can be specifically excluded from an embodiment.

[0110] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), "characterized by" (and any form of including, such as "characterized as") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0111] The compositions and methods for their use can "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this application. The phrase "consisting of" excludes any elements, steps, or components not specified. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of the term "comprising" can also be practiced in the context of the term "consisting of" or "consisting essentially of."

[0112] Any method in the context of a therapeutic, diagnostic or physiological purpose or effect may also be described in "use" claim language, such as the "use of" any compound, composition or agent discussed herein to achieve or carry out the stated therapeutic, diagnostic or physiological purpose or effect.

[0113] The use of one or more sequences or compositions can be utilized according to any of the methods described herein.Other aspects are discussed throughout this application.Any aspect discussed with respect to one aspect of this disclosure also applies to other aspects of this disclosure, and vice versa.

[0114] It is specifically contemplated that any limitation discussed with respect to one embodiment of the present invention may be applied to any other embodiment of the present invention.Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention.Aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in different examples or elsewhere in this application, for example in the Summary of the Invention, Detailed Description of the Embodiments, Claims, and Figure Legend Descriptions.

[0115] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0116] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0117] [Figure 1] Identification of SARS-Cov-2 membrane glycoprotein (MGP) HLA-A0201-restricted peptides by MHC immunoprecipitation (IP) method. [Diagram 2] SARS-Cov-2 HLA-A0201 restricted peptide (MGP-65, FVLAAVYRI - SEQ ID NO:22) CTL generation. [Diagram 3] FIG. 3A-C: Functional validation of MGP-65 peptide-specific CTL cell lines. [Figure 4] MGP-65 CTL cell line recognition specificity detection by non-radioactive target inhibition assay. [Figure 5-1] Figure 5: Functional validation of MGP-65-specific CTL cell lines using the intracellular cytokine staining (ICS) method. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 6] SARS CoV-2 specific TCR-T production. [Figure 7] Figure 7A-C: Functional validation of SARS-CoV-2 specific TCR-T. (A) Ability of SARS-CoV-2 specific TCR-T to lyse target cells (Cr51 release assay, CRA). T2 cells pulsed with various concentrations of SARS-CoV-2 peptides were used as targets. Effector to target (E:T) ratio is 20:1. (B, C) Ability of SARS-CoV-2 TCR-T to lyse different targets (CRA). A375, Mel624 cell lines overexpressing eGFP or SARS-Cov-2 full-length protein were used as targets. [Figure 8-1] Figure 8A-L: SARS-CoV-2 derived HLA class-I peptide identification by mass spectrometry. (A) Schematic diagram of HLA-I peptide identification. MS / MS spectra from immunopeptidome and proteome analyses were searched against Swiss-Prot human and viral protein databases and filtered at 1% FDR. [Figure 8-2]Figure 8A-L: SARS-CoV-2 derived HLA class-I peptide identification by mass spectrometry. (B) MS / MS annotation of NSP13-400 peptide (VYIGDPAQL- SEQ ID NO:37). (C-E) PRM analysis of NSP13-448 peptide (IVDTVSALVY - SEQ ID NO:34), NSP13-242 peptide (TLVPQEHYV - SEQ ID NO:35) and MGP-65 peptide (FVLAAVYRI - SEQ ID NO:22). MS1 XIC area and MS / MS of each target peptide were plotted using Skyline software. [Figure 8-3] Figure 8A-L: SARS-CoV-2 derived HLA class-I peptide identification by mass spectrometry. (C-E) PRM analysis of NSP13-448 peptide (IVDTVSALVY - SEQ ID NO:34), NSP13-242 peptide (TLVPQEHYV - SEQ ID NO:35) and MGP-65 peptide (FVLAAVYRI - SEQ ID NO:22). MS1 XIC area and MS / MS of each target peptide were plotted using Skyline software. [Figure 8-4] Figure 8A-L: SARS-CoV-2 derived HLA class-I peptide identification by mass spectrometry. (F-J) Multiple sequence alignment of five candidate peptide sequences against all coronaviruses known to infect humans. [Figure 8-5] Figure 8A-L: SARS-CoV-2 derived HLA class-I peptide identification by mass spectrometry. (F-J) Multiple sequence alignment of five candidate peptide sequences against all coronaviruses known to infect humans. [Figure 8-6] Figure 8A-L: SARS-CoV-2-derived HLA class-I peptide identification by mass spectrometry. (K) Adapted from the Nextstrain project (http: / / nextstrain.org). Five candidate SARS-CoV-2 peptide sequences were located in conserved regions of the SARS-CoV-2 genome. [Figure 8-7]Figure 8A-L: SARS-CoV-2 derived HLA class-I peptide identification by mass spectrometry. (L) Four mutations were reported in the NSP13 protein: E341D, A368V (P.1 lineage), K460R (B.1.1.7 lineage) and T588I (B.1.351 lineage). The following abbreviations are used: VAR: variant, dot (.) indicates the same amino acid at that position. [Figure 9-1] Figure 9A-G: T cell generation and functional validation of membrane glycol-protein (MGP)-derived HLA-A0201 peptide MGP-65. (A) Mature dendritic cells (DCs) derived from HLA-A0201 healthy donors were pulsed with MGP-65 peptide (FVLAAVYRI - SEQ ID NO:22) and co-cultured with autologous PBMCs. After two rounds of stimulation, a small CD8+ and MGP-65 tetramer+ population was observed (left). CD8+ and MGP-65 tetramer positive cells were then sorted and expanded using a standard rapid expansion protocol (REP). After 2 weeks of expansion, highly pure CTLs (>90% tetramer+ population) were generated (right). (B) 51Cr-labeled T2 cells pulsed with various concentrations of MGP-65 peptide were co-cultured with MGP-65-specific CTLs at an effector-to-target (E:T) ratio of 20:1. The lytic ability of MGP-65-specific CTLs was detected using a standard 51Cr-release assay (CRA). Data are shown as the mean of triplicates. (C, D) 51Cr-labeled MGP or GFP-forced HLA-A0201 cell lines A375 (A375-MGP, A375-GFP) and Mel624 (Mel624-MGP, Mel624-GFP) were co-cultured with MGP-65-specific CTLs at various E:T ratios (from 40:1 to 1.25:1). The lytic ability of MGP-65-specific CTLs against different targets was detected by a standard CRA. Data are shown as the mean of triplicates. [Figure 9-2]Figure 9A-G: T cell generation and functional validation of membrane glycolipid protein (MGP)-derived HLA-A0201 peptide MGP-65. (E, F) Non-radioactive target inhibition assay. 51Cr-labeled A375-MGP and Mel624-MGP were the radioactive targets. Non-radioactive labelled T2 cells pulsed with MGP-65 peptide or M26 irrelevant peptide were the non-radioactive targets. The ratio of non-radioactive target:radioactive target was 10:1 or 20:1. MGP-65-specific CTLs were co-cultured with radioactive targets alone or with radioactive targets and with non-radioactive targets at an E:radioactive T ratio of 20:1. The lytic ability of MGP-65-specific CTLs was detected by standard CRA. Data are shown as the mean of triplicates. [Figure 9-3] Figure 9A-G: T cell generation and functional validation of membrane glycolipid protein (MGP)-derived HLA-A0201 peptide MGP-65. (G) Intracellular cytokine staining (ICS) assay. MGP-65-specific CTLs were co-cultured overnight with T2 pulsed with MGP-65 peptide or M26 irrelevant peptide, as well as A375-MGP, A375-GFP, Mel624-MGP, and Mel624-GFP in the presence of brefeldin A (BFA) at an E:T ratio of 10:1. After incubation, the levels of IFN-γ and TNF-α, as well as downstream activation markers of the TCR pathway, CD137 and CD69, were detected using flow cytometry. [Figure 10-1]Figure 10A-G: T cell generation and functional validation of HLA-A0201 peptide NSP13-242 derived from nonstructural protein NSP13. (A) PBMCs from HLA-A0201 healthy donors were co-cultured with autologous DCs pulsed with NSP13-242 peptide (TLVPQEHYV - SEQ ID NO:35). After two rounds of stimulation, CD8+ and NSP13-242 tetramer positive T cells were derived (left). CD8+ and tetramer+ T cells were sorted and subsequently expanded in REP for 2 weeks to generate highly pure NSP13-242 specific CTLs (right). (B) 51Cr-labeled T2 cells pulsed with various concentrations of NSP13-242 peptide were co-cultured with NSP13-242 specific CTLs at an E:T ratio of 20:1. The lytic ability of NSP13-242-specific CTLs was detected by standard CRA. Data are shown as the mean of triplicates. (C, D) 51Cr-labeled NSP13- or GFP-forced HLA-A0201 cell lines A375 (A375-NSP13, A375-GFP) and Mel624 (Mel624-NSP13, Mel624-GFP) were cocultured with NSP13-242-specific CTLs at various E:T ratios (from 40:1 to 1.25:1). The lytic ability of NSP13-242-specific CTLs against different targets was detected by standard CRA. Data are shown as the mean of triplicates. [Figure 10-2] Figure 10A-G: T cell generation and functional validation of HLA-A0201 peptide NSP13-242 derived from nonstructural protein NSP13. (E, F) Cold target inhibition assay. 51Cr-labeled A375-NSP13 and Mel624-NSP13 were the radioactive targets. Nonradiolabeled T2 cells pulsed with NSP13-242 peptide or M26 irrelevant peptide were the cold target. The cold target:radioactive target ratio was 10:1 or 20:1. NSP13-242-specific CTLs were co-cultured with radioactive targets alone or with radioactive targets and cold targets at an E:radioactive T ratio of 20:1. The lytic ability of NSP13-242-specific CTLs was detected by standard CRA. Data are shown as the mean of triplicates. [Figure 10-3]Figure 10A-G: T cell generation and functional validation of HLA-A0201 peptide NSP13-242 derived from nonstructural protein NSP13. (G) Intracellular cytokine staining (ICS) assay. NSP13-242-specific CTLs were co-cultured overnight with T2 pulsed with NSP13-242 peptide or M26 irrelevant peptide, as well as A375-NSP13, A375-GFP, Mel624-NSP13, and Mel624-GFP in the presence of BFA at an E:T ratio of 10:1. After incubation, the levels of IFN-γ and TNF-α, as well as downstream activation markers of the TCR pathway, CD137 and CD69, were detected using flow cytometry. [Figure 11-1] Figure 11A-G: T cell generation and functional validation of HLA-A0101 peptide NSP13-448 derived from nonstructural protein NSP13. (A) PBMCs from HLA-A0101 healthy donors were co-cultured with autologous DCs pulsed with NSP13-448 peptide (VYIGDPAQL - SEQ ID NO:37). After two rounds of stimulation, CD8+ and NSP13-448 tetramer positive T cells were derived (left). CD8+ and tetramer+ T cells were sorted and subsequently expanded in REP for 2 weeks to generate highly pure NSP13-448 specific CTLs (right). (B) 51Cr-labeled A375 cells pulsed with various concentrations of NSP13-448 peptide were co-cultured with NSP13-448 specific CTLs at an E:T ratio of 20:1. The lytic ability of NSP13-448-specific CTLs was detected by standard CRA. Data are shown as the mean of triplicates. (C, D) 51Cr-labeled NSP13- or GFP-forced HLA-A0101 cell lines A375 (A375-NSP13, A375-GFP) and RPMI-7951 (RPMI-7951-NSP13, RPMI-7951-GFP) were cocultured with NSP13-448-specific CTLs at various E:T ratios (from 40:1 to 1.25:1). The lytic ability of NSP13-448-specific CTLs against different targets was detected by standard CRA. Data are shown as the mean of triplicates. [Figure 11-2]Figure 11A-G: T cell generation and functional validation of HLA-A0101 peptide NSP13-448 derived from nonstructural protein NSP13. (E, F) Cold target inhibition assay. 51Cr-labeled A375-NSP13 and RPMI-7951-NSP13 served as the radioactive targets. Nonradiolabeled A375 cells pulsed with NSP13-448 peptide or irrelevant HLA-A0101 peptide served as the cold target. The cold target:radioactive target ratio was 10:1 or 20:1. NSP13-448-specific CTLs were co-cultured with radioactive targets alone or with radioactive targets and cold targets at an E:radioactive T ratio of 20:1. The lytic ability of NSP13-448-specific CTLs was detected by standard CRA. Data are shown as the mean of triplicates. [Figure 11-3] Figure 11A-G: T cell generation and functional validation of HLA-A0101 peptide NSP13-448 derived from nonstructural protein NSP13. (G) Intracellular cytokine staining (ICS) assay. NSP13-448-specific CTLs were co-cultured overnight with NSP13-448 peptide or irrelevant HLA-A0101 peptide, and A375 pulsed with A375-NSP13, A375-GFP, RPMI-7951-NSP13, RPMI-7951-GFP in the presence of BFA at an E:T ratio of 10:1. After incubation, the levels of IFN-γ and TNF-α, as well as downstream activation markers of the TCR pathway, CD137 and CD69, were detected using flow cytometry. [Figure 12-1]Figure 12A-E: T cell generation and functional validation of HLA-A0301 peptide NSP13-134 derived from nonstructural protein NSP13. (A) NSP13-134 peptide (KLFAAETLK - SEQ ID NO:36) pulsed autologous DCs were co-cultured with autologous PBMCs of HLA-A0301 healthy donors. After two rounds of stimulation, CD8+ and NSP13-134 tetramer positive T cells were induced (left). CD8+ and tetramer+ T cells were sorted and subsequently expanded in REP for 2 weeks to generate highly pure NSP13-134 specific CTLs (right). (B) 51Cr-labeled HLA-A0301 cell line Hs-578T pulsed with various concentrations of NSP13-134 peptide were co-cultured with NSP13-134 specific CTLs at an E:T ratio of 20:1. The lytic ability of NSP13-134-specific CTLs was detected by standard CRA. Data are shown as the mean of triplicates. (C) 51Cr-labeled NSP13 or GFP-forced HLA-A0301 cell line Hs-578T (Hs-578T-NSP13, Hs-578T-GFP) was co-cultured with NSP13-134-specific CTLs at various E:T ratios (from 40:1 to 1.25:1). The lytic ability of NSP13-134-specific CTLs against different targets was detected by standard CRA. Data are shown as the mean of triplicates. (D) Cold target inhibition assay. 51Cr-labeled Hs-578T-NSP13 cells were used as the radioactive targets. Non-radiolabeled Hs-578T cells pulsed with NSP13-134 peptide or irrelevant HLA-A0301 peptide were used as the cold targets. The ratio of cold target: radioactive target was 10:1 or 20:1. NSP13-134-specific CTLs were co-cultured with the radioactive target alone or with the cold target at an E:T ratio of 20:1. The lytic ability of NSP13-134-specific CTLs was detected by standard CRA. Data are presented as the mean of triplicates. [Figure 12-2]Figure 12A-E: T cell generation and functional validation of HLA-A0301 peptide NSP13-134 derived from nonstructural protein NSP13. (E) Intracellular cytokine staining (ICS) assay. NSP13-134-specific CTLs were co-cultured overnight with NSP13-134 peptide or irrelevant HLA-A0301 peptide, as well as Hs-578T-NSP13, Hs-578T pulsed with Hs-578T-GFP, in the presence of BFA at an E:T ratio of 10:1. After incubation, the levels of IFN-γ and TNF-α, as well as downstream activation markers of the TCR pathway, CD137 and CD69, were detected using flow cytometry. [Figure 13-1] Figure 13A-G: T cell generation and functional validation of HLA-A2402 peptide NSP13-400 derived from nonstructural protein NSP13. (A) PBMCs from HLA-A2402 healthy donors were co-cultured with autologous DCs pulsed with NSP13-400 peptide (VYIGDPAQL - SEQ ID NO:37). After two rounds of stimulation, CD8+ and NSP13-400 tetramer positive T cells were induced (left). After sorting and REP of CD8+ and tetramer+ T cells for 2 weeks, highly pure NSP13-400 specific CTLs were expanded (right). (B) 51Cr-labeled HLA-A2402 cell line M14 pulsed with various concentrations of NSP13-400 peptide were co-cultured with NSP13-400 specific CTLs at an E:T ratio of 20:1. The lytic ability of NSP13-400-specific CTLs was detected by standard CRA. Data are shown as the mean of triplicates. (C, D) 51Cr-labeled NSP13- or GFP-forced HLA-A2402 cell lines Hs-578T (Hs-578T-NSP13, Hs-578T-GFP) and M14 (M14-NSP13, M14-GFP) were cocultured with NSP13-400-specific CTLs at various E:T ratios (from 40:1 to 1.25:1). The lytic ability of NSP13-400-specific CTLs against different targets was detected by standard CRA. Data are shown as the mean of triplicates. [Figure 13-2]Figure 13A-G: T cell generation and functional validation of HLA-A2402 peptide NSP13-400 derived from nonstructural protein NSP13. (E, F) Cold target inhibition assay. 51Cr-labeled Hs-578T-NSP13 and M14-NSP13 served as the radioactive targets. Nonradiolabeled M14 cells pulsed with NSP13-400 peptide or irrelevant HLA-A2402 peptide served as the cold target. The cold target:radioactive target ratio was 10:1 or 20:1. NSP13-400-specific CTLs were co-cultured with radioactive targets alone or with radioactive targets and cold targets at an E:radioactive T ratio of 20:1. The lytic ability of NSP13-242-specific CTLs was detected by standard CRA. Data are presented as the mean of triplicates. [Figure 13-3] Figure 13A-G: T cell generation and functional validation of HLA-A2402 peptide NSP13-400 derived from nonstructural protein NSP13. (G) Intracellular cytokine staining (ICS) assay. NSP13-400-specific CTLs were co-cultured overnight with NSP13-400 peptide or irrelevant HLA-A2402 peptide, and M14 pulsed with Hs-578T-NSP13, Hs-578T-GFP, M14-NSP13, M14-GFP in the presence of BFA at an E:T ratio of 10:1. After incubation, the levels of IFN-γ and TNF-α, as well as downstream activation markers of the TCR pathway, CD137 and CD69, were detected using flow cytometry. [Figure 14-1]Figure 14A-E: Generation and functional validation of MGP-65 peptide-specific T cell receptor engineered T cells (TCR-T). (A) Full-length MGP-65 TCR α and β chains linked with FP2A peptide were inserted into retroviral vector pMSGV1, and then this recombinant retroviral vector was used to infect OKT3-activated HLA-A0201 allo-PBMCs. After approximately 5 days of infection, CD8+ and MGP-65-tetramer+ T cell populations were analyzed by flow cytometry (middle). CD8+ and MGP-65 tetramer-positive cells were then sorted and expanded in REP. After 2 weeks of expansion, highly pure CTLs (>90% tetramer+ population) were generated (right). (B) Peptide titration assay of MGP-65-specific TCR-T. 51Cr-labeled T2 cells pulsed with various concentrations of MGP-65 peptides were co-cultured with MGP-65-specific TCR-T. Lysis levels of MGP-65-specific TCR-T were analyzed by standard CRA. Data are shown as the mean of triplicates. (C, D) Antigen-specific cytolysis analysis of MGP-65-specific TCR-T. 51Cr-labeled A375-MGP, A375-GFP, Mel624-MGP or Mel624-GFP were co-cultured with MGP-65-specific TCR-T at various E:T ratios. Lysis levels of MGP-65-specific TCR-T against different targets were analyzed by standard CRA. Data are shown as the mean of triplicates. [Figure 14-2] Figure 14A-E: Generation and functional validation of MGP-65 peptide-specific T cell receptor engineered T cells (TCR-T). (E) Intracellular cytokine staining (ICS) assay. MGP-65-specific TCR-T were co-cultured with T2 pulsed with MGP-65 peptide or M26 irrelevant peptide, as well as A375-MGP, A375-GFP, Mel624-MGP, and Mel624-GFP in the presence of BFA at an E:T ratio of 10:1 overnight. After incubation, the levels of IFN-γ and TNF-α, as well as downstream activation markers of the TCR pathway, CD137 and CD69, were detected using flow cytometry. [Figure 15]SARS-CoV-2 MHC-binding peptide identification and specific T cell generation workflow. Schematic diagram of the SARS-CoV-2 antigen discovery platform. (1) SARS-CoV-2 structural protein membrane glycolipid protein (MGP) and nonstructural protein helicase (NSP13) gene-expressing cell lines with high frequency HLA alleles were established. For immunopeptidome measurements, expanded MGP or NSP13-expressing cells were lysed, and MHC-peptide complexes were immunoprecipitated with anti-MHC antibody (W6 / 32) and eluted with acid. The eluted MHC-binding peptides were then analyzed by LC-MS / MS. (2) An endogenous T cell (ETC) generation workflow was deployed to generate peptide-specific CTLs. (3) Validation of the generated SARS-CoV-2-specific CTLs with SARS-CoV-2 target recognition. (4) Development of SARS-CoV-2 functional T cell receptor (TCR)-engineered T cells (TCR-T) based on the validation of specific CTLs. [Figure 16]T cell generation and functional validation of predicted SARS-CoV-2 HLA-A0201-restricted peptides. Three predicted membrane glycol protein (MGP) HLA-A0201 peptides MGP-53 (FLWLLWPVTL - SEQ ID NO:63), MGP-56 (LLWPVTLACFV - SEQ ID NO:67), MGP-89 (GLMWLSYFI - SEQ ID NO:43), and four predicted spike protein (SP) HLA-A0201 peptides SP-424 (KLPDDFTGCV - SEQ ID NO:78), SP-821 (LLFNKVTLA - SEQ ID NO:79), SP-983 (RLDKVEAEV - SEQ ID NO:80), SP-995 (RLITGRLQSL - SEQ ID NO:81) were selected for antigen-specific T cell generation using the endogenous T cell (ETC) generation workflow (left). Antigen-specific cell lysis was analyzed by standard 51Cr-release assay (CRA) using MGP-, SP- or GFP-forced HLA-A0201 cell lines (A375-MGP, A375-SP, A375-GFP, Mel624-MGP, Mel624-GFP, SK-MEL-5-SP, SK-MEL-5-GFP) as targets (right). [Figure 17] PRM screening of HLA-A0301 peptides predicted from NSP13. Ten highly potent HLA-A0301 binding peptides predicted from NSP13 were selected and analyzed by PRM method. [Figure 18-1] Figure 18: Generation of MGP-65 specific T cells using ETC workflow. Mature dendritic cells (DCs) derived from HLA-A0201 healthy donors were pulsed with MGP-65 peptide (FVLAAVYRI - SEQ ID NO:22) and co-cultured with autologous PBMCs in one 48-well plate. After two rounds of stimulation, flow cytometry was used to detect CD8+ and MGP-65 tetramer+ populations in individual wells. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 19-1]Figure 19: Generation of NSP13-242 specific T cells using ETC workflow. Mature dendritic cells (DCs) derived from HLA-A0201 healthy donors were pulsed with NSP13-242 peptide (TLVPQEHYV - SEQ ID NO:35) and co-cultured with autologous PBMCs in a single 48-well plate. After two rounds of stimulation, flow cytometry was used to detect CD8+ and NSP13-242 tetramer+ populations in individual wells. [Figure 19-2] This is a continuation of Figure 19-1. [Figure 20-1] Figure 20: Generation of NSP13-448 specific T cells using ETC workflow. Mature dendritic cells (DCs) derived from HLA-A0101 healthy donors were pulsed with NSP13-448 peptide (IVDTVSALVY - SEQ ID NO:34) and co-cultured with autologous PBMCs in one 48-well plate. After two rounds of stimulation, CD8+ and NSP13-134 tetramer+ populations were detected in individual wells using flow cytometry. [Figure 20-2] This is a continuation of Figure 20-1. [Figure 21-1] Figure 21: Generation of NSP13-134 specific T cells using ETC workflow. Mature dendritic cells (DCs) derived from HLA-A0301 healthy donors were pulsed with NSP13-134 peptide (KLFAAETLK - SEQ ID NO:36) and co-cultured with autologous PBMCs in one 48-well plate. After two rounds of stimulation, flow cytometry was used to detect CD8+ and NSP13-134 tetramer+ populations in individual wells. [Figure 21-2] This is a continuation of Figure 21-1. [Figure 22-1] Figure 22: Generation of NSP13-400 specific T cells using ETC workflow. Mature dendritic cells (DCs) derived from HLA-A0201 healthy donors were pulsed with NSP13-400 peptide (VYIGDPAQL - SEQ ID NO:37) and co-cultured with autologous PBMCs in a single 48-well plate. After two rounds of stimulation, flow cytometry was used to detect CD8+ and NSP13-400 tetramer+ populations in individual wells. [Figure 22-2] This is a continuation of Figure 22-1. [Diagram 23] Generation of NSP13-134 peptide-specific TCR-T cells. Full-length TCR α and β chains were inserted into the retroviral vector pMSGV3, and then the recombinant retroviral vector was used to infect PBMCs. After infection, the CD8+ / tetramer+ population was detected by flow cytometry. After tetramer-induced selection and expansion, highly pure TCR-T cells were generated. [Figure 24] Figure 24A-C: Target killing assay of NSP13-134 peptide-specific TCR-T cells. (A) T2 cells pulsed with various concentrations of NSP13-134 peptide were used as targets. The lytic ability of NSP13-134-specific TCR-T cells was detected by Cr51 release assay (CRA). The effector-to-target (E:T) ratio used was 20:1. (B, C) SK-MES-1 (HLA-A0301+), Hs-578T (HLA-A0301+) cell lines with forced expression of GFP or SARS-Cov-2 NSP13 peptide were used as targets and co-cultured with NSP13-134-specific TCR-T cells. The effector-to-target (E:T) ratio used was 40:1 to 1.25:1. The lytic ability of NSP13-134 TCR-T cells against different targets was detected by Cr51 release assay (CRA).NSP13-134 TCR-T cells show significantly higher killing levels against positive targets than against negative controls. [Figure 25-1]Figure 25: Functional validation of NSP13-134 TCR-T cells by intracellular cytokine staining (ICS) assay. NSP13-134-specific TCR-T cells were co-cultured with SK-MES-1 + control A0301 peptide, SK-MES-1 + NSP13-134, SK-MES-1-GFP, SK-MES-1-NSP13, Hs-578T-GFP and Hs-578T-NSP13 (E:T=10:1). After overnight co-culture, downstream activation markers of the TCR pathway, CD137, CD69, IFN-γ and TNF-α, were detected by ICS assay. The levels of CD137, CD69, IFN-γ and TNF-α of NSP13-134-specific TCR-T were significantly enhanced when co-cultured with positive targets compared to negative control targets. [Figure 25-2] This is a continuation of Figure 25-1. [Figure 26] Generation of NSP13-242 peptide-specific TCR-T cells. Full-length TCR α and β chains were inserted into the retroviral vector pMSGV3, and then the recombinant retroviral vector was used to infect PBMCs. After infection, the CD8+ / tetramer+ population was detected by flow cytometry. After tetramer-induced selection and expansion, highly pure TCR-T cells were generated. [Figure 27] Figure 27A-C: Target killing assay of NSP13-242 peptide-specific TCR-T cells. (A) T2 cells pulsed with various concentrations of NSP13-242 peptide were used as targets. The lytic ability of NSP13-242-specific TCR-T cells was detected by Cr51 release assay (CRA). The effector-to-target (E:T) ratio used was 20:1. (B, C) A375 (HLA-A0201+), RPMI-7951 (HLA-A0201+) cell lines with forced expression of GFP or SARS-Cov-2 NSP13 were used as targets and co-cultured with NSP13-242-specific TCR-T cells. The effector-to-target (E:T) ratio used was 40:1 to 1.25:1. The lytic ability of NSP13-242 TCR-T against different targets was detected by Cr51 release assay (CRA).NSP13-242 TCR-T showed significantly higher killing levels against positive targets than against negative controls. [Figure 28-1] Figure 28: Functional validation of NSP13-242 TCR-T by intracellular cytokine staining (ICS) assay. NSP13-242-specific TCR-T cells were co-cultured with T2+M26, T2+NSP13-242, A375-GFP, A375-NSP13, RPMI-7951-GFP and RPMI-7951-NSP13 (E:T=10:1). After overnight co-culture, downstream activation markers of the TCR pathway, CD137, CD69, IFN-γ and TNF-α, were detected by ICS assay. The levels of CD137, CD69, IFN-γ and TNF-α of NSP13-242-specific TCR-T cells were significantly enhanced when co-cultured with positive targets compared to negative control targets. [Figure 28-2] This is a continuation of Figure 28-1. [Figure 29] Generation of NSP13-400 antigen-specific TCR-T cells. The α-chain and β-chain TCR genes were cloned from one of the NSP13-400-specific CTL cell lines. The full-length TCR α-chain and β-chain were inserted into the retroviral vector pMSGV3, and then the recombinant retroviral vector was used to infect PBMCs. After infection, both CD8+ / tetramer+ and CD4+ / tetramer+ populations were observed. After tetramer-induced selection and expansion, highly pure CD8+ and CD4+ TCR-T cells were generated. [Figure 30-1]Figure 30A-F: Validation of the specificity of MGP-65-specific TCR-T cells by killing assay. (A, D) M14 cells pulsed with various concentrations of NSP13-400 peptide were used as targets. The lytic ability of NGP13-400 CD8+ and CD4+ specific TCR-T cells was detected by Cr51 release assay (CRA). The effector to target (E:T) ratio was 20:1. (B, C, E, F) Hs-578T (HLA-A2402+), M14 (HLA-A2402+) cell lines with forced expression of GFP or SARS-Cov-2 NSP13 were used as targets and co-cultured with NSP13-400-specific TCR-T cells. The effector to target (E:T) ratios used were from 40:1 to 1.25:1. The lytic ability of NSP13-400-specific TCR-T cells against different targets was detected by Cr51 release assay (CRA). [Figure 30-2] Figure 30A-F: Validation of the specificity of MGP-65-specific TCR-T cells by killing assay. (A, D) M14 cells pulsed with various concentrations of NSP13-400 peptide were used as targets. The lytic ability of NGP13-400 CD8+ and CD4+ specific TCR-T cells was detected by Cr51 release assay (CRA). The effector to target (E:T) ratio was 20:1. (B, C, E, F) Hs-578T (HLA-A2402+), M14 (HLA-A2402+) cell lines with forced expression of GFP or SARS-Cov-2 NSP13 were used as targets and co-cultured with NSP13-400-specific TCR-T cells. The effector to target (E:T) ratios used were from 40:1 to 1.25:1. The lytic ability of NSP13-400-specific TCR-T cells against different targets was detected by Cr51 release assay (CRA). [Figure 31-1]Figure 31: Functional validation of NSP13-400 TCR-T by intracellular cytokine staining (ICS) assay. NSP13-400-specific CD8+ and CD4+ TCR-T were co-cultured with M14+ control peptide, M14+NSP13-400, Hs-578T-GFP, Hs-578T-NSP13, M14-GFP and M14-NSP13 (E:T=10:1). After overnight co-culture, downstream activation markers of the TCR pathway, CD137, CD69, IFN-γ and TNF-α, were detected by ICS assay. The levels of CD137, CD69, IFN-γ and TNF-α of NSP13-400-specific CTL cell lines were significantly enhanced when co-cultured with positive targets compared to negative control targets. [Figure 31-2] This is a continuation of Figure 31-1. [Figure 31-3] This is a continuation of Figure 31-1. [Figure 31-4] This is a continuation of Figure 31-1. [Diagram 32] Generation of NSP13-448 peptide-specific TCR-T cells. Full-length TCR α and β chains were inserted into the retroviral vector pMSGV3, and then the recombinant retroviral vector was used to infect PBMCs. After infection, the CD8+ / tetramer+ population was detected by flow cytometry. After tetramer-induced selection and expansion, highly pure TCR-T cells were generated. [Diagram 33]Figure 33A-C: Target killing assay of NSP13-448 peptide-specific TCR-T cells. (A) A375 cells pulsed with various concentrations of NSP13-448 peptide were used as targets. The lytic ability of NSP13-448-specific TCR-T was detected by Cr51 release assay (CRA). The effector-to-target (E:T) ratio was 20:1. (B, C) A375 (HLA-A0101+) and RPMI-7951 (HLA-A0101+) cell lines overexpressing GFP or SARS-Cov-2 NSP13 were used as targets and co-cultured with NSP13-448-specific TCR-T. The effector-to-target (E:T) ratio was 40:1 to 1.25:1. The lytic ability of NSP13-448 TCR-T against different targets was detected by Cr51 release assay (CRA). NSP13-448 TCR-T shows significantly higher killing levels against positive targets than against negative controls. [Figure 34-1] Figure 34: Functional validation of NSP13-448 TCR-T cells by intracellular cytokine staining (ICS) assay. NSP13-448-specific TCR-T were co-cultured with A375+control A0101 peptide, A375+NSP13-448, A375-GFP, A375-NSP13, RPMI-7951-GFP and RPMI-7951-NSP13 (E:T=10:1). After overnight co-culture, downstream activation markers of the TCR pathway, CD137, CD69, IFN-γ and TNF-α, were detected by ICS assay. The levels of CD137, CD69, IFN-γ and TNF-α of NSP13-448-specific TCR-T were significantly enhanced when co-cultured with positive targets compared to negative control targets. [Figure 34-2] This is a continuation of Figure 34-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0118] Detailed Description of the Invention SARS-CoV-2 infection induces both humoral and cellular immune responses. It is becoming increasingly clear that T cell responses are as important, if not more important, than humoral responses in mediating recovery and immune protection for the prevention and treatment of COVID19, the disease caused by SARS-CoV-2. One of the major challenges in the development of T cell-based therapeutics for infectious and malignant diseases has been to identify immunogenic epitopes that can induce meaningful T cell responses. Traditionally, this has been achieved using advanced in silico methods that predict putative epitopes inferred from binding affinity or consensus data. We found that “immunodominant” SARS-CoV-2 peptides thus defined fail to induce T cell responses that recognize naturally presented SARS-CoV-2 epitopes. We hypothesized that immunogenic epitopes of SARS-CoV-2 would best be defined empirically by directly analyzing peptides eluted from naturally processed peptide-MHC complexes and validating immunogenicity by determining whether such peptides can elicit T cells that recognize cells expressing SARS-CoV-2 antigens. Using tandem mass spectrometry, we identified epitopes of SARS-CoV-2 derived from structural as well as nonstructural genes in regions highly conserved among SARS-CoV-2 strains, including recently recognized variants. Finally, no TCR sequences have been reported that, when incorporated into a recombinant vector, can redirect T cell specificity to recognize and kill SARS-CoV-2 target cells. We report here, for the first time, several novel SARS-CoV-2 epitopes defined by mass spectrometry of MHC-eluted peptides, provide empirical evidence for their immunogenicity, and demonstrate killing by engineered TCR redirection.

[0119] The peptides described herein can be used to detect immune responses in patients with SARS-Cov-2 infection (COVID19) or for vaccination. This will lead to a better understanding of COVID19 immunity, which will have a direct impact on the management of patients with COVID19 and allow for the evaluation of immune responses to vaccines. Furthermore, the peptide cytotoxic T cells and engineered TCRs can be used to generate SARS-Cov-2 specific T cells against HLA-matched targets, thus providing an off-the-shelf T cell therapy for patients with COVID19 disease.

[0120] I. Immunotherapy Using the Peptides of the Disclosure The peptides described herein (e.g., peptides of SEQ ID NOs:22-81) may be used in immunotherapy to treat viral infections. For example, a peptide of one of SEQ ID NOs:22-81 may be contacted with or used to stimulate a population of T cells to induce proliferation of T cells that recognize or bind the peptide. In other aspects, the peptides of the present disclosure may be administered to a subject, such as a human patient, to enhance the subject's immune response to SARS-Cov-2 infection.

[0121] The peptides of the present disclosure may be included in active immunotherapy (e.g., vaccine) or passive immunotherapy (e.g., adoptive immunotherapy). Active immunotherapy includes immunizing a subject with purified peptide antigen or immunodominant peptide (natural or modified); alternatively, antigen-presenting cells pulsed with the peptides of the present disclosure (or transfected with a gene encoding an antigen comprising the peptide) may be administered to the subject. The peptides may be modified or may include one or more mutations, such as substitution mutations. Passive immunotherapy includes adoptive immunotherapy. Adoptive immunotherapy generally involves administering to a subject cells (e.g., cytotoxic T cells) that have been sensitized in vitro to the peptides of the present disclosure (see, e.g., US 7910109).

[0122] In some aspects, flow cytometry may be used in adoptive immunotherapy for rapid isolation of human tumor antigen-specific T cell clones, for example, by using a proliferation assay based on T cell receptor (TCR) Vβ antibodies in combination with carboxyfluorescein succinimidyl ester (CFSE). See, for example, Lee et al., J. Immunol. Methods, 331:13-26, 2008, which is incorporated by reference for all purposes. In some aspects, tetramer-guided cell sorting may be used, for example, the method described in Pollack, et al., J Immunother Cancer. 2014; 2: 36, which is incorporated by reference for all purposes. Various culture protocols are also known for adoptive immunotherapy and may be used in the aspects of the present disclosure. In some aspects, cells may be cultured in conditions that do not require the use of antigen-presenting cells (e.g., Hida et al., Cancer Immunol. Immunotherapy, 51:219-228, 2002, incorporated by reference). In other aspects, T cells may be expanded under culture conditions that utilize antigen-presenting cells such as dendritic cells (Nestle et al., 1998, incorporated by reference), and in some aspects, artificial antigen-presenting cells may be used for this purpose (Maus et al., 2002, incorporated by reference). Further methods of adoptive immunotherapy are disclosed in Dudley et al. (2003), incorporated by reference, which may be used in aspects of the present disclosure. Various methods are known and may be used for cloning and expanding human antigen-specific T cells (see, e.g., Riddell et al., 1990, incorporated by reference herein).

[0123] In certain aspects, the following protocol may be used to generate T cells that selectively recognize the peptides of the present disclosure. Peptide-specific T cell lines may be generated from normal donors or HLA-restricted normal donors and patients using previously reported methods (Hida et al., 2002). ENREF 32 Briefly, PBMCs (1 × 10 5 Cells / well) can be stimulated with about 10 μg / ml of each peptide in quadruplicate in 96-well U-bottom microculture plates (Corning Incorporated, Lowell, MA) in about 200 μl of culture medium. The culture medium can consist of 50% AIM-V medium (Invitrogen), 50% RPMI1640 medium (Invitrogen), 10% human AB serum (Valley Biomedical, Winchester, VA) and 100 IU / ml interleukin-2 (IL-2). Cells can be restimulated with the corresponding peptide about every 3 days. After five stimulations, T cells from each well can be washed and incubated with T2 cells in the presence or absence of the corresponding peptide. After about 18 hours, production of interferon (IFN)-γ can be determined in the supernatant by ELISA. T cells secreting large amounts of IFN-γ can be further expanded by rapid expansion protocols (Riddell et al., 1990; Yee et al., 2002b).

[0124] In some aspects, immunotherapy may utilize the peptides of the present disclosure in association with cell penetrating agents, such as liposomes or cell penetrating peptides (CPPs). Antigen-presenting cells (such as dendritic cells) pulsed with peptides may be used to enhance anti-tumor immunity (Celluzzi et al., 1996; Young et al., 1996). Liposomes and CPPs are described in more detail below. In some aspects, immunotherapy may utilize nucleic acids encoding the peptides of the present disclosure, where the nucleic acids are delivered, for example, in viral or non-viral vectors.

[0125] II. Cell-penetrating peptides The peptides of the present disclosure may also be associated with or covalently linked to a cell-penetrating peptide (CPP). Cell-penetrating peptides that may be covalently linked to the peptides of the present disclosure include, for example, HIV Tat, herpes virus VP22, Drosophila antennapedia homeobox gene product, signal sequence, fusion sequence, or protegrin I. Covalently linking a peptide to a CPP can prolong presentation of the peptide by dendritic cells, thus enhancing anti-tumor immunity (Wang and Wang, 2002). In some aspects, a peptide of the present disclosure (e.g., contained within a peptide or polyepitope string) can be covalently linked (e.g., via a peptide bond) to a CPP to create a fusion protein. In other aspects, a peptide according to the present disclosure or a nucleic acid encoding a peptide can be encapsulated in or associated with a liposome, such as a multilamellar, vesicular, or multivesicular liposome.

[0126] As used herein, "association" refers to a physical association, a chemical association, or both. For example, the association can involve covalent bonding, hydrophobic interactions, encapsulation, surface adsorption, and the like.

[0127] As used herein, "cell penetrating agent" refers to a composition or compound that enhances the intracellular delivery of peptide / polyepitope string to antigen-presenting cells. For example, cell penetrating agent may be a lipid that enhances its ability to cross plasma membrane when associated with peptide. Alternatively, cell penetrating agent may be a peptide. Cell penetrating peptides (CPPs) are known in the art and include, for example, the Tat protein of HIV (Frankel and Pabo, 1988), the VP22 protein of HSV (Elliott and O'Hare, 1997) and fibroblast growth factor (Lin et al., 1995).

[0128] Cell-penetrating peptides (or "protein transduction domains") have been identified from the third helix of the Drosophila Antennapedia homeobox gene (Antp), HIV Tat, and herpesvirus VP22, all of which contain positively charged domains rich in arginine and lysine residues (Schwarze et al., 2000; Schwarze et al., 1999). Hydrophobic peptides derived from signal sequences have also been identified as cell-penetrating peptides (Rojas et al., 1996; Rojas et al., 1998; Du et al., 1998). Conjugation of these peptides to marker proteins such as β-galactosidase has been shown to confer efficient internalization of the marker protein into cells, and chimeric in-frame fusion proteins containing these peptides have been used to deliver proteins to a wide range of cell types both in vitro and in vivo (Drin et al., 2002). The fusion of these cell-penetrating peptides to the peptides of the present disclosure can enhance cellular uptake of the polypeptide.

[0129] In some aspects, cellular uptake is promoted by the addition of lipids such as stearate or myristilate. Lipid addition has been shown to enhance the passage of peptides into cells. The addition of lipid moieties is another way in which the present invention increases the uptake of polypeptides by cells.

[0130] The peptide of the present invention can be included in a liposomal vaccine composition. For example, the liposomal composition can be or include a proteoliposome composition. Methods for producing proteoliposome compositions that can be used with the present invention are described, for example, in Neelapu et al. (2007) and Popescu et al. (2007). In some aspects, the proteoliposome composition can be used to treat melanoma.

[0131] By enhancing uptake of the polypeptides of the present disclosure, it may be possible to reduce the amount of protein or peptide required for treatment. This in turn can significantly reduce treatment costs and increase supply of therapeutic agents. Lower dosages can also minimize potential immunogenicity of peptides and limit toxic side effects.

[0132] In some aspects, the peptides of the present disclosure may interact with nanoparticles to form nanoparticle-polypeptide complexes. In some aspects, the nanoparticles are liposomes or other lipid-based nanoparticles, such as lipid-based vesicles (e.g., DOTAP:cholesterol vesicles). In other aspects, the nanoparticles are iron oxide-based superparamagnetic nanoparticles. Superparamagnetic nanoparticles, with diameters ranging from about 10-100 nm, are small enough to avoid sequestration by the spleen, but large enough to avoid clearance by the liver. Particles of this size can penetrate very small capillaries and can be effectively distributed throughout body tissues. Superparamagnetic nanoparticle-polypeptide complexes can be used as MRI contrast agents to identify and track cells that take up the peptide. In some aspects, the nanoparticles are semiconductor nanocrystals or semiconductor quantum dots, both of which can be used in optical imaging. In further aspects, the nanoparticles can be nanoshells, including a layer of gold on a silica core. One advantage of nanoshells is that the polypeptide can be conjugated to the gold layer using standard chemistry. In other aspects, the nanoparticles can be fullerenes or nanotubes (Gupta et al., 2005).

[0133] Peptides are rapidly removed from circulation by the kidney and are also susceptible to degradation by proteases in serum. By interacting peptides with nanoparticles, the nanoparticle-polypeptide complexes of the present invention can protect against degradation and / or reduce clearance by the kidney. This can increase the serum half-life of the polypeptide, thereby reducing the polypeptide dose required for effective therapy. Furthermore, this can lower the cost of treatment and minimize immunological problems and toxic reactions of the therapy.

[0134] III. Polyepitope Strings In some aspects, the peptide is encompassed or included in a polyepitope string. A polyepitope string is a peptide or polypeptide that contains multiple antigenic epitopes from one or more antigens linked together. A polyepitope string can be used to induce an immune response in a subject, such as a human subject. Polyepitope strings have been used previously to target malaria and other pathogens (Baraldo et al., 2005; Moorthy et al., 2004; Baird et al., 2004). A polyepitope string can refer to a nucleic acid (e.g., a nucleic acid that encodes multiple antigens, including the peptides of the present disclosure) or a peptide or polypeptide (e.g., that contains multiple antigens, including the peptides of the present disclosure). A polyepitope string can be included in a vaccine composition.

[0135] IV. Application of antigen peptides Various aspects are directed to the development and use of antigen peptides that are useful for treating and preventing certain viral infections.In many aspects, antigen peptides are produced by chemical synthesis or by molecular expression in host cells.Peptides can be purified and used in a variety of applications, including (but not limited to) the assay for determining peptide immunogenicity, the assay for determining T cell recognition, peptide vaccines for treating viral infections, the development of modified TCRs on T cells, and the development of antibodies.

[0136] Peptides can be chemically synthesized by several methods. One common method is to use solid phase peptide synthesis (SPPS). In general, SPPS is performed by building up the peptide from C-terminus to N-terminus through alternating cycles of N-terminal deprotection and coupling reactions. The C-terminus of the first amino acid is coupled to the resin, then the amine is removed, followed by coupling with the free acid of the second amino acid. This cycle is repeated until the peptide is synthesized.

[0137] Peptides can also be synthesized using molecular tools and host cells. Nucleic acid sequences corresponding to antigenic peptides can be synthesized. In some aspects, synthetic nucleic acids are synthesized in an in vitro synthesizer (e.g., phosphoramidite synthesizer), bacterial recombinant system, or other suitable methods. Furthermore, the synthesized nucleic acid can be purified and lyophilized, or can be kept in storage in a biological system (e.g., bacteria, yeast, etc.). For use in a biological system, the synthetic nucleic acid molecule can be inserted into a plasmid vector, etc. The plasmid vector can also be an expression vector in which a suitable promoter and a suitable 3'-poly A tail are combined with the transcript sequence.

[0138] Aspects are also directed to expression vectors and systems that produce antigenic peptides or proteins. These expression systems can incorporate expression vectors to express transcripts and proteins in appropriate expression systems. Typical expression systems include bacterial (e.g., E. coli), insect (e.g., SF9), yeast (e.g., S. cerevisiae), animal (e.g., CHO) or human (e.g., HEK 293) cell lines. RNA and / or protein molecules can be purified from these systems using standard biotechnology production procedures.

[0139] Assays for determining immunogenicity and / or TCR binding can be performed. One such is the dextramer flow cytometry assay. Typically, custom-made HLA-matched MHC class I dextramer:peptide (pMHC) complexes are developed or purchased (Immudex, Copenhagen, Denmark). T cells from peripheral blood mononuclear cells (PBMCs) or tumor infiltrating lymphocytes (TILs) are incubated with the pMHC complexes, stained, and then run through a flow cytometer to determine whether the peptides can bind to the TCR of the T cells.

[0140] The peptides of the present disclosure can also be used to isolate and / or identify T cell receptors that bind to the peptides. T cell receptors contain two distinct polypeptide chains, called the T cell receptor alpha (TCRα) and beta (TCRβ) chains, linked by disulfide bonds. These alpha:beta heterodimers are very similar in structure to the Fab fragments of immunoglobulin molecules and are responsible for antigen recognition by most T cells. A small number of T cells have a different, but structurally similar, receptor composed of a pair of distinct polypeptide chains, called gamma and delta. Both types of T cell receptors differ from membrane-bound immunoglobulins that function as B cell receptors: T cell receptors have only one antigen-binding site, whereas B cell receptors have two, and T cell receptors are never secreted, whereas immunoglobulins can be secreted as antibodies.

[0141] Both chains of the T cell receptor have an amino-terminal variable (V) region with homology to immunoglobulin V domains, a constant (C) region with homology to immunoglobulin C domains, and a short hinge region containing cysteine ​​residues that form interchain disulfide bonds. Each chain spans the lipid bilayer by a hydrophobic transmembrane domain and terminates in a short cytoplasmic tail.

[0142] The three-dimensional structure of the T cell receptor has been determined. This structure is certainly similar to that of an antibody Fab fragment, as inferred from previous studies of the genes encoding the T cell receptor. The chains of the T cell receptor fold almost identically to those of the Fab fragment, although the final structure appears to be a bit shorter and wider. However, there are some clear differences between the T cell receptor and the Fab fragment. The most striking difference is in the Cα domain, which folds differently from any other immunoglobulin-like domain. The half of the domain juxtaposed with the Cβ domain forms a β-sheet similar to those found in other immunoglobulin-like domains, while the other half of the domain is formed of loosely packed chains and short segments of α-helices. An intramolecular disulfide bond, which normally links two β-strands in immunoglobulin-like domains, links the β-strand to this segment of α-helix in the Cα domain.

[0143] There are also differences in the way the domains interact. The interface between the V and C domains of both T cell receptor chains is more extensive than in antibodies, which may reduce the mobility of the hinge junction between the domains. And the interaction between the Cα and Cβ domains is distinctive in that it is carbohydrate-assisted, with sugar groups from the Cα domain making several hydrogen bonds to the Cβ domain. Finally, comparison of the variable binding sites shows that the complementarity determining region (CDR) loops are aligned fairly closely with the loops of the antibody molecule, but with some displacement compared to the loops of the antibody molecule. This displacement is particularly pronounced in the Vα CDR2 loop, which is oriented at almost a right angle to the equivalent loop of the antibody V domain, as a result of a shift of the β strand from one face of the domain that anchors one end of the loop to the other. The strand displacement also causes a change in the orientation of the Vβ CDR2 loop in two of the seven Vβ domains whose structures are known. So far, the crystal structures of seven T cell receptors have been solved at this level of resolution.

[0144] Aspects of the disclosure relate to engineered T cell receptors that bind to a peptide of the disclosure, such as one of SEQ ID NOs:22-81. The term "engineered" refers to a T cell receptor having a TCR variable region grafted onto a TCR constant region to create a chimeric polypeptide that binds to a peptide and antigen of the disclosure. In certain aspects, the TCR is used for cloning, enhancing expression, detection, or therapeutic control of the construct, but includes intervening sequences not present in the endogenous TCR, such as multiple cloning sites, linkers, hinge sequences, modified hinge sequences, modified transmembrane sequences, detection polypeptides or molecules, or therapeutic controls that may allow for selection or screening of cells containing the TCR.

[0145] In some aspects, the TCR comprises non-TCR sequences.Thus, certain aspects relate to TCRs with sequences that are not derived from TCR genes.In some aspects, the TCR is chimeric in that it comprises sequences that are normally found in TCR genes, but from at least two TCR genes that are not necessarily found together in nature.

[0146] V. Antibodies An aspect of the present disclosure relates to an antibody or fragment thereof that targets the peptide of the present disclosure.The term "antibody" refers to any isotype of intact immunoglobulin or fragment thereof that can compete with intact antibody for specific binding to target antigen, including chimeric antibody, humanized antibody, fully human antibody, and bispecific antibody.As used herein, the term "antibody" or "immunoglobulin" is used interchangeably and refers to any of several classes of structurally related proteins that function as part of animal immune response, including IgG, IgD, IgE, IgA, IgM and related proteins, as well as polypeptides that contain antibody CDR domains that retain antigen binding activity.

[0147] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen can possess one or more epitopes capable of interacting with different antibodies.

[0148] The term "epitope" includes any region or portion of a molecule capable of eliciting an immune response by binding to an immunoglobulin or T-cell receptor. Epitopic determinants include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antibody specific for a particular target antigen selectively recognizes an epitope on the target antigen within a complex mixture.

[0149] Epitope regions of a given polypeptide can be identified using many different epitope mapping techniques well known in the art, including X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, and protein display arrays, see, for example, Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY. Such techniques are known in the art, see, for example, U.S. Patent No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). Furthermore, antigenic regions of a protein can also be predicted and identified using standard antigenicity and hydrophobicity plots.

[0150] The term "immunogenic sequence" refers to a molecule that contains the amino acid sequence of at least one epitope such that the molecule is capable of stimulating the production of antibodies in an appropriate host. The term "immunogenic composition" refers to a composition that contains at least one immunogenic molecule (e.g., an antigen or carbohydrate).

[0151] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but may contain fewer chains in some cases, such as antibodies naturally occurring in camelids, which may contain only heavy chains. The antibodies disclosed herein may be derived from only a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. For example, the variable or CDR regions may be derived from a rat or mouse source, while the constant regions are derived from different animal sources, such as humans. Antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes its derivatives, variants, fragments, and muteins, examples of which are described below (Sela-Culang et al. Front Immunol. 2013; 4: 302; 2013).

[0152] The term "light chain" includes full-length light chains and fragments thereof that have sufficient variable region sequence to confer binding specificity. Full-length light chains have a molecular weight of around 25,000 daltons and contain a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (κ) and lambda (λ). The term "VL fragment" refers to a fragment of the light chain of a monoclonal antibody that contains all or a portion of the light chain variable region, including the CDRs. The VL fragment may further contain a light chain constant region sequence. The light chain variable region domain is at the amino terminus of the polypeptide.

[0153] The term "heavy chain" includes full-length heavy chains and fragments thereof that have sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of around 50,000 daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term "VH fragment" refers to a fragment of a heavy chain of a monoclonal antibody that includes all or a portion of the heavy chain variable region, including the CDRs. A VH fragment may further include a heavy chain constant region sequence. The number of heavy chain constant region domains will depend on the isotype. The VH domain is at the amino terminus of the polypeptide, the CH domain is at the carboxy terminus, and CH3 is closest to the -COOH terminus. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE, and is defined by the presence of five heavy chain classifications: mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, respectively. IgG has several subtypes, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0154] VI. Antibody Conjugates An aspect of the present disclosure relates to an antibody against the peptide of the present invention, generally of monoclonal type, which is linked to at least one agent to form an antibody conjugate. To enhance the efficacy of the antibody molecule as a diagnostic or therapeutic agent, it is usual to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules with desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules added to antibodies include toxins, antitumor agents, therapeutic enzymes, radiolabeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands, e.g., biotin.

[0155] Any antibody with sufficient selectivity, specificity, or affinity can be used as the basis for antibody conjugates. Such properties can be evaluated using conventional immunological screening methods known to those skilled in the art. The binding sites for biologically active molecules in antibody molecules include, in addition to the standard antigen binding site, sites located within the variable domain that can bind to pathogens, B cell superantigens, T cell co-receptor CD4, and the HIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann et al., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993; Kreier et al., 1991). In addition, the variable domain is involved in the self-binding of antibodies (Kang et al., 1988) and contains the epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

[0156] A particular example of an antibody conjugate is a conjugate in which the antibody is linked to a detectable label. "Detectable labels" are compounds and / or elements that can be detected by their special functional properties and / or chemical characteristics and can be used to detect and / or, if necessary, even quantify the antibody to which they are attached. Another such example is the formation of a conjugate that includes an antibody linked to a cytotoxic or anticellular agent, which can be referred to as an "immunotoxin."

[0157] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall into two classes: those for use in in vitro diagnostic methods, such as various immunoassays, and / or those for use in in vivo diagnostic protocols, commonly known as "antibody-directed imaging."

[0158] Many suitable imaging agents are known in the art, and methods for attaching them to antibodies are also known (see, for example, U.S. Patent Nos. 5,021,236; 4,938,948; and 4,472,509, each of which is incorporated herein by reference).The imaging moiety used can be a paramagnetic ion; a radioisotope; a fluorescent dye; an NMR detection substance; X-ray imaging.

[0159] In the case of paramagnetic ions, by way of example, ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III) may be mentioned, with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II) and, in particular, bismuth (III).

[0160] In the case of radioisotopes for therapeutic and / or diagnostic applications, astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, Copper 67 , 152 Eu, gallium 67 , 3 Hydrogen, Iodine 123 , iodine 125 , iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, technicium 99m and / or yttrium 90 may be mentioned. 125 I is often preferred for use in certain situations, whereas technetium 99m and / or indium 111are also often preferred due to their low energy nature and suitability for wide range detection. Radiolabeled monoclonal antibodies of the invention may be produced according to methods well known in the art. For example, monoclonal antibodies may be iodinated by contact with sodium iodide and / or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be iodinated with technetium by a ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium on a Sephadex column and applying the antibody to the column. 99m Alternatively, direct labeling techniques can be used, for example, by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium potassium phthalate solution, and the antibody. Intermediary functional groups often used to attach radioisotopes that exist as metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0161] Fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renografin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0162] Another type of antibody conjugate contemplated in the present invention is an antibody conjugate intended primarily for in vitro use, in which the antibody is linked to an enzyme (enzyme tag) and / or secondary binding ligand that produces a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and / or avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference.

[0163] Yet another known method of site-specific attachment of molecules to antibodies involves the reaction of the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids in the antigen-binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous, as it may result in reduced antigen binding by the antibody conjugate.

[0164] Molecules containing azide groups can also be used to form covalent bonds to proteins through reactive nitrene intermediates generated by low intensity ultraviolet light (Potter & Haley, 1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-specific photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azido nucleotides have also been used to map nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; and Dholakia et al., 1989) and can be used as antibody binding agents.

[0165] Several methods are known in the art for the attachment or conjugation of antibodies to conjugate moieties. Some attachment methods require the use of metal chelate complexes, such as organic chelating agents, such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3, attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948, each of which is incorporated herein by reference). Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents, such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using a monoclonal antibody, and a detectable imaging moiety is attached to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0166] In another aspect, it is intended to derivatize immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulins, using reaction conditions that do not alter the antibody binding site. It has been disclosed that antibody conjugates produced according to this method show improved longevity, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific addition of effector or reporter molecules, in which the reporter or effector molecule is conjugated to a sugar residue in the Fc region, has also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce antibodies with diagnostic and therapeutic potential, which are currently undergoing clinical evaluation.

[0167] In another aspect of the invention, the antibody may be linked to a semiconductor nanocrystal, such as those described in U.S. Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357, all of which are incorporated herein in their entireties; as well as PCT Publication No. 99 / 26299, published May 27, 1999. In particular, exemplary materials for use as semiconductor nanocrystals in the biological and chemical assays of the present invention include, but are not limited to, the materials described above, including II-VI, III-V, and IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge, and Si, and ternary and quaternary mixtures thereof. Methods for linking semiconductor nanocrystals to antibodies are described in U.S. Patent Nos. 6,630,307 and 6,274,323.

[0168] In yet a further aspect, the present invention relates to immunodetection methods for binding, purifying, removing, quantifying, and / or otherwise generally detecting biological components, such as T cells that selectively bind or recognize the peptides of the present disclosure. In some aspects, tetramer assays can be used with the present invention. Tetramer assays generally involve making soluble peptide-MHC tetramers that can bind to antigen-specific T lymphocytes, and methods for tetramer assays are described, for example, in Altman et al. (1996). Some immunodetection methods that can be used include, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluorescent immunoassay, chemiluminescence assay, bioluminescence assay, tetramer assay, and Western blot. Various useful steps of immunodetection methods are described in the scientific literature, e.g., Doolittle and Ben-Zeev, 1999; Gulbis and Galand, 1993; De Jager et al., 1993; and Nakamura et al., 1987, each of which is incorporated herein by reference.

[0169] VII. MHC Polypeptides Aspects of the present disclosure relate to compositions comprising MHC polypeptides. In some aspects, the MHC polypeptide comprises at least two, three or four MHC polypeptides, which may be expressed as separate polypeptides or as fusion proteins. Presentation of antigens to T cells is mediated by two distinct classes of molecules, MHC class I (MHC-I) and MHC class II (MHC-II) (also identified herein as "pMHC"), which use different antigen processing pathways. Peptides derived from intracellular antigens are presented to CD8+ T cells by MHC class I molecules, which are expressed on nearly all cells, whereas peptides derived from extracellular antigens are presented to CD4+ T cells by MHC-II molecules. In certain aspects, specific antigens are identified and presented in antigen-MHC complexes under appropriate MHC class I or II polypeptides. In certain aspects, the genetic makeup of the subject may be evaluated to determine which MHC polypeptide should be used for a particular patient and a particular set of peptides. In certain aspects, the MHC class 1 polypeptide comprises all or a portion of an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or CD-1 molecule. In aspects where the MHC polypeptide is an MHC class II polypeptide, the MHC class II polypeptide can comprise all or a portion of an HLA-DR, HLA-DQ, or HLA-DP.

[0170] Non-classical MHC polypeptides are also contemplated for use in the MHC complexes of the invention. Non-classical MHC polypeptides are non-polymorphic, conserved across species, and possess narrow and deep hydrophobic ligand-binding pockets. These binding pockets can present glycolipids and phospholipids to natural killer T (NKT) cells or to specific subsets of CD8+ T cells, such as Qa1, HLA-E-restricted CD8+ T cells, or MAIT cells. NKT cells represent a unique lymphocyte population that co-expresses NK cell markers and a semi-invariant T cell receptor (TCR). They are involved in regulating immune responses associated with a wide range of diseases.

[0171] VIII. Further Agents In some aspects, the method further comprises administering an additional agent. In some aspects, the additional agent is an immunostimulant. The term "immunostimulant" as used herein refers to a compound that can stimulate immune response in a subject, and can include adjuvants. In some aspects, an immunostimulant is a drug that does not constitute a specific antigen, but can increase the strength and useful longevity of the immune response to the antigen. Such immunostimulants are activators of pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLRs), inorganic salts, such as alum, alum in combination with monophosphoryl lipid (MPL) A of enterobacteria, such as Escherichia coli, Salmonella minnesota, Salmonella typhimurium or Shigella flexneri, or in particular alum in combination with MPL® (ASO4), alum in combination with MPL A of the above bacteria separately, saponins, such as QS-21, Quil-A, ISCOM, ISCOMATRIX, MF59, Montanide, ISA 51 and ISA 720, AS02 These may include, but are not limited to, emulsions such as (QS21 + squalene + MPL), liposomes and liposomal formulations such as AS01, synthetic or specially prepared microparticles and microcarriers such as outer membrane vesicles (OMVs) derived from bacteria such as N. gonorrhea, Chlamydia trachomatis, or depot forming agents such as chitosan particles, pluronic block copolymers, specially modified or prepared peptides such as muramyl dipeptide, aminoalkyl glucosaminide 4-phosphate such as RC529, or proteins such as bacterial toxoids or toxin fragments.

[0172] In some aspects, the additional agent comprises an agonist for a pattern recognition receptor (PRR), including but not limited to a Toll-like receptor (TLR), specifically TLR 2, 3, 4, 5, 7, 8, 9 and / or a combination thereof. In some aspects, the additional agent comprises an agonist for Toll-like receptor 3, an agonist for Toll-like receptors 7 and 8, or an agonist for Toll-like receptor 9; preferably, the cited immunostimulant comprises an imidazoquinoline; for example, R848; an adenine derivative, such as those disclosed in U.S. Pat. No. 6,329,381, U.S. Published Patent Application No. 2010 / 0075995, or WO 2010 / 018132; an immunostimulatory DNA; or an immunostimulatory RNA.In some aspects, the additional agent may also be, but is not limited to, dsRNA, poly I:C or poly I:poly C12U (available as Ampligen®, both poly I:C and poly I:poly C12U are known TLR3 stimulators), and / or other agents as described in F. Heil et al., "Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer et al., "Immune modulation by chemically modified ribonucleosides and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway" WO 2007062107 A2; E. Uhlmann et al., "Immunotropic agents and their applications in a wide variety of therapeutic applications," including, but not limited to, dsRNA, poly I:C or poly I:poly C12U (available as Ampligen®, both poly I:C and poly I:poly C12U are known TLR3 stimulators), and / or other agents as described in F. Heil et al., "Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8" Science 303(5663), 1526-1529 (2004); J. Vollmer et al., "Immune modulation by chemically modified ribonucleosides and oligoribonucleotides" WO 2008033432 A2; A. Forsbach et al., "Immunostimulatory oligoribonucleotides containing specific sequence motif(s) and targeting the Toll-like receptor 8 pathway" WO 200706 al., "Modified oligoribonucleotide analogs with enhanced immunostimulatory activity," U.S. Patent Application Publication No. US 2006241076; G. Lipford et al., "Immunostimulatory viral RNA oligonucleotides and use for treating cancer and infections," WO 2005097993 A2; G. Lipford et al., "Immunostimulatory G,U-containing oligoribonucleotides, compositions, and screening methods," WO 2003086280 A2.In some aspects, the additional agent may be a TLR-4 agonist, such as bacterial lipopolysaccharide (LPS), VSV-G, and / or HMGB-1. In some aspects, the additional agent may include a TLR-5 agonist, such as flagellin, including but not limited to those disclosed in U.S. Patent Nos. 6,130,082, 6,585,980, and 7,192,725, or portions or derivatives thereof.

[0173] In some aspects, the additional agent can be a proinflammatory stimulus (e.g., urate crystals) released from necrotic cells. In some aspects, the additional agent can be an activated component of the complement cascade (e.g., CD21, CD35, etc.). In some aspects, the additional agent can be an activated component of an immune complex. The additional agent also includes a complement receptor agonist, such as a molecule that binds to CD21 or CD35. In some aspects, the complement receptor agonist induces endogenous complement opsonization of the synthetic nanocarrier. In some aspects, the immunostimulant is a cytokine, a small protein or biological factor (in the range of 5 kD to 20 kD) that is released by cells and exerts a specific effect on cell-cell interactions, communication, and the behavior of other cells. In some aspects, the cytokine receptor agonist is a small molecule, an antibody, a fusion protein, or an aptamer.

[0174] IX. Engineered T Cell Receptors The T cell receptor contains two distinct polypeptide chains, called the T cell receptor α (TCRα) and β (TCRβ) chains, linked by disulfide bonds. These α:β heterodimers are very similar in structure to the Fab fragments of immunoglobulin molecules and are responsible for antigen recognition by most T cells. A small number of T cells have a different, but structurally similar, receptor composed of a pair of distinct polypeptide chains, called γ and δ. Both types of T cell receptors differ from membrane-bound immunoglobulins that function as B cell receptors: T cell receptors have only one antigen-binding site, whereas B cell receptors have two, and T cell receptors are never secreted, whereas immunoglobulins can be secreted as antibodies.

[0175] Both chains of the T cell receptor have an amino-terminal variable (V) region with homology to immunoglobulin V domains, a constant (C) region with homology to immunoglobulin C domains, and a short hinge region containing cysteine ​​residues that form interchain disulfide bonds. Each chain spans the lipid bilayer by a hydrophobic transmembrane domain and terminates in a short cytoplasmic tail.

[0176] The three-dimensional structure of the T cell receptor has been determined. This structure is certainly similar to that of an antibody Fab fragment, as inferred from previous studies of the genes encoding the T cell receptor. The chains of the T cell receptor fold almost identically to those of the Fab fragment, although the final structure appears to be a bit shorter and wider. However, there are some clear differences between the T cell receptor and the Fab fragment. The most striking difference is in the Cα domain, which folds differently from any other immunoglobulin-like domain. The half of the domain juxtaposed with the Cβ domain forms a β-sheet similar to those found in other immunoglobulin-like domains, while the other half of the domain is formed of loosely packed chains and short segments of α-helices. An intramolecular disulfide bond, which normally links two β-strands in immunoglobulin-like domains, links the β-strand to this segment of α-helix in the Cα domain.

[0177] There are also differences in the way the domains interact. The interface between the V and C domains of both T cell receptor chains is more extensive than in antibodies, which may reduce the mobility of the hinge junction between the domains. And the interaction between the Cα and Cβ domains is distinctive in that it is carbohydrate-assisted, with sugar groups from the Cα domain making several hydrogen bonds to the Cβ domain. Finally, comparison of the variable binding sites shows that the complementarity determining region (CDR) loops are aligned fairly closely with the loops of the antibody molecule, but with some displacement compared to the loops of the antibody molecule. This displacement is particularly pronounced in the Vα CDR2 loop, which is oriented at almost a right angle to the equivalent loop of the antibody V domain, as a result of a shift of the β strand from one face of the domain that anchors one end of the loop to the other. The strand displacement also causes a change in the orientation of the Vβ CDR2 loop in two of the seven Vβ domains whose structures are known. So far, the crystal structures of seven T cell receptors have been solved at this level of resolution.

[0178] Aspects of the present disclosure relate to engineered T cell receptors. The term "engineered" refers to T cell receptors that have TCR variable regions grafted onto TCR constant regions to create chimeric polypeptides that bind to the peptides and antigens of the present disclosure. In certain aspects, TCRs are used for cloning, enhancing expression, detection, or therapeutic control of constructs, but contain intervening sequences that are not present in endogenous TCRs, such as multiple cloning sites, linkers, hinge sequences, modified hinge sequences, modified transmembrane sequences, detection polypeptides or molecules, or therapeutic controls that may allow the selection or screening of cells that contain TCRs.

[0179] In some aspects, the TCR comprises non-TCR sequences.Thus, certain aspects relate to TCRs with sequences that are not derived from TCR genes.In some aspects, the TCR is chimeric in that it comprises sequences that are normally found in TCR genes, but from at least two TCR genes that are not necessarily found together in nature.

[0180] In some aspects, the engineered TCRs of the present disclosure include the aspects set forth below.

[0181] Table 1. TCR aspects TIFF2024514477000001.tif212160TIFF2024514477000002.tif220160TIFF2024514477000003.t if216160TIFF2024514477000004.tif203160TIFF2024514477000005.tif225160TIFF20245144770 00006.tif212160TIFF2024514477000007.tif230160TIFF2024514477000008.tif212160TIFF202 4514477000009.tif230160TIFF2024514477000010.tif203160TIFF2024514477000011.tif221160

[0182] X. Proteinaceous Compositions As used herein, "protein", "peptide" or "polypeptide" refers to a molecule that comprises at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, a wild type version of a protein or polypeptide is utilized, but in many aspects of the present disclosure, a modified protein or polypeptide is utilized to generate an immune response. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is modified relative to the wild type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be modified with respect to one activity or function, but may retain wild type activity or function in other respects, such as immunogenicity.

[0183] When a protein is specifically referred to herein, it generally refers to a natural (wild type) or recombinant (modified) protein, or a protein with any signal sequence removed, if desired. A protein can be directly isolated from an organism in which it is naturally occurring, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof). The term "recombinant" may be used in conjunction with the name of a polypeptide or a specific polypeptide, but generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or is the product of replication of such a molecule.

[0184] In certain aspects, the size of a protein or polypeptide (wild type or modified) is determined by: TIFF2024514477000012.tif49163 amino acid or nucleic acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein, but are not limited to such. It is contemplated that the polypeptides may be mutated by truncation to be shorter than their corresponding wild-type forms, and may be modified by fusing or conjugating heterologous proteins or polypeptide sequences with specific functions (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).

[0185] Polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the disclosure may contain 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, or 50 (or any range derivable therein) or more variant amino acid or nucleic acid substitutions, or at least one of SEQ ID NOs: 1-141. TIFF2024514477000013.tif9316One or more consecutive amino acids or nucleic acids, or any range derivable therein, or at most TIFF2024514477000014.tif9316 may be at least 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% or 100% (or any range derivable therein) similar, identical, or homologous to one or more contiguous amino acids or nucleic acids, or any range derivable therein. In certain aspects, the peptide or polypeptide is a human sequence or is based on a human sequence. In certain aspects, the peptide or polypeptide is non-naturally occurring and / or in a combination of peptides or polypeptides.

[0186] In some aspects, the protein, polypeptide or nucleic acid comprises one of the amino acids or nucleotides of SEQ ID NOs: 1-141, TIFF2024514477000015.tif114166 (or any range derivable therein).

[0187] In some aspects, a peptide or polypeptide of one of SEQ ID NOs: 1-141 The amino acid at position TIFF2024514477000016.tif152161 is substituted with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0188] In some aspects, the protein, polypeptide or nucleic acid is selected from the group consisting of SEQ ID NOs: 1-141. TIFF2024514477000017.tif122163 (or any range derivable therein) consecutive amino acids or nucleic acids.

[0189] In some aspects, the polypeptide, protein or nucleic acid has at least 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%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150%, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 1 3%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein), at most 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%, 100%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 1 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein), or exactly 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% or 100% (or any range derivable therein) similar, identical or homologous to at least one of SEQ ID NOs: 1-141. TIFF2024514477000018.tif114163 consecutive amino acids (or any range derivable therein), or at most TIFF2024514477000019.tif114163 consecutive amino acids (or any range derivable therein), or exactly TIFF2024514477000020.tif114163 (or any range derivable therein) consecutive amino acids.

[0190] In some aspects, any of SEQ ID NOs: 1-141. TIFF2024514477000021.tif196162TIFF2024514477000022.tif160162 and at least one of SEQ ID NO: 1 to 141 TIFF2024514477000023.tif86161TIFF2024514477000024.tif246162TIFF2024514477000025.tif27162 (or any range derivable therein) consecutive amino acids or nucleotides, or at most TIFF2024514477000026.tif86161TIFF2024514477000027.tif246162TIFF2024514477000028.tif27162 (or any range derivable therein) consecutive amino acids or nucleotides, or exactly There is a nucleic acid molecule or polypeptide that comprises TIFF2024514477000029.tif86161TIFF2024514477000030.tif246162TIFF2024514477000031.tif27162 (or any range derivable therein) consecutive amino acids or nucleotides.

[0191] Nucleotide and protein, polypeptide, and peptide sequences of various genes have been disclosed previously and can be found in recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information's Genbank and GenPept databases (ncbi.nlm.nih.gov / on the World Wide Web) and the Universal Protein Resource (UniProt; uniprot.org on the World Wide Web). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or as known to those skilled in the art.

[0192] It is contemplated that in the compositions of the present disclosure, there is about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per ml. The concentration of protein in the composition may be about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein), at least about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein), or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0193] The following is a discussion of altering the amino acid subunits of proteins to create equivalent or possibly improved second generation variant polypeptides or peptides. For example, certain amino acids can be substituted for other amino acids in a protein or polypeptide sequence with or without significant loss of interactive binding ability with structures such as, for example, the antigen-binding region of an antibody or a binding site on a substrate molecule. Since it is the interacting ability and properties of a protein that define its functional activity, certain amino acid substitutions can be made in a protein sequence and in its corresponding DNA coding sequence, and still produce a protein with similar or desirable properties. Thus, the inventors contemplate that various changes can be made in the DNA sequence of a gene encoding a protein without significant loss of its biological usefulness or activity.

[0194] The term "functionally equivalent codons" is used herein to refer to codons that code for the same amino acid, such as the six different codons for arginine. "Neutral substitutions" or "neutral mutations," which refer to alterations of codons that code for biologically equivalent amino acids, are also contemplated.

[0195] The amino acid sequence variants of the disclosure can be substitution, insertion, or deletion variants. Mutations in the polypeptides of the disclosure can affect 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, or more (or any range derivable therein) non-contiguous or contiguous amino acids of a protein or polypeptide compared to the wild type. Variants can include amino acid sequences that are at least 50%, 60%, 70%, 80%, or 90% identical to any sequence provided or referenced herein, including all values ​​and ranges in between. Variants can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substituted amino acids.

[0196] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and still be essentially identical as set forth in one of the sequences disclosed herein, so long as they meet the above criteria, including the maintenance of the biological protein activity to which expression of the protein is associated. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region.

[0197] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues may be deleted, or several adjacent amino acids may be deleted. Stop codons may be introduced (by substitution or insertion) into the coding nucleic acid sequence to generate truncated proteins.

[0198] Insertional mutants typically involve the addition of amino acid residues at non-terminal points in the polypeptide. This can include the insertion of one or more amino acid residues. Terminal additions can also be made, and these can include fusion proteins that are multimers or concatamers of one or more peptides or polypeptides described or referenced herein.

[0199] Substitution variants typically involve the exchange of one amino acid for another at one or more sites within a protein or polypeptide, and may be designed to modify one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, i.e., one amino acid may be replaced with an amino acid of similar chemical properties. A "conservative amino acid substitution" may involve the exchange of a member of one amino acid class with another member of the same class. Conservative substitutions are well known in the art and include, for example, changing alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine.Conservative amino acid substitutions may include amino acid residues that do not occur in nature, which are usually incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics or other reversed or inverted amino acid moieties.

[0200] Alternatively, the substitution may be "non-conservative" such that the function or activity of the polypeptide is affected. Non-conservative changes typically involve the replacement of one amino acid residue with a chemically different residue, such as a polar or charged amino acid replacing a non-polar or uncharged amino acid and vice versa. Non-conservative substitutions may involve the exchange of a member of one amino acid class for a member from another class.

[0201] Those skilled in the art can use well-known techniques to determine suitable variants of polypeptides as described herein. Those skilled in the art can identify suitable regions of molecules that can be altered without destroying activity by targeting regions that are not believed to be important for activity. Those skilled in the art can also identify amino acid residues and parts of molecules that are conserved between similar proteins or polypeptides. In a further aspect, regions that may be important for biological activity or for structure can be subject to conservative amino acid substitutions without significantly changing biological activity or adversely affecting protein or polypeptide structure.

[0202] In making such changes, the hydropathic index of the amino acids may be taken into consideration. The hydropathic profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then averaging these values ​​iteratively along the peptide chain. Each amino acid is assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resulting protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It is also known that certain amino acids can be substituted for other amino acids having similar hydropathic indexes or scores and still retain similar biological activity. When making changes based on hydropathic index, in certain aspects, substitutions of amino acids with hydropathic indexes within ±2 are included. In some aspects of the invention, within ±1 are included, and in other aspects of the invention, within ±0.5 are included.

[0203] It is also understood in the art that similar amino acids can be effectively substituted based on hydrophilicity.In U.S. Patent No. 4,554,101, which is incorporated herein by reference, it is stated that the greatest local average hydrophilicity of a protein, which is governed by the hydrophilicity of adjacent amino acids, correlates with the biological properties of the protein.In certain aspects, the greatest local average hydrophilicity of a protein, which is governed by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigen binding, i.e., as the biological properties of the protein. These amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). Making changes based on similar hydrophilicity values ​​includes, in certain aspects, substitution of amino acids with hydrophilicity values ​​within ±2, in other aspects within ±1, and in still other aspects within ±0.5. In some cases, epitopes can be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions." It is understood that an amino acid can be substituted with another amino acid with a similar hydrophilicity value and still result in a biologically equivalent and immunologically equivalent protein.

[0204] Moreover, one skilled in the art can review structure-function studies that identify residues in similar polypeptides or proteins that are important for activity or structure. In view of such comparisons, one can predict the importance of amino acid residues in the protein that correspond to amino acid residues that are important for the activity or structure of the similar protein. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0205] The skilled artisan can also analyze the three-dimensional structure and amino acid sequence in relation to its structure in a similar protein or polypeptide. In view of such information, the skilled artisan can predict the alignment of the amino acid residues of the antibody with respect to its three-dimensional structure. The skilled artisan may choose not to modify amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. In addition, the skilled artisan can generate test variants containing a single amino acid substitution at each desired amino acid residue position. These variants can then be screened using standard assays for binding and / or activity, thus obtaining information gathered from such routine experiments, which allows the skilled artisan to determine amino acid positions where further substitutions should be avoided, either alone or in combination with other mutations. Various tools available for determining secondary structure can be found on the World Wide Web at expasy.org / proteomics / protein_structure.

[0206] In some aspects of the present invention, amino acid substitutions are made that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) modify binding affinity to form protein complexes, (4) modify ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties to such polypeptides. For example, single or multiple amino acid substitutions (in certain aspects, conservative amino acid substitutions) can be made in naturally occurring sequences. Substitutions can be made in that portion of the antibody that is outside the domain that forms intermolecular contacts. In such aspects, conservative amino acid substitutions that do not substantially change the structural features of the protein or polypeptide (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes natural antibodies) can be used.

[0207] XI. Nucleic acids In certain aspects, the nucleic acid sequence can be present in various instances, such as: an isolated segment of an integrated sequence or recombinant polynucleotide and a recombinant vector encoding one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; a polynucleotide sufficient for use as a hybridization probe, PCR primer, or sequencing primer for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide; an antisense nucleic acid for inhibiting expression of a polynucleotide; and a complementary sequence of the foregoing described herein. Nucleic acids encoding epitopes for certain antibodies provided herein are also provided. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Nucleic acids can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids).

[0208] The term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from total genomic nucleic acid. Included within the scope of the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. A polynucleotide, in some aspects, includes a regulatory sequence that is substantially isolated from a naturally occurring gene or protein coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may or may not be present in a polynucleotide.

[0209] In this regard, the terms "gene," "polynucleotide," or "nucleic acid" are used to refer to a nucleic acid (including any sequences required for proper transcription, post-translational modification, or localization) that encodes a protein, polypeptide, or peptide. As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and variants. A nucleic acid that encodes all or a portion of a polypeptide can include a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide can be encoded by a nucleic acid that includes variants that have slightly different nucleic acid sequences but still encode the same or substantially similar proteins.

[0210] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein; the variants include at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, including all values ​​and ranges between, compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis with standard parameters). In some aspects, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95% and more identity to the amino acid sequences described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide.

[0211] Regardless of the length of the coding sequence itself, the nucleic acid segment may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and therefore the total length may vary considerably. The nucleic acids can be of any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can include one or more additional sequences, e.g., regulatory sequences, and / or can be part of a larger nucleic acid, e.g., a vector. It is therefore contemplated that nucleic acid fragments of almost any length can be used, with the total length being preferably limited by the ease of purification and by the intended use in the recombinant nucleic acid protocol. In some cases, the nucleic acid sequence can encode a polypeptide sequence with additional heterologous coding sequences, for example, to allow purification, transport, secretion, post-translational modification of the polypeptide, or to allow therapeutic utility such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides can be added to the sequence encoding the modified polypeptide, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0212] Hybridization A nucleic acid that hybridizes to another nucleic acid under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. For example, see Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a pre-wash solution containing 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), a hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as those that use a hybridization temperature of 42°C and contain about 50% formamide), and washing conditions of 60°C in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions involve hybridization in 6×SSC at 45° C., followed by one or more washes in 0.1×SSC, 0.2% SDS at 68° C. Moreover, one of skill in the art can manipulate the hybridization and / or wash conditions to increase or decrease the stringency of hybridization such that nucleic acids containing nucleotide sequences that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other will typically remain hybridized to each other.

[0213] Parameters influencing the selection of hybridization conditions and guidance for devising suitable conditions are described, for example, by Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are incorporated by reference in their entireties for all purposes), and can be readily determined by one of skill in the art based, for example, on the length and / or base composition of the DNA.

[0214] B. Mutation Changes can be introduced into nucleic acids by mutation, thereby resulting in changes in the amino acid sequence of the polypeptide (e.g., antibody or antibody derivative) that the nucleic acid encodes. Any technique known in the art can be used to introduce mutations. In one aspect, one or more specific amino acid residues can be changed, for example, using a site-directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed, for example, using a random mutagenesis protocol. However created, mutant polypeptides can be expressed and screened for desired properties.

[0215] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively alters the biological activity of the polypeptide it encodes. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, mutations can quantitatively or qualitatively alter biological activity. Examples of quantitative changes include increasing, reducing, or eliminating activity. Examples of qualitative changes include altering the antigen specificity of an antibody.

[0216] C. Probe In another aspect, the nucleic acid molecule is suitable for use as a primer or hybridization probe for the detection of a nucleic acid sequence. The nucleic acid molecule can comprise a portion of a nucleic acid sequence encoding a full-length polypeptide, e.g., a fragment that can be used as a probe or primer, or only a fragment that encodes an active portion of a given polypeptide.

[0217] In another aspect, the nucleic acid molecule can be used as a probe or PCR primer for a specific antibody sequence. For example, the nucleic acid molecule probe can be used in a diagnostic method, or the nucleic acid molecule PCR primer can be used to amplify a region of DNA that can be used to isolate a nucleic acid sequence for use in the production of a variable domain of an antibody, among others. See, for example, Gaily Kivi et al., BMC Biotechnol. 16:2 (2016). In a preferred aspect, the nucleic acid molecule is an oligonucleotide. In a more preferred aspect, the oligonucleotide is derived from the highly variable regions of the heavy and light chains or alpha and beta chains of the antibody or TCR of interest. In an even more preferred aspect, the oligonucleotide encodes all or part of one or more CDRs or TCRs.

[0218] A probe based on the desired sequence of the nucleic acid can be used to detect the nucleic acid or a similar nucleic acid, for example, a transcript that encodes a polypeptide of interest. The probe can contain a label group, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such a probe can be used to identify cells that express the polypeptide.

[0219] XII. Polypeptide Expression In some aspects, there are nucleic acid molecules (e.g., TCR genes) that encode the polypeptides or peptides of the present disclosure. These can be made by methods known in the art, such as by isolating from B cells of immunized and isolated mice, expressing them as phage displays in any suitable recombinant expression system, and allowing them to assemble to form antibody molecules, or by recombinant methods.

[0220] Expression The nucleic acid molecule may be used to express large amounts of the polypeptide. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, the nucleic acid molecule may be used for humanization of the TCR gene.

[0221] B. Vector In some aspects, expression vectors containing nucleic acid molecules encoding a polypeptide of desired sequence or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains) are contemplated. Expression vectors containing nucleic acid molecules may encode heavy chains, light chains, alpha chains, beta chains, or antigen-binding portions thereof. In some aspects, expression vectors containing nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0222] To express the polypeptide or peptide of the present disclosure, the DNA encoding the polypeptide or peptide is inserted into an expression vector such that the gene region is operably linked to transcriptional and translational control sequences. In some aspects, the vector encodes a functionally complete human CH or CL immunoglobulin or TCR sequence with suitable restriction sites engineered so that any variable region sequence can be easily inserted and expressed. In some aspects, the vector encodes a functionally complete human TCR alpha or TCR beta sequence with suitable restriction sites engineered so that any variable sequence or CDR1, CDR2 and / or CDR3 can be easily inserted and expressed. Typically, the expression vector used in any of the host cells contains sequences for plasmid or virus maintenance and for cloning and expressing exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences," typically include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding an expressed polypeptide, and a selectable marker element. Such sequences and methods for their use are well known in the art.

[0223] C. Expression Systems There are many expression systems that contain at least some or all of the above expression vectors. Prokaryotic and / or eukaryotic based systems can be used in one aspect to produce the nucleic acid sequence, or its cognate polypeptide, protein and peptide. Commercially and widely available systems include, but are not limited to, bacterial, mammalian, yeast and insect cell systems. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of expressed foreign proteins. Those skilled in the art can express the vector using a suitable expression system to produce the nucleic acid sequence, or its cognate polypeptide, protein or peptide.

[0224] D. Methods of Gene Transfer Suitable methods for nucleic acid delivery to result in expression of the composition are expected to include virtually any method by which a nucleic acid (e.g., DNA, including viral and non-viral vectors) can be introduced into a cell, tissue or organism, as described herein or known to those of skill in the art. Such methods include microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, incorporated herein by reference), by injection (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by the use of DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, each of which is incorporated herein by reference); by stirring with silicon carbide fibers (Kaeppler et al., each of which is incorporated herein by reference);, 1990; U.S. Patent Nos. 5,302,523 and 5,464,765); by Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or by PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patent Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); direct delivery of DNA, such as by desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0225] host cell In another aspect, the use of a host cell into which a recombinant expression vector has been introduced is contemplated. Antibodies can be expressed in a variety of cell types. An expression construct encoding an antibody can be transfected into a cell by a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may utilize control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. In certain aspects, an antibody expression construct can be placed under the control of a promoter associated with T cell activation, such as one controlled by NFAT-1 or NF-κB, both transcription factors that can be activated upon T cell activation. Control of antibody expression allows T cells, such as tumor-targeting T cells, to sense their surroundings and perform real-time regulation of cytokine signaling in both the T cell itself and surrounding endogenous immune cells. Those skilled in the art will understand the conditions under which host cells are incubated to maintain them and allow replication of the vector. Techniques and conditions that allow large-scale production of vectors, as well as production of the nucleic acid encoded by the vector and its cognate polypeptide, protein, or peptide, are also understood and known.

[0226] It is known that for stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small proportion of cells can integrate foreign DNA into their genome.To identify and select these integrants, a selectable marker (e.g., for antibiotic resistance) is generally introduced into host cells together with the gene of interest.The cells that have stably transfected the introduced nucleic acid can be identified by drug selection (e.g., the cells that have integrated the selectable marker gene survive, while other cells die), among other methods known in the art.

[0227] 2. Isolation The nucleic acid molecules encoding the whole or variable regions of the heavy, light, alpha and beta chains of an antibody or TCR can be obtained from any source that produces antibodies. Methods for isolating mRNA encoding an antibody are well known in the art. See, for example, Sambrook et al., supra. The sequences of human heavy and light chain constant region genes are also known in the art. See, for example, Kabat et al., 1991, supra. The nucleic acid molecules encoding the full-length heavy and / or light chains can then be expressed in cells into which they have been introduced, and the antibody can be isolated.

[0228] XIII. Cell Formulation and Culture In certain aspects, the cells of the present disclosure may be specially formulated and / or they may be cultured in a specific medium. The cells may be formulated in such a way that they are suitable for delivery to a recipient without adverse effects.

[0229] In certain aspects, the medium may be prepared using a medium used to culture animal cells as the basal medium, such as AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, 199 Medium, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fisher's medium, and any combination thereof, but the medium may not be particularly limited thereto, as long as it can be used to culture animal cells. In particular, the medium may be xeno-free or chemically defined.

[0230] The medium can be serum-containing or serum-free, or xeno-free. From the aspect of preventing contamination by heterologous animal-derived components, serum can be derived from the same animal as that of stem cells. Serum-free medium refers to a medium that does not have raw or unpurified serum, and therefore can include medium that has purified blood-derived components or animal tissue-derived components (such as growth factors).

[0231] The medium may or may not contain any substitute for serum. Serum substitutes may include materials that suitably contain albumin (such as albumin substitutes such as lipid-rich albumin, bovine albumin, recombinant albumin or humanized albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Serum substitutes may be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679, which is incorporated herein in its entirety. Alternatively, for more convenience, any commercially available material may be used. Commercially available materials include knockout serum replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0232] In certain aspects, the medium may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more of the following: biotin; DL-alpha tocopherol acetate; DL-alpha-tocopherol; vitamins such as vitamin A (acetate); BSA (bovine serum albumin) or human albumin, fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; proteins such as superoxide dismutase; corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2HCl; sodium selenite; and / or other components such as T3 (triiodo-I-thyronine). In certain aspects, one or more of these may be explicitly excluded.

[0233] In some aspects, the medium further comprises vitamins. In some aspects, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, vitamin B12 (and any range derivable therein), or the medium comprises combinations thereof or salts thereof. In some aspects, the medium comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, nicotinamide folate, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some aspects, the vitamin comprises or consists essentially of biotin, DL-alpha tocopherol acetate, DL-alpha-tocopherol, vitamin A, or combinations or salts thereof. In some aspects, the medium further comprises a protein. In some aspects, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some aspects, the medium further comprises one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, or combinations thereof. In some aspects, the medium comprises one or more of B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or combinations thereof. In some aspects, the medium comprises or further comprises amino acids, simple sugars, inorganic ions.In some aspects, the amino acid comprises arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof.In some aspects, the inorganic ion comprises sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or a combination or salt thereof.In some aspects, the medium further comprises one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or a combination thereof. In certain aspects, the medium comprises or consists essentially of one or more vitamins discussed herein, and / or one or more proteins discussed herein, and / or one or more of the following: corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triiodo-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, amino acids (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In certain aspects, one or more of these may be explicitly excluded.

[0234] The medium may also contain one or more exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts, of which in certain aspects one or more may be explicitly excluded.

[0235] One or more of the medium components has a concentration of at least 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein, and at most 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 It may be added at a concentration of about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml or any range derivable therein.

[0236] In certain aspects, the cells of the present disclosure are specially formulated. They may or may not be formulated as a cell suspension. In certain cases, they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin, and certain formulations include 2.5% human albumin. The cells may be specially formulated for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In certain aspects, the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from the time of thawing.

[0237] In certain aspects, the cells of the present disclosure comprise an exogenous TCR, which may be of defined antigen specificity. In some aspects, the TCR can be selected based on lack or reduced alloreactivity to the intended recipient. In the example where the exogenous TCR is non-allo-reactive, during T cell differentiation, the exogenous TCR suppresses rearrangement and / or expression of the endogenous TCR locus through a developmental process called allelic exclusion, resulting in T cells that only express non-allo-reactive exogenous TCRs and thus become non-allo-reactive. In some aspects, the selection of the exogenous TCR may not necessarily be defined based on lack of alloreactivity. In some aspects, the endogenous TCR gene is modified by genome editing so that it does not express protein. Methods of gene editing, such as those using the CRISPR / Cas9 system, are known in the art and are described herein.

[0238] In some aspects, the cells of the present disclosure further comprise one or more chimeric antigen receptors (CARs). Examples of tumor cell antigens to which the CAR can be directed include, for example, at least 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, ErbB2 (HER2), EGFR family including EGFRvIII, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, EPCAM, EphA2, EpCAM, folate receptor-a, FAP, FBP, fetal AchR, FRα, GD2, G250 / CAIX, GD3, glypican-3 (GPC3), Her2, IL-13Rα2, Lambda, Lewis-Y, Kappa, KDR, MAGE, MCSP, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSC1, PSCA, PSMA, ROR1, SP17, Survivin, TAG72, TEM, Carcinoembryonic antigen, HMW-MAA, AFP, CA-125, ETA, Tyrosinase, MAGE, Laminin receptor, HPV Examples of antigens that may be used include E6, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PAME, SSX-2, Melan-A / MART-1, GP100 / pmel17, TRP-1 / -2, P. polypeptide, MC1R, prostate specific antigen, β-catenin, BRCA1 / 2, CML66, fibronectin, MART-2, TGF-βRII, or VEGF receptor (e.g., VEGFR2). CARs may be first, second, third, or higher generation CARs. CARs may be bispecific for any two non-identical antigens, or specific for three or more non-identical antigens.

[0239] XIV. Administration of Therapeutic Compositions The therapeutic methods provided herein may include the administration of a combination of therapeutic agents, such as a first antiviral therapeutic and a second antiviral therapeutic. The therapeutic methods may be administered in any suitable manner known in the art. For example, the first and second antiviral treatments may be administered sequentially (at different times) or simultaneously (at the same time). In some aspects, the first and second antiviral treatments are administered in separate compositions. In some aspects, the first and second antiviral treatments are in the same composition.

[0240] Aspects of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapies can be administered in one composition, or in two or more compositions, such as two, three or four compositions. Various combinations of drugs can be utilized.

[0241] The therapeutic compositions of the present disclosure may be administered by the same or different routes of administration. In some aspects, the peptide, polypeptide, engineered TCR, engineered T cell, nucleic acid, antiviral therapy, or pharmaceutical composition is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some aspects, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0242] Treatment may include various "unit doses". A unit dose is defined as containing a predetermined amount of therapeutic composition. The amount to be administered, as well as the specific route and formulation, are within the skill of those skilled in the art in the clinical field to determine. A unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. In some aspects, a unit dose includes a single administrable dose.

[0243] Precise amounts of a therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dosage include the physical and clinical condition of the patient, the route of administration, the intended treatment goal (palliation of symptoms vs. cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other treatments the subject may be undergoing.

[0244] XV. Sample Preparation In certain aspects, the method involves obtaining a sample from a subject. The methods provided herein may include biopsy methods such as fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, incisional biopsy, excision biopsy, punch biopsy, shave biopsy or skin biopsy. In certain aspects, the sample is obtained from a biopsy from ovarian or endometrial tissue by any of the aforementioned biopsy methods. The sample may be obtained from any other source, including but not limited to blood, serum, plasma, sweat, hair follicle, cheek tissue, tears, menses, feces, or saliva. In certain aspects of the method, any medical professional, such as a doctor, nurse, or medical technician, may obtain a biological sample for testing. Furthermore, the biological sample may be obtained without the assistance of a medical professional.

[0245] The sample may include, but is not limited to, tissue, cells, or biological material derived from cells or from the subject's cells. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known in the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods, including, but not limited to, skin or cervical scraping, buccal swabbing, saliva collection, urine collection, fecal collection, menstrual, tear, or semen collection.

[0246] The sample may be obtained by methods known in the art. In certain aspects, the sample is obtained by biopsy. In other aspects, the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other method known in the art. In some cases, the sample may be obtained, stored, or transported using the components of the kit of the method. In some cases, multiple samples, such as multiple plasma or serum samples, may be obtained for diagnosis by the method described herein. In other cases, multiple samples, such as one or more samples from one tissue type (e.g., ovary or related tissue) and one or more samples from another specimen (e.g., serum), may be obtained for diagnosis by the method. The samples may be obtained at different times and stored and / or analyzed by different methods. For example, the samples may be obtained and analyzed by routine staining or any other cytological analysis method.

[0247] In some aspects, the biological sample may be obtained by a doctor, nurse, or other medical professional, such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or pulmonologist. The medical professional may prescribe the appropriate test or assay to be performed on the sample. In certain aspects, the molecular profiling operator may advise on which assay or test is most appropriately prescribed. In further aspects of the method, the patient or subject may obtain the biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0248] In other cases, samples are obtained by invasive procedures, including but not limited to biopsy, needle aspiration, blood draw, endoscopy, or phlebotomy. Methods of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some aspects, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0249] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, the entirety of which is incorporated herein by reference, describe general methods for biopsy and cytological methods.

[0250] In some aspects of the method, the molecular profiling entity may obtain the biological sample directly from the subject, from a medical professional, from a third party, or from a kit provided by the molecular profiling entity or a third party. In some cases, the biological sample may be obtained by the molecular profiling entity after the subject, a medical professional, or a third party obtains the biological sample and sends it to the molecular profiling entity. In some cases, the molecular profiling entity may provide appropriate containers and excipients for transport and storage of the biological sample to the molecular profiling entity.

[0251] In some aspects of the methods described herein, it is not necessary for a medical professional to be involved in the initial diagnosis or specimen collection. An individual may instead obtain a sample by use of an over-the-counter (OTC) kit. The OTC kit may include a means for obtaining a sample as described herein, a means for storing the sample for testing, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the purchase price of the kit. In other cases, molecular profiling services are charged separately. A sample suitable for use by a molecular profiling entity may be any material including tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of the individual to be tested. Methods are provided for determining the suitability and / or suitability of a sample.

[0252] In some aspects, the subject may be referred to a specialist, such as an oncologist, surgeon, endocrinologist, etc. The specialist may also obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of a biological sample. In some cases, a medical professional may refer the subject to a testing center or laboratory for submission of a biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling entity may obtain the sample.

[0253] XVI. Detection and Vaccination Kits The peptide or antibody of the present disclosure may be included in a kit. The peptide or antibody in the kit may be detectably labeled or immobilized on the surface of a support substrate that is also included in the kit. The peptide or antibody may be provided in the kit in a suitable form, such as, for example, sterile, lyophilized, or both.

[0254] The support substrate included in the kit of the invention may be selected based on the method to be performed. As non-limiting examples, the support substrate may be a multi-well plate or microplate, a membrane, a filter, paper, an emulsion, a bead, a microbead, a microsphere, a nanobead, a nanosphere, a nanoparticle, an ethosome, a liposome, a niosome, a transferosome, a dipstick, a card, a celluloid strip, a glass slide, a microslide, a biosensor, a lateral flow device, a microchip, a comb, a silica particle, a magnetic particle, or a self-assembled monolayer.

[0255] As appropriate for the method to be carried out, the kit may further comprise one or more devices for delivering the composition to a subject or for otherwise handling the composition of the present invention.As non-limiting examples, the kit may comprise a device that is a syringe, an eye dropper, a ballistic particle applicator (e.g., the applicator disclosed in U.S. Patent Nos. 5,797,898, 5,770,219 and 5,783,208 and U.S. Patent Application No. 2005 / 0065463), a medicine spoon, a microslide cover, a test paper holder or cover, etc.

[0256] The detection reagent for labeling the components of the kit may be optionally included in the kit for carrying out the method of the present invention.In certain aspects, the label or detection reagent is selected from the group including the reagents commonly used in the art, including but not limited to radioactive elements, enzymes, molecules that absorb light in the UV range, and fluorophores such as fluorescein, rhodamine, auramine, Texas Red, AMCA Blue and Lucifer Yellow.In other aspects, a kit is provided that includes one or more container means and a BST protein agent that is already labeled with a detection reagent selected from the group including radioactive elements, enzymes, molecules that absorb light in the UV range, and fluorophores.

[0257] When the reagents and / or components constituting the kit are provided in lyophilized form (lyophilizate) or as a dry powder, the lyophilizate or powder can be reconstituted by adding a suitable solvent. In certain aspects, the solvent can be a sterile, pharma- ceutically acceptable buffer and / or other diluent. It is also contemplated that such a solvent can be provided as part of the kit.

[0258] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution may be, by way of non-limiting example, a sterile aqueous solution. The composition may be formulated into an administration composition. In this case, the container means may itself be a syringe, pipette, topical applicator, or the like, from which the formulation may be applied to an affected area of ​​the body, injected into a subject, and / or applied to or mixed with other components of the kit.

[0259] XVII. Sequence (Table 1) TIFF2024514477000032.tif115164 EXAMPLES

[0260] XVIII. Working Examples The following examples are included to demonstrate preferred aspects of the present disclosure. Those skilled in the art will recognize that the procedures disclosed in the following examples are procedures that the inventors have found to work well in the practice of the present disclosure and therefore may be considered to constitute preferred modes for its practice. However, those skilled in the art will recognize in light of the present disclosure that many changes may be made in the specific aspects disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0261] Example 1: SARS-Cov-2 Membrane Glycoprotein-65 (MGP-65) Peptides, CTL and TCR The HLA-A2 restricted SARS-Cov-2 epitope of SEQ ID NO:22 can be used to detect immune responses in patients with COVID-19 infection or for vaccination of patients to prevent the disease. This peptide will enable research leading to a better understanding of COVIDl 9 immunity and will have a direct impact on the management of patients with COVID-19 infection. Furthermore, this peptide, CTLs, and corresponding TCRs can be used to generate third-patty SARS-Cov-2 specific T cells against HLA-matched targets, thus providing an off-the-shelf T cell therapy to patients with COVIDl9 disease. The ability to bank off-the-shelf allogeneic T cell therapy for the treatment of a life-threatening viral disease is unprecedented and can be easily applied to COVID 19 specific T cell therapy.

[0262] A375 cell line (HLA-A0201+) overexpressing MGP was lysed using NP-40 buffer, and the lysate was incubated with Sepharose Fast Flow beads coupled with anti-MHC class I antibody (W6 / 32). After washing unbound proteins, MHC-bound peptides were eluted using acetic acid. The eluted peptide solution was concentrated and analyzed using tandem mass spectrometry. One peptide hit of the MGP sequence (MGP-65, FVLAAVYRI (SEQ ID NO:22) was found (Figure 1).

[0263] MGP-65 (FVLAAVYRI - SEQ ID NO:22) peptide was pulsed into HLA-A0201 mature dendritic cells (MDCs) from healthy donors, followed by co-culture with autologous cells in 48-well plates. After two rounds of stimulation, a portion of T cells was harvested from each well for flow cytometric detection with MGP-65 tetramer and anti-CD8 staining. A small CD8+ / tetramer+ population was observed after stimulation. T cells in wells showing tetramer+ / CD8+ populations were pooled, and the tetramer+ / CD8+ population was sorted and expanded by rapid expansion protocol (REP). After 2 weeks of REP, highly pure CTLs (>90% tetramer+ population) were detected (Figure 2).

[0264] T2 cells pulsed with various concentrations of MGP-65 peptides were used as targets for cell lysis experiments. The lytic ability of MGP-65-specific CTL cell lines was detected by Cr51 release assay (CRA). The effector-to-target (E:T) ratio used was 20:1 (Figure 3A). A375 (HLA-A0201+). Mel624 (HLA-A0201+) cell lines with forced expression of eGFP or SARS-Cov-2 MGP were used as targets and co-cultured with MGP-65-specific CTL cell lines. The effector-to-target (E:T) ratio used ranged from 40:1 to 1.25:1. The lytic ability of MGP-65 CTL cell lines against different targets was detected by Cr51 release assay (CRA) (Figure 3B-C). MGP-65 CTL cell lines show significantly higher killing levels against positive targets than against negative controls (Figure 3).

[0265] A375-MGP and Mel624-MGP were used as "radioactive" target cells and were labeled with Cr51. T2 cells pulsed with MGP-65 peptide without Cr51 labeling corresponded to "non-radioactive" targets. T2 cells pulsed with irrelevant peptide M26 were used as control "non-radioactive" targets. E:T used was 20:1. Ratios of non-radioactive target:radioactive target used were 10:1 or 20:1. Inhibition of non-radioactive target killing against MGP-65 CTL was detected by CRA. When T2 pulsed with Hormad1-56 peptide was used as a non-radioactive target, killing of MGP-65 CTL against radioactive target was significantly inhibited compared to the group with only radioactive target. T2 pulsed with M26 negative control did not inhibit killing of MGP-65 CTL against radioactive target (Figure 4).

[0266] MGP-65-specific CTL cell lines were co-cultured with T2+M26, T2+MGP-65, A375-eGFP, A375-MGP, Mel624-eGFP and Mel624-MGP (E:T=10:1). After overnight co-culture, downstream activation markers of the TCR pathway, CD137, CD69, IFN-γ and TNF-α, were detected by ICS assay. The levels of CD137, CD69, IFN-γ and TNF-α of MGP-65-specific CTL cell lines were significantly enhanced when co-cultured with positive targets compared to negative control targets (Figure 5).

[0267] T cell receptors (TCRs), including α and β chains, were cloned using the 5-RACE PCR protocol. Sequences were annotated using the IMGT website tool (IMGT / V-QUEST) (Tables 1-3).

[0268] (Table 1) TIFF2024514477000033.tif52160

[0269] (Table 2) TIFF2024514477000034.tif52160

[0270] (Table 3) TIFF2024514477000035.tif65166

[0271] The TCR α and β chains were cloned and inserted into a retroviral vector. The recombinant retroviral vector was used to infect PBMCs. A CD8+ / tetramer+ population was detected after infection. After tetramer-induced selection and expansion, a highly pure TCR-T population was generated (Figure 6). Using a similar method, TCR-T cell populations were also generated against the NSP13-134 peptide (KLFAAETLK - SEQ ID NO:36) (Figure 23), the NSP13-242 peptide (TLVPQEHYV - SEQ ID NO:35) (Figure 26), the NSP13-400 peptide (VYIGDPAQL - SEQ ID NO:37) (Figure 29) and the NSP13-448 peptide (IVDTVSALVY - SEQ ID NO:34) (Figure 32).

[0272] The ability of SARS-CoV-2 specific TCR-T to lyse target cells was tested by Cr51 release assay. As shown in Figures 7, 24, 27, 30 and 33, SARS-CoV-2 TCR-T shows significantly higher lysis of positive targets compared to negative controls. NSP13-134 specific TCR-T was co-cultured with SK-MES-1 + control A0301 peptide, SK-MES-1 + NSP13-134, SK-MES-1-GFP, SK-MES-1-NSP13, Hs-578T-GFP and Hs-578T-NSP13 (E:T=10:1) (Figure 25). NSP13-242-specific TCR-T were co-cultured with T2+M26, T2+NSP13-242, A375-GFP, A375-NSP13, RPMI-7951-GFP and RPMI-7951-NSP13 (E:T=10:1) (Figure 28). NSP13-400-specific CD8+ and CD4+ TCR-T were co-cultured with M14+control peptide, M14+NSP13-400, Hs-578T-GFP, Hs-578T-NSP13, M14-GFP and M14-NSP13 (E:T=10:1) (Figure 31). NSP13-448-specific TCR-T were co-cultured with A375 + control A0101 peptide, A375 + NSP13-448, A375-GFP, A375-NSP13, RPMI-7951-GFP and RPMI-7951-NSP13 (E:T=10:1) (Figure 34). After overnight co-culture, downstream activation markers of the TCR pathway, CD137, CD69, IFN-γ and TNF-α, were detected by ICS assay. The levels of CD137, CD69, IFN-γ and TNF-α of peptide-specific TCR-T were significantly enhanced when co-cultured with positive targets compared to negative control targets (Figures 25, 28, 31 and 34).

[0273] In conclusion, we have generated SARS-CoV-2-specific CTLs, cloned TCR and TCR engineered T cells (TCR-T) and functionally validated the CTLs and TCR T cells. Cell therapy using TCR-T can be adopted as an optional therapeutic approach for the treatment of severe SARS-CoV-2 infected patients. The identified SARS-CoV-2 peptides or coding sequences can be loaded into presenting cells and co-cultured with T cells to generate antigen-specific CTL cell lines or clones. Autologous or allogeneic CTL cell lines or clones can be used for adoptive immunotherapy for HLA-matched patients with SARS-CoV-2 infection.

[0274] Example 2: Identification of immunogenic SARS-CoV-2 epitopes and cognate TCRs by mass spectrometry for T cell-based therapy of COVID19 disease A. Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a highly contagious respiratory virus responsible for the outbreak of the COVID-19 pandemic, continues to have a significant and lasting impact on global public health, raising the urgent need to develop accurate immunodiagnostics and effective treatment strategies (Hui et al., 2020; Wu et al., 2020b). The rapid dissemination of the SARS-CoV-2 genome sequence, first revealed by Dr. Zhang Yongzhen, led to extensive efforts around the world to develop protective vaccines that could elicit humoral (antibody) and cellular (T cell) responses (Wu et al., 2020a). Identifying immunogenic epitopes of SARS-CoV-2 recognized by the human immune system is therefore crucial for rational vaccine development.

[0275] Using in silico prediction algorithms, several investigators have collected extensive panels of class I and class II restricted epitopes to explore SARS-CoV-2 specific T cell responses, in some cases combining these with overlapping “megapools” spanning conserved regions of the genome ( Campbell et al., 2020 ; Grifoni et al., 2020a ). These peptides have been used to track responses in infected and convalescent individuals (Kar et al., 2020; Peng et al., 2020), to design multi-epitope vaccines, and have been used directly or indirectly to measure the spread and severity of COVID19 disease (Braun et al., 2020; Grifoni et al., 2020b; Kar et al., 2020; Le Bert et al., 2020; Nolan et al., 2020; Snyder et al., 2020; Weiskopf et al., 2020). Although these studies have revealed insights into the T cell immunobiology of COVID19, the accuracy of in silico predicted responses and T cell responses using overlapping long peptide (OLP) pools is diminished by not considering whether such epitopes are immunogenic. An immunogenic epitope in this sense is defined as a peptide known to be presented by self-MHC and capable of eliciting T cells of sufficient affinity to recognize target cells endogenously expressing the antigen and presenting antigen-derived peptides in the context of MHC complexes with sufficient surface density to sensitize the target cells to peptide-specific T cell-mediated recognition. In essence, the identification of SARS-CoV-2 immunogenic epitopes requires both direct sequencing of peptides presented by MHC and empirical validation of T cell immunogenicity.

[0276] To our knowledge, this study is the first use of tandem MS to identify T cell epitopes of SARS-CoV-2 following peptide elution from the MHC complex of SARS-CoV-2-expressing cells and the first to empirically validate the immunogenicity of SARS-CoV-2-specific CTLs by in vitro generation. We applied a technique developed by our group that allows the isolation of rare tumor-reactive T cells from a population with very low precursor frequency in peripheral blood (Chapuis et al., 2017). We present data on the identification of five immunogenic epitopes in highly conserved regions of membrane glycoproteins and in nonstructural protein regions of the SARS-CoV-2 genome, and demonstrate that such MGP65- and NSP13-specific CTLs recognize and kill SARS-CoV-2 antigen-expressing target cells; we further demonstrate that specificity can be transferred by sequencing the TCR α and β chains and manipulating the expression of this TCR in polyclonal lymphocytes.

[0277] B. Results 1. SARS-CoV-2 peptides defined "in silico" fail to induce T cells that recognize SARS-CoV-2 antigen-expressed targets As an initial screen of known predicted epitopes for immunogenicity, we selected class I-restricted peptides against the SARS-CoV-2 spike protein and membrane glycoprotein (MGP) based on a literature search of studies in which such "in silico" predicted peptides were described as "immunodominant". These peptides have previously been reported to be “immunodominant” based on their ability to generate high-level peptide-specific responses from PBMCs of COVID19+ patients and, surprisingly, in some healthy donors as well (apparently as a result of cross-reactive responses from T cells previously elicited against non-pathogenic SARS viruses) (Agerer et al., 2021; Ahmed et al., 2020; Gao et al., 2020; Grifoni et al., 2020a; Kar et al., 2020; Safavi et al., 2020; Shomuradova et al., 2020; Sohail et al., 2021; V. Gauttier, 2020; William Chour, 2020). We have synthesized four of these spike protein peptides and three membrane glycoprotein (MGP) peptides. Using an endogenous T cell (ETC) generation workflow (see Methods section), we generated individual T cell cultures against all four spike peptides and all three MGP peptides (Figure 16). However, when these highly enriched (>80% tetramer+) T cell cultures were tested against HLA-matched target cells engineered to express the relevant SARS-CoV-2 spike protein or membrane glycoprotein, no evidence of target cell killing was observed (Figure 16). We hypothesized that a more accurate means of identifying these in silico predicted peptides that are not endogenously presented and endogenously presented immunogenic epitopes would be desirable and could be achieved by directly eluting and sequencing peptides from the MHC of SARS-CoV-2 expressing cells.

[0278] 2. Profiling MHC class-I restricted epitopes of SARS-CoV-2 The antigen discovery platform for SARS-CoV-2 consists of four stages: (1) peptide elution and mass spectrometry (MS) identification of SARS-CoV-2 targets; (2) endogenous T cell (ETC) generation workflow to elicit peptide-specific CTLs; (3) empirical validation of antigen-specific CTLs against SARS-CoV2 targets; and (4) development of SARS-CoV-2-specific T cell receptor (TCR) engineered T cells (TCR-T) (Figure 15). To elute and sequence MHC-binding peptides derived from SARS-CoV-2, SARS-CoV-2 genes were overexpressed in target cells with distinct HLA allele expression. Lentiviral expression vectors spanning highly conserved regions of SARS-CoV-2 domains: membrane glycolipid protein (MGP) or nonstructural protein helicase (NSP13) (Keller et al., 2020; Le Bert et al., 2020) were constructed and used to infect A375 (HLA-A0101 / 0201), Mel624 (HLA-A0201), RPMI-7951 (HLA-A0101 / 0201), Hs-578T (HLA-A0301 / A2402) and M14 (HLA-A1101 / 2402). After puromycin selection and expansion of MGP or NSP13 stable expressing cell lines, purity of >90% was achieved (data not shown). MGP- or NSP13-expressing cell lines were expanded to 300–500 million, harvested, lysed in NP40 detergent lysis buffer, and subjected to total HLA class I immunoprecipitation (anti-HLA-A, B, C) and acid elution followed by tandem mass spectrometry (MS) to analyze HLA-binding peptides.

[0279] We first analyzed the eluted HLA-binding peptides derived from the SARS-CoV-2 targets established above by using data-dependent analysis liquid chromatography tandem mass spectrometry (DDA MS / MS). The eluted spectra were searched against the Swiss-Prot human proteome database (version 2020_05) followed by the viral proteome database (version 2020_05) using the Mascot search engine node (version 2.6) in the Proteome Discoverer (version 2.3) processing workflow. To reduce false positive hits from the human proteome, the "Spectrum Confidence Filter" node in the Proteome Discoverer processing workflow filtered out all spectra with high confidence of spectral matches with peptides annotated from the human proteome. The remaining spectra were further searched against the viral proteome (Figure 8A). In total, 12,770 MS / MS were acquired and 9731 Peptide-Spectrum-Match (PSM) were annotated, resulting in 357 peptides with Mascot Ions Score ≥25. Among the 357 peptides, a peptide derived from NSP13 (NSP13-400, VYIGDPAQL - SEQ ID NO:37) was annotated with Mascot Ions Score=27 (Figure 8B). This peptide was eluted from M14-NSP13 cells (HLA-A1101 / A2402). From the HLA binding assay (IEDB tools), the NSP13-400 peptide recorded high predicted binding affinity to the HLA-A2402 allele (Table 2), suggesting that the NSP13-400 peptide is likely presented by HLA-A2402.

[0280] To enable more comprehensive profiling of potential HLA class I restricted peptides from SARS-CoV-2, we further analyzed the eluted peptides by parallel reaction monitoring mass spectrometry (PRM-MS) to focus on predicted high probability HLA binding peptides derived from SARS-CoV-2 that were not successfully detected by the DDA approach. Prior to PRM-MS, we selected 10 predicted high probability HLA-A0101, HLA-0201 or HLA-A0301 binding peptides from MGP or NSP13, respectively (Table S1). For peptides eluted from A375-NSP13, a precursor ion inclusion list of 10 potential peptides was generated using Skyline, and we targeted and monitored these 10 peptides using nanoflow LC-PRM-MS with high mass accuracy and resolution. Pierce™ Peptide Retention Time Calibration Mixture peptides were used to monitor retention time drift and calibrate the scheduled PRM method. We first created a spectral library using synthetic peptides, and we defined a normalized dimensionless peptide characteristic value iRT using both synthetic peptides and Pierce™ Peptide Retention Time Calibration Mixture peptides to accurately predict the retention time of each target peptide. We detected IVDTVSALVY (SEQ ID NO:34) (NSP13-448) with a mean product ion ppm error of -0.7 ppm (Figure 8C) and TLVPQEHYV (SEQ ID NO:35) (NSP13-242) with a mean product ion ppm error of -0.7 ppm (Figure 8D). Applying the same rule, from A375-MGP, we detected FVLAAVYRI (SEQ ID NO:22) (MGP-65) with a mean product ion ppm error of -0.3 ppm (Figure 8E). These XIC MS1 analyses reported sufficient chromatographic separation and high-resolution MS1 ​​detection in a narrow m / z window, suggesting that these target peptides were present in the eluted peptide samples.Additionally, one potential peptide, KLFAAETLK (SEQ ID NO:36) (NSP13-134), was selected from the PRM-MS screening (FIG. 17) for further immunogenicity functional assays (MS / MS not shown).

[0281] To assess whether these five candidate SARS-CoV-2 HLA Class-I restricted peptides identified by DDA or PRM-MS are homologous to other coronaviruses, including SARS-CoV, Middle East Respiratory Syndrome coronavirus (MERS-CoV), and four other coronaviruses 229E, NL63, OC43, and HKU1, multiple sequence alignment (MSA) analysis was performed. NSP13-242 (Figure 8G), NSP13-134 (Figure 8H), and MGP-65 (Figure 8J) show a high degree of homology to sequences of SARS-CoV. NSP13-400 (Figure 8I) shows a high degree of homology to sequences of SARS-CoV, HCoV-OC43, and HCoV-HKU1 (red underline). NSP13-448 (Figure 8F) shows a high degree of homology to sequences of SARS-CoV, HCoV-OC43 (red underline). To assess whether these five candidates have homology to non-coronavirus species, the peptides were analyzed by using BLAST searches to identify all potential source proteins. The top 250 hits for each target sequence reported up to 88.99% identity or 100% identity, but the identity with related coronaviruses was up to 88.99% or 100% coverage, indicating that these five candidate peptides show homology only to coronaviruses and not to other species (data not shown).

[0282] 3. The newly defined epitope is found in a highly conserved region of SARS-CoV-2 and SARS-CoV2 variants Olvera et al. recently described the development of a COVID19 vaccine using overlapping SARS-CoV-2 consensus sequences (Olvera et al., 2020). This paper utilized entropy-based calculations on over 1700 viral genome entries in NCBI to encompass all described SARS-CoV-2 open reading frames (ORFs), including recently described frameshift and length variant ORFs. The Nextstrain project (found online at nextstrain.org) is an open-source project that provides a continuously updated view of publicly available data, along with powerful analytical and visualization tools to aid epidemiological understanding and improve outbreak response, providing a means to analyze genetic diversity across the SARS-CoV-2 genome. Using both of these sources, we validated that these five peptides are located in highly conserved regions of the SARS-CoV-2 genome (Figure 8K). Recently, genetic variants of SARS-CoV-2 have emerged on a global scale, for example, the mutation 23403A>G-(D614G) located on the spike protein is thought to make SARS-CoV-2 more infectious (Garcia-Beltran et al., 2021; Tzou et al., 2020). To investigate whether the recently reported variants are among these five SARS-CoV-2 HLA class-I restricted epitope candidates, we focused on variants of concern first described in the UK (B.1.1.7), Denmark (B.1.1.298), the US (B.1.429), Brazil and Japan (P.2 and P.1), and South Africa (B.1.351). For the NSP-13 region, there are two mutations E341Y and A368V present in the P.1 variant, one mutation K460R present in the B.1.1.7 variant, and one mutation T588I present in the B.1.351 variant, but none of these four NSP-13 HLA class-I restricted peptides overlap. No variant strains from the membrane glycol protein (MGP) region have been reported (Figure 8L).

[0283] 4. MGP-65 peptide-specific cytotoxic T cells generated from peripheral blood recognize SARS-CoV-2-MGP-expressing target cells After leukapheresis, HLA-A0201 healthy donor PBMCs were stimulated with MGP-65 peptide (FVLAAVYRI - SEQ ID NO:22)-pulsed autologous DCs. After two rounds of stimulation, MGP-65-A2 tetramer-positive staining populations were detected (Figure 9A). Approximately 33 wells from one 48-well plate showed distinct MGP-65 peptide tetramer-positive CD8+ T cell populations (Figure 8), suggesting that MGP-65 peptide-specific T cells are readily expanded by cognate peptide stimulation, even in PBMCs from healthy donors with no history of SARS-CoV-2 infection. Expanded MGP-65 CTLs were then cultured using standard 51 Functional testing was performed using a Cr release assay (CRA). T2 cells (HLA-A0201) pulsed with titrated amounts of MGP-65 peptide induced CTL recognition and killing at peptide concentrations as low as 10 pM (Figure 9B), suggesting that MGP-65 CTLs have a very high recognition affinity for the cognate peptide.

[0284] To verify that MGP-65-specific CTLs recognized the endogenously presented cognate peptide, HLA-A0201+ target cells were engineered to express the SARS-CoV-2 MGP gene (A375-MGP, Mel624-MGP). MGP-65-specific CTLs were able to lyse the A375-MGP and Mel624-MGP cell lines, but were unable to lyse the A375-GFP and Mel624-GFP control cell lines (Figures 9C and 9D). To further confirm that the target recognition of MGP-65-specific CTLs was due to the engagement of the endogenously presented cognate peptide, a non-radioactive target inhibition assay was performed. 51 Cr-pulsed MGP expressing cells 51Cr, whereas T2 cells pulsed with MGP-65 or M26 irrelevant peptide were left unlabeled and used as cold or control cold targets, respectively. When cold targets were added at both cold to radioactive target ratios (C:H) of 20:1 and 10:1, the cytotoxicity of MGP-65-specific CTLs against radiolabeled MGP-65 peptide targets was significantly inhibited (Figures 9E and 9F). However, no inhibition was observed when control cold targets were added, indicating that MGP-65 CTLs can lyse MGP-65 targets via recognition of the endogenously presented cognate peptide. This data provided further evidence that the MGP-65 peptide is a natural endogenously presented MHC peptide.

[0285] To further evaluate the function of MGP-65-specific CTLs, intracellular staining (ICS) assays were performed to detect IFN-γ and TNF-α production. Co-culture of MGP-65-specific CTLs with target cells pulsed with MGP-65 peptide or engineered with MGP demonstrated specific recognition by the produced IFN-γ and TNF-α compared to control targets pulsed with an irrelevant peptide or engineered to express control GFP (Figure 9G). Upon encountering relevant SARS-CoV-2 targets, proportional increases in T cell activation markers CD137 and CD69 were also specifically and significantly elevated compared to the control groups (Figure 9G).

[0286] 5. NSP13-242 peptide-specific cytotoxic T cells generated from peripheral blood recognize SARS-CoV-2-NSP13-expressing target cells In contrast to structural proteins such as MGP and spike proteins, nonstructural proteins of SARS-CoV-2 are not expressed on the virion surface and are therefore less likely to induce humoral responses or neutralizing antibodies. However, nonstructural proteins in SARS-CoV-2-infected cells can be presented as MHC-binding peptides and induce cellular immune responses that can last for a long time. Here, using the same workflow, the HLA-A0201-restricted peptide NSP13-242 (TLVPQEHYV - SEQ ID NO:35), derived from the NSP13 helicase, was identified by MS / MS. Similar to MGP-65, NSP13-242-specific T cells were readily generated using the ETC workflow (Figure 10A). Surprisingly, after stimulation with NSP13-242 peptide, all 48 wells from one 48-well plate showed distinct NSP13-242 peptide tetramer-positive CD8+ T cell populations (Figure 19), suggesting that the NSP13-242 peptide may be highly immunogenic.

[0287] Cytotoxicity assays also demonstrated that NSP13-242-specific CTLs could recognize cognate peptides as low as 100 pM (Figure 10B), indicating the expression of high-affinity TCR. More importantly, NSP13-242-specific CTLs were able to lyse NSP13-expressing targets A375-NSP13 and Mel624-NSP13, but not control targets, even at a very low E:T ratio (2.5:1) (Figures 10C and 10D), indicating that NSP13-242-specific CTLs could recognize endogenously presented peptides of NSP13 protein. Similar to MGP-65 CTLs, nonradioactive target inhibition assays also demonstrated that addition of nonradioactive target significantly inhibited the lytic ability of NSP13-242-specific CTLs against radioactive targets A375-NSP13 and Mel624-NSP13 (Figures S10E and S10F), further confirming that NSP13-242-specific CTLs lyse targets via recognition of endogenously presented cognate peptides.

[0288] ICS assays demonstrated that NSP13-242-specific CTLs produced higher levels of the proinflammatory cytokines IFN-γ and TNF-α and expressed higher levels of the antigen-driven activation markers CD137 and CD69 when cocultured with NSP13-242 peptide-pulsed or NSP13-expressing targets compared to control targets (Figure 10G). Thus, similar to MGP-65-specific CTLs, NSP13-242-specific CTLs also initiate specific cellular immune responses upon encountering SARS-CoV-2.

[0289] 6. NSP13-448 peptide-specific cytotoxic T cells generated from peripheral blood recognize SARS-CoV-2-NSP13-expressing target cells The HLA-A0201 allele is expressed in approximately 45% of Caucasians and Asians (Kessler et al., 2003). Given the global spread of COVID19, it would be desirable to specifically target T cells to other highly prevalent HLA-A alleles of SARS-CoV-2. Using the same workflow for MGP65 and NSP13-242 peptides, we identified an HLA-A0101-restricted peptide, NSP13-448 (IVDTVSALVY - SEQ ID NO:34), derived from NSP13 protein by MS / MS; this allele encompasses approximately 26% of Caucasians and 7% of Asians (Kessler et al., 2003). Similar to NSP13-242, NSP13-448-specific T cells were readily generated using the ETC workflow (Figure 11A). However, after stimulation with NSP13-448 peptide, only one well from one 48-well plate showed a distinct NSP13-448 peptide tetramer-positive CD8+ T cell population (Figure 20), suggesting that NSP13-448 peptide may not be highly immunogenic.

[0290] Cytotoxicity assays also demonstrated that NSP13-448-specific CTLs could recognize the cognate peptide as low as 100 nM (Figure 11B), indicating moderate to low TCR affinity. Interestingly, NSP13-448-specific CTLs were able to lyse the NSP13-expressing targets A375-NSP13 and RPMI-7951-NSP13, but not the control targets, even at a very low E:T ratio (2.5:1) (Figures 11C and 11D), indicating that NSP13-448-specific CTLs could recognize endogenously presented peptides of the NSP13 protein. Nonradioactive target inhibition assays also demonstrated that addition of nonradioactive target significantly inhibited the lytic ability of NSP13-448-specific CTLs against radioactive targets A375-NSP13 and RPMI-7951-NSP13 (Figures 6E and 6F), further confirming that NSP13-448-specific CTLs lyse these targets via recognition of the endogenously presented cognate peptide.

[0291] ICS assays demonstrated that NSP13-448-specific CTLs produced higher levels of the proinflammatory cytokines IFN-γ and TNF-α and expressed higher levels of the antigen-driven activation markers CD137 and CD69 when cocultured with NSP13-448 peptide-pulsed or NSP13-expressing targets compared with control targets ( Fig. 12G ).

[0292] 7. NSP13-134 peptide-specific cytotoxic T cells generated from peripheral blood recognize SARS-CoV-2-NSP13-expressing target cells In addition to HLA-A0101 and HLA-A0201 alleles, HLA-A0301 alleles cover approximately 22% of Caucasians and approximately 13% of Africans (Kessler et al., 2003). Using the same workflow, we identified an HLA-A0301-restricted peptide, NSP13-134 (KLFAAETLK - SEQ ID NO:36) derived from NSP13 protein. After in vitro stimulation using the ETC workflow, 11 of 48 wells showed a clear NSP13-134 peptide tetramer-positive CD8+ T cell population (Figure 21), suggesting that the NSP13-134 peptide is sufficiently immunogenic to induce T cell responses in healthy donors without previous SARS-CoV-2 infection. After sorting and expansion, highly pure CD8+ and tetramer+ NSP13-134 CTLs were generated (Figure 12A). Peptide titration assays showed that NSP13-134-specific CTLs could recognize cognate peptides as low as 100 pM (Figure 12B), suggesting the expression of a relatively high affinity TCR. Similar to MGP-65- and NSP13-242-specific CTLs, NSP13-134-specific CTLs were able to lyse the NSP13-expressing target Hs-578T-NSP13, even at a low E:T ratio (2.5:1), but not the control target (Figure 12C). Similar to MGP-65 and NSP13-242 CTLs, non-radioactive target inhibition assays confirmed the specific recognition of endogenously presented cognate peptides (Figure 12D). ICS assays also confirmed the specific recognition of NS13-expressing targets (Figure 12E).

[0293] 8. Expansion proliferation and functional assay of NSP13-400 peptide-specific cytotoxic T cells from peripheral blood of healthy donors In addition to HLA-A0101, HLA-A0201 and HLA-A0301, the HLA-A2402 allele covers an additional 40% of Asians and 20% of Caucasians (Kessler et al., 2003). Using the same workflow, we identified the HLA-A2402-restricted peptide of NSP13, NSP13-400 (VYIGDPAQL - SEQ ID NO:37). After in vitro stimulation with 9 of 48 wells, a clear NSP13-400 peptide tetramer-positive CD8+ T cell population was demonstrated (Figure 22). After sorting and expansion, highly pure CD8+ and tetramer+ NSP13-400 CTLs were generated (Figure 13A). Similar to MGP-65 CTLs, NSP13-400-specific CTLs showed very high recognition affinity for the cognate peptide at concentrations as low as 10 pM in peptide titration assays (Figure 13B), and showed very high specific lysis of NSP13-expressing targets Hs-578T-NSP13 and M14-NSP13 even at E:T ratios as low as 1.25:1 according to peptide titration assays (Figures 13C and 13D). Similarly, non-radioactive target inhibition assays showed specific levels of NSP13-400 CTLs against radioactive targets Hs-578T-NSP13 and M14-NSP13, which were significantly inhibited by the addition of non-radioactive targets (Figures 13E and 13F), further confirming that NSP13-400-specific CTLs lyse SARS-CoV-2 targets via recognition of endogenously presented cognate peptides.

[0294] ICS assays also confirmed specific recognition of NS13-expressing targets (Figure S13G). IFN-γ and TNF-α levels were significantly elevated compared to other SARS-CoV-2 CTL target assays, suggesting high density endogenous presentation.

[0295] In summary, using our MHC IP elution and MS identification workflow, we discovered five HLA class-I restricted peptides derived from the structural protein MGP and nonstructural protein NSP13 of SARS-CoV-2 that are presented by several HLA alleles (HLA-A0101, HLA-A0201, HLA-A0301 and HLA-A2402) covering approximately 80% of Caucasian and Asian populations. All five peptides were highly immunogenic and could readily elicit T cell responses among healthy COVID19 negative donors. All five SARS-CoV-2 specific CTLs recognized the endogenously presented cognate peptides and specifically lysed SARS-CoV-2+ targets.

[0296] 9. MGP-65-specific T cell receptor engineered T cells (TCR-T) recognize SARS-CoV-2 MGP-65-expressing target cells As proof of principle that these findings could lead to the development of an "off-the-shelf" SARS-CoV-2-specific T cell therapy for COVID19 patients, we sequenced and cloned the TCR α and β chains from MGP-65-specific T cells and determined whether it was possible to transfer specificity and function to peripheral blood lymphocytes (PBLs). Sequence annotation revealed an α chain (TCR-α) belonging to the TRAV17*01F / TRAJ50*01F subtype and a β chain (TCR-β) belonging to TRBV9*02F / TRBJ2-1*01F / TRBD1*01F (Table 3). A retroviral vector, pMSGV1, containing full-length TCR α and β chains linked with the truncated peptides Furin and P2A was constructed and used to infect OKT3-activated allogeneic PBLs from another HLA-A0201 healthy donor. Five days after infection, a population of approximately 37% CD8+tetramer+ T cells was observed (Figure 14A), indicating successful exogenous TCR pairing in allogeneic PBLs. After selection and expansion, highly pure MGP-65-specific TCR-T cells were generated (Figure 14A).

[0297] To evaluate the function and specificity of MGP-65-specific TCR-T, 51Cr release assay (CRA) and intracellular staining (ICS) assays were performed and compared with parental MGP-65-specific CTL lines. MGP-65 TCR-T was able to recognize peptide-pulsed targets titrated at peptide concentrations as low as 10 pM (Figure 14B), indicating that this TCR-T also exhibits high affinity recognition of the cognate peptide. Furthermore, MGP-65 TCR-T specifically lysed MGP-expressing targets A375-MGP and M624-MGP (Figures 14C and 14D), correlating with ICS assays demonstrating that MGP-65-specific TCR-T produced higher levels of IFN-γ and TNF-α, as well as activation markers CD137 and CD69, when co-cultured with MGP-expressing or MGP-65 peptide-pulsed targets, compared with parental CTLs (Figure 14E).

[0298] C. Discussion To date, about 1,500 predicted class I epitopes of SARS-CoV2 have been identified by in silico prediction methods and, in some cases, “validated” by eliciting T cell responses using PBMCs from patients with COVID19 (Campbell et al., 2020; Grifoni et al., 2020a). These peptides have been widely used to evaluate T cell responses of patients, and sometimes healthy donors, to COVID19 and COVID19 vaccines, and are increasingly being used to develop T cell-based therapeutics. What remains to be demonstrated, however, is whether any of these 1,500 predicted peptides actually represent natural epitopes processed and presented by SARS-CoV2+ cells and recognized by T cells. A preliminary screen of predicted SARS-CoV2 epitopes considered “immunodominant” in widely cited reports seems to support this premise: we found that seven of these eight predicted peptides failed to elicit T cell responses leading to recognition of SARS-CoV2+ targets, suggesting that responses to these peptides may be artifactual or at best cross-reactive (Figure 15) (Agerer et al., 2021; Ahmed et al., 2020; Gao et al., 2020; Grifoni et al., 2020a; Kar et al., 2020; Safavi et al., 2020; Shomuradova et al., 2020; Sohail et al., 2021; V. Gauttier, 2020; William Chour, 2020). To date, there is no empirical validation of the immunogenicity of SARS-CoV2 epitopes.

[0299] We hypothesize that immunogenic epitopes of SARS-CoV-2 are best defined empirically by directly analyzing peptides eluted from MHC and then validating immunogenicity by determining whether such peptides can elicit T cells that recognize SARS-CoV-2 antigen-expressing targets. Mass spectrometry (MS) is an ideal analytical approach to accurately identify naturally expressed antigenic epitopes, allowing researchers to address the complexities associated with differential expression and processing of antigenic proteins by infected cells. Based on immunoaffinity capture of MHC-antigen peptide complexes from cells engineered to express SARS-CoV2 genes, this approach allowed for direct profiling and identification of the SARS-CoV2 immune peptidome. By eliciting T cell responses against these candidate epitopes, we confirmed the empirical recognition of SARS-CoV2+ cells and the endogenous presentation of these peptides.

[0300] In this study, we identified and validated five class I-restricted SARS-CoV2 epitopes expressed by structural (MGP) and nonstructural (NSP13) genes that are presented by class I alleles (HLA-A*0101, A*0201, A*0301, HLA-A*2402) prevalent among >75% of the general population. Using recombinant vectors encoding these alleles, we engineered expression of highly conserved regions of the SARS-CoV2 membrane glycoprotein (MGP) and nonstructural protein-13 (NSP13) genes, harvested MHC, eluted peptides, and applied data-dependent analysis liquid chromatography tandem mass spectrometry (DDA MS / MS) to obtain >12,000 spectra, which were then deconvoluted and filtered for a small number of candidate peptide epitopes. The immunogenicity of the five peptides was verified based on their ability to induce peptide-specific T cells capable of recognizing and killing target cells expressing SARS-CoV2, and in one example, redirecting the specificity of peripheral blood lymphocytes bearing engineered TCRs to SARS-CoV2 MGP.

[0301] The importance of eliciting meaningful antiviral T cell responses is well documented; SARS- and MERS-responsive T cells were found to have a protective role (Poland et al., 2020). The emergence of SARS-CoV-2-specific T cell responses was recently shown to be associated with sustained viral clearance, highlighting the importance of vaccine development to promote cell-mediated immunity against SARS-CoV-2 (Gallais et al., 2021; Long et al., 2020; Sekine et al., 2020).

[0302] Recently, the emergence of mutant escape variants of SARS-CoV2 has led to global concerns about the possible compromise of viral protection following immunization with current vaccines that primarily induce serological responses (Abdool Karim and de Oliveira, 2021; Agerer et al., 2021; Darby and Hiscox, 2021; Kuzmina et al., 2021; Plante et al., 2021). By targeting a non-surface, non-structural protein, in this case nsp13 encoding the viral helicase, escape variants are unlikely to arise; indeed, none of the known variants carry mutations among the epitope sequence...

Claims

1. a T cell receptor (TCR)-a polypeptide and a TCR-b polypeptide, Optionally, the TCR-b polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3, and the TCR-a polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3; An engineered TCR, The TCR-a polypeptide is (i) a CDR3 having an amino acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:7; or (ii) a CDR3 having an amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:14; Including, And the TCR-b polypeptide is CDR3 having an amino acid sequence of SEQ ID NO:21 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:21 Including, Engineered TCR.

2. (a) CDR1 of the TCR-a polypeptide, (i) having an amino acid sequence of SEQ ID NO:5 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:5; or (ii) having an amino acid sequence of SEQ ID NO:12 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:12; and / or the CDR1 of the TCR-b polypeptide is having an amino acid sequence of SEQ ID NO:19 or having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:19; and / or (b) CDR2 of the TCR-a polypeptide (i) having an amino acid sequence of SEQ ID NO:6 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:6; or (ii) having an amino acid sequence of SEQ ID NO:13 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:13; and / or the CDR2 of the TCR-b polypeptide is having an amino acid sequence of SEQ ID NO:20 or having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:20; and / or (c) CDR1, CDR2, and CDR3 of the TCR-a polypeptide (i) the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively; or (ii) the amino acid sequences of SEQ ID NOs: 12, 13, and 14, respectively; and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs: 19, 20, and 21, respectively; and / or (d) a TCR-a polypeptide, (i) the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:3; or (ii) the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:10 and the TCR-b polypeptide comprises the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:

17. The TCR of claim 1.

3. a T cell receptor (TCR)-a polypeptide and a TCR-b polypeptide, Optionally, the TCR-b polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3, and the TCR-a polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3; An engineered TCR, the TCR-a polypeptide comprises a CDR3 having an amino acid sequence of SEQ ID NO:92 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:92, and the TCR-b polypeptide comprises a CDR3 having an amino acid sequence of SEQ ID NO:99 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:99; Engineered TCR.

4. (a) CDR1 of the TCR-a polypeptide, having an amino acid sequence of SEQ ID NO:90 or having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:90; and / or CDR1 of the TCR-b polypeptide is having an amino acid sequence of SEQ ID NO:97 or having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:97; and / or (b) CDR2 of the TCR-a polypeptide having an amino acid sequence of SEQ ID NO:91 or having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:91; and / or CDR2 of the TCR-b polypeptide having an amino acid sequence of SEQ ID NO:98 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:98; and / or (c) the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs:90, 91, and 92, respectively, and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs:97, 98, and 99, respectively; and / or (d) the TCR-a polypeptide comprises an amino acid sequence of SEQ ID NO:88 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:88, and the TCR-b polypeptide comprises an amino acid sequence of SEQ ID NO:95 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:95; The TCR of claim 3.

5. a T cell receptor (TCR)-a polypeptide and a TCR-b polypeptide, Optionally, the TCR-b polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3, and the TCR-a polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3; An engineered TCR, An engineered TCR, wherein the TCR-a polypeptide comprises a CDR3 having an amino acid sequence of SEQ ID NO:106 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:106, and the TCR-b polypeptide comprises a CDR3 having an amino acid sequence of SEQ ID NO:113 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

113.

6. (a) CDR1 of the TCR-a polypeptide, having an amino acid sequence of SEQ ID NO:104 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:104; and / or CDR1 of the TCR-b polypeptide is having an amino acid sequence of SEQ ID NO:111 or having an amino acid sequence having at least 80% sequence identity to SEQ ID NO:111; and / or (b) CDR2 of the TCR-a polypeptide having an amino acid sequence of SEQ ID NO:105 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:105; and / or CDR2 of the TCR-b polypeptide having an amino acid sequence of SEQ ID NO: 112 or having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 112; and / or (c) the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs: 104, 105, and 106, respectively, and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs: 111, 112, and 113, respectively; and / or (d) the TCR-a polypeptide comprises an amino acid sequence of SEQ ID NO:102, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:102, and the TCR-b polypeptide comprises an amino acid sequence of SEQ ID NO:109, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:109; The TCR of claim 5.

7. a T cell receptor (TCR)-a polypeptide and a TCR-b polypeptide, Optionally, the TCR-b polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3, and the TCR-a polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3; An engineered TCR, An engineered TCR, wherein the TCR-a polypeptide comprises a CDR3 having an amino acid sequence of SEQ ID NO:120 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:120, and the TCR-b polypeptide comprises a CDR3 having an amino acid sequence of SEQ ID NO:127 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

127.

8. (a) CDR1 of the TCR-a polypeptide, having the amino acid sequence of SEQ ID NO:118 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:118; and / or CDR1 of the TCR-b polypeptide is having an amino acid sequence of SEQ ID NO:125 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:125; and / or (b) CDR2 of the TCR-a polypeptide having the amino acid sequence of SEQ ID NO:119 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:119; and / or CDR2 of the TCR-b polypeptide having the amino acid sequence of SEQ ID NO:126 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:126; and / or (c) the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs: 118, 119, and 120, respectively, and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs: 125, 126, and 127, respectively; and / or (d) the TCR-a polypeptide comprises an amino acid sequence of SEQ ID NO:116, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:116, and the TCR-b polypeptide comprises an amino acid sequence of SEQ ID NO:123, or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:123; The TCR of claim 7.

9. a T cell receptor (TCR)-a polypeptide and a TCR-b polypeptide, Optionally, the TCR-b polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3, and the TCR-a polypeptide comprises a variable region comprising CDR1, CDR2, and CDR3; An engineered TCR, An engineered TCR, wherein the TCR-a polypeptide comprises a CDR3 having an amino acid sequence of SEQ ID NO:134 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:134, and the TCR-b polypeptide comprises a CDR3 having an amino acid sequence of SEQ ID NO:141 or an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

141.

10. (a) CDR1 of the TCR-a polypeptide, having the amino acid sequence of SEQ ID NO:132 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:132; and / or CDR1 of the TCR-b polypeptide is having an amino acid sequence of SEQ ID NO:139 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:139; and / or (b) CDR2 of the TCR-a polypeptide having an amino acid sequence of SEQ ID NO:133 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:133; and / or CDR2 of the TCR-b polypeptide having an amino acid sequence of SEQ ID NO:140 or having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:140; and / or (c) the CDR1, CDR2, and CDR3 of the TCR-a polypeptide comprise the amino acid sequences of SEQ ID NOs: 132, 133, and 134, respectively, and the CDR1, CDR3, and CDR3 of the TCR-b polypeptide comprise the amino acid sequences of SEQ ID NOs: 139, 140, and 141, respectively; and / or (d) the TCR-a polypeptide comprises an amino acid sequence of SEQ ID NO:130 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:130, and the TCR-b polypeptide comprises an amino acid sequence of SEQ ID NO:137 or an amino acid sequence having at least 70% sequence identity to SEQ ID NO:137; The TCR of claim 9.

11. (a) contains modifications or is chimeric; and / or (b) the TCR-b polypeptide and the TCR-a polypeptide are operably linked; A TCR according to any one of claims 1 to 10.

12. The TCR of claim 11(b), wherein the TCR-b polypeptide and the TCR-a polypeptide are functionally linked by a peptide bond.

13. (a) is a single chain TCR, or (b) the TCR-b polypeptide and the TCR-a polypeptide are on the same polypeptide, and the TCR-b is amino-proximal to the TCR-a; or (c) the TCR-b polypeptide and the TCR-a polypeptide are on the same polypeptide, and the TCR-a is amino-proximal to the TCR-b; Optionally, a linker between the TCR-a polypeptide and the TCR-b polypeptide, optionally wherein the linker comprises glycine and serine residues; The TCR of claim 12.

14. A peptide comprising or consisting of one of SEQ ID NOs:22-38, or comprising or consisting of a peptide comprising at least 60% sequence identity to one of SEQ ID NOs:22-38.

15. (a) SEQ ID NO:22, or a peptide containing at least 60% sequence identity to SEQ ID NO:22 (b) one of SEQ ID NOs:23-25, or a peptide comprising at least 60% sequence identity to one of SEQ ID NOs:23-25; (c) one of SEQ ID NOs: 26-33, or a peptide comprising at least 60% sequence identity to one of SEQ ID NOs: 26-33. (d) SEQ ID NO:34, or a peptide containing at least 60% sequence identity to SEQ ID NO:

34. (e) SEQ ID NO:35, or a peptide containing at least 60% sequence identity to SEQ ID NO:

35. (f) SEQ ID NO:36, or a peptide containing at least 60% sequence identity to SEQ ID NO:

36. (g) SEQ ID NO:37, or a peptide containing at least 60% sequence identity to SEQ ID NO:37; or (h) SEQ ID NO:38, or a peptide containing at least 60% sequence identity to SEQ ID NO:

38.

15. The peptide of claim 14, comprising or consisting of:

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