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WO2025179050A2 - Compositions de vaccin et méthodes d'utilisation - Google Patents

Compositions de vaccin et méthodes d'utilisation

Info

Publication number
WO2025179050A2
WO2025179050A2 PCT/US2025/016645 US2025016645W WO2025179050A2 WO 2025179050 A2 WO2025179050 A2 WO 2025179050A2 US 2025016645 W US2025016645 W US 2025016645W WO 2025179050 A2 WO2025179050 A2 WO 2025179050A2
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
seq
prame
nucleic acid
recombinant nucleic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/016645
Other languages
English (en)
Other versions
WO2025179050A3 (fr
Inventor
Kendra FOLEY
Vikram JUNEJA
Christina M. ARIETA
Shirisha MEDA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biontech US Inc
Original Assignee
Biontech US Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biontech US Inc filed Critical Biontech US Inc
Publication of WO2025179050A2 publication Critical patent/WO2025179050A2/fr
Publication of WO2025179050A3 publication Critical patent/WO2025179050A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001189—PRAME
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K40/427—PRAME
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/876—Skin, melanoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • a method of treating a subject with a disease or condition comprising administering to the subject a therapy comprising (i) a multiepitopic polypeptide, (ii) a recombinant nucleic acid encoding the multiepitopic polypeptide, or (iii) a cell comprising the multiepitopic polypeptide or the recombinant nucleic acid encoding the multiepitopic polypeptide, wherein the multiepitopic polypeptide does not comprise a full- length PRAME polypeptide and comprises a first PRAME amino acid sequence comprising a first PRAME epitope sequence and a second PRAME amino acid sequence comprising a second PRAME epitope sequence, wherein the first PRAME epitope sequence and
  • the method comprises administering to the subject a T-cell receptor (TCR) or a recombinant nucleic acid encoding the TCR.
  • TCR T-cell receptor
  • the first PRAME epitope sequence and the second PRAME epitope sequence is separated by a linker.
  • WSGR Docket No.: 50401-787.601 the first PRAME amino acid sequence is the first epitope sequence and/or the second PRAME amino acid sequence is the second epitope sequence.
  • the first PRAME amino acid sequence consists of the first epitope sequence, and/or the second PRAME amino acid sequence consists of the second epitope sequence.
  • the first PRAME epitope sequence and/or the second PRAME epitope sequence consists of from 7 to 12 consecutive amino acids from the full-length PRAME polypeptide. [0010] In some embodiments, the first PRAME epitope sequence and/or the second PRAME epitope sequence consists of from 13 to 25 consecutive amino acids from the full-length PRAME polypeptide. [0011] In some embodiments, the first PRAME amino acid sequence and the second PRAME amino acid sequence is separated by a linker.
  • the first PRAME amino acid sequence comprises the first PRAME epitope sequence and one or more residues flanking the N-terminus or the C- terminus of the first PRAME epitope sequence from the full-length PRAME polypeptide.
  • the second PRAME amino acid sequence comprises the second PRAME epitope sequence and one or more residues flanking the N-terminus or the C- terminus of the second PRAME epitope sequence from the full-length PRAME polypeptide.
  • the multiepitopic polypeptide does not comprise more than 12 or more consecutive amino acids from the full-length PRAME polypeptide, or the multiepitopic polypeptide does not comprise more than 25 or more consecutive amino acids from the full-length PRAME polypeptide.
  • the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the first or the second PRAME epitope sequence, and (ii) a human MHC encoded by an HLA allele.
  • Also provided herein is a method of treating a subject with a disease or condition comprising administering to the subject (a) a PRAME polypeptide, (b) a recombinant nucleic acid encoding the PRAME polypeptide, or (c) a cell comprising the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide, wherein the PRAME polypeptide comprises a PRAME epitope sequence, wherein the subject has been previously administered a T-cell receptor (TCR) or a recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC WSGR Docket No.: 50401-787.601 complex comprising (i) the PRAME epitope sequence, and (ii) a human MHC encoded by an HLA allele.
  • TCR T-cell receptor
  • Also provided herein is a method of treating a subject with a disease or condition comprising administering to the subject a T-cell receptor (TCR) or a recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) a PRAME epitope sequence, and (ii) a human MHC encoded by an HLA allele, wherein the subject has been previously administered (a) a PRAME polypeptide, (b) a recombinant nucleic acid encoding the PRAME polypeptide, or (c) a cell comprising the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide, wherein the PRAME polypeptide comprises the PRAME epitope sequence.
  • TCR T-cell receptor
  • a recombinant nucleic acid encoding the TCR wherein the TCR recognizes and binds to
  • Also provided herein is a method of treating a subject with a disease or condition comprising, (a) administering to the subject (i) a PRAME polypeptide, (ii) a recombinant nucleic acid encoding the PRAME polypeptide, or (iii) a cell comprising the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide, wherein the PRAME polypeptide comprises a PRAME epitope sequence; and (b) administering to the subject a TCR or a recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the PRAME peptide sequence, and (ii) a human MHC encoded by an HLA allele.
  • administering in (a) is performed concurrently with administering in (b). [0020] In some embodiments, administering in (a) is prior to administering in (b). [0021] In some embodiments, administering in (a) is subsequent to administering in (b). [0022] In some embodiments, the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 1 day, 2 days, 5 days, 10 days, 20 days, 30 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years or more after the subject has been administered the TCR or the recombinant nucleic acid encoding the TCR.
  • the TCR or the recombinant nucleic acid encoding the TCR is administered at least 1 day, 2 days, 5 days, 10 days, 20 days, 30 days, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years or more after the subject has been administered the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide.
  • the T-cell receptor (TCR) or the recombinant nucleic acid encoding the TCR is expressed by an immune cell.
  • the TCR is a soluble TCR.
  • the method further comprises administering two or more different TCRs or recombinant nucleic acids encoding the two or more different TCRs, and wherein the two or more different TCRs comprise a first TCR and a second TCR.
  • the two or more different TCRs are expressed on surface of two different immune cells.
  • the first TCR and the second TCR bind to different peptide:MHC complexes, each peptide:MHC complex comprising (i) an epitope sequence and (ii) a human MHC encoded by an HLA allele.
  • the two or more different TCRs or recombinant nucleic acids encoding the two or more different TCRs are administered separately or co-administered in a same mixture.
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 78 and an MHC encoded by an HLA-A*02:01 allele and the second TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 79 and an MHC encoded by an HLA-B*07:02 allele;
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 78 and an MHC encoded by an HLA-A*02:01 allele and the second TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 80 or
  • the PRAME polypeptide does not comprise a full-length PRAME protein sequence.
  • the PRAME epitope sequence comprises a sequence of SEQ ID NO: 78.
  • the HLA allele is an HLA-A*02:01 allele.
  • the PRAME epitope sequence comprises a sequence of SEQ ID NO: 79.
  • the HLA allele is an HLA-B*07:02 allele.
  • the PRAME epitope sequence comprises a sequence of SEQ ID NO: 80 or 108.
  • the HLA allele is an HLA-A*24:02 allele.
  • WSGR Docket No.: 50401-787.601 the PRAME polypeptide comprises a multiepitopic polypeptide, and wherein the multiepitopic polypeptide does not comprise a full-length PRAME polypeptide and comprises two or more different PRAME epitope sequences.
  • the multiepitopic polypeptide comprises at least 3, 4, 5, or more different PRAME epitope sequences.
  • the two or more different PRAME epitope sequences are separated by linker sequences.
  • antigen presenting cells of the subject administered the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide comprising the multiepitopic polypeptide present more of a PRAME epitope sequence as a peptide:MHC complex compared to the APCs of a subject administered a full-length PRAME polypeptide or a recombinant nucleic acid encoding the full-length PRAME polypeptide.
  • T cells of the subject administered the multiepitopic polypeptide or a recombinant nucleic acid encoding the multiepitopic polypeptide exhibit increased expansion compared to T cells of a subject administered a full-length PRAME polypeptide or a recombinant nucleic acid encoding the full-length PRAME polypeptide.
  • the multiepitopic polypeptide comprises a first PRAME amino acid sequence comprising a first PRAME epitope sequence and a second PRAME amino acid sequence comprising a second PRAME epitope sequence, and wherein the first PRAME epitope sequence and the second PRAME epitope sequence are different.
  • the first PRAME epitope sequence and the second PRAME epitope sequence is separated by a linker.
  • the first amino acid sequence is the first epitope sequence and/or the second amino acid sequence is the second epitope sequence.
  • the first amino acid sequence consists of the first epitope sequence, and/or the second amino acid sequence consists of the second epitope sequence.
  • the first PRAME epitope sequence and/or the second PRAME epitope sequence consists of from 7 to 12 consecutive amino acids from the full-length PRAME polypeptide.
  • the first PRAME epitope sequence and/or the second PRAME epitope sequence consists of from 13 to 25 consecutive amino acids from the full-length PRAME polypeptide.
  • the first amino acid sequence and the second amino acid sequence is separated by a linker.
  • the first amino acid sequence comprises the first PRAME epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the first PRAME epitope sequence from the full-length PRAME polypeptide.
  • the second amino acid sequence comprises the second PRAME epitope sequence and one or more residues flanking the N-terminus or the C- terminus of the second PRAME epitope sequence from the full-length PRAME polypeptide.
  • the multiepitopic polypeptide does not comprise more than 12 or more consecutive amino acids from the full-length PRAME polypeptide, or the multiepitopic polypeptide does not comprise more than 25 or more consecutive amino acids from the full-length PRAME polypeptide.
  • the first PRAME epitope sequence and the second PRAME epitope sequence are presentable by different HLA alleles, are presented by different HLA alleles, bind to different HLA alleles, are predicted to bind to different HLA alleles, or are predicted to be presented by different HLA alleles.
  • a first PRAME epitope sequence (i) binds to or is predicted to bind to a first HLA allele with a KD of less than 100 nM and (ii) to binds to or is predicted to bind to a second HLA allele with a KD of more than 500 nM.
  • the first PRAME epitope sequence is operably linked to the second PRAME epitope sequence via a linker.
  • the linker comprises a sequence selected from the group consisting of SEQ ID NOs: 86-96, and 97.
  • the multiepitopic polypeptide comprises a first PRAME epitope sequence, operably linked to a second PRAME epitope sequence, operably linked to a third PRAME epitope sequence.
  • the first PRAME epitope sequence is presentable by HLA- A*02:01 allele, is presented by HLA-A*02:01 allele, binds to HLA-A*02:01 allele, is predicted to bind to HLA-A*02:01 allele, or is predicted to be presented by HLA-A*02:01 allele
  • the second PRAME epitope sequence is presentable by HLA-A*24:02 allele, is presented by HLA-A*24:02 allele, binds to HLA-A*24:02 allele, is predicted to bind to HLA-A*24:02 allele, or is predicted to be presented by HLA-A*24:02 allele
  • the third PRAME epitope sequence is presentable by HLA-B*07:02 allele, is presented by HLA- B*07:02, binds to HLA-B*07:02, is predicted to bind to HLA-B*
  • the linker sequence is a cleavable linker.
  • the linker sequence comprises a sequence of selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 18, 19, 20, 21, 29, 30, 31, 32, 43, 47, 49, 54, WSGR Docket No.: 50401-787.601 56, 58, 45, 68, 69, 70, 114, 116, 118, 120, 122, 124, 126, 130, 132, 134, 136, 138, 146, 147, 148, 149, 150, 151, 152, and 153.
  • the peptide:MHC complex comprises the PRAME epitope sequence having at least 7 consecutive amino acids of a sequence according to SEQ ID NO: 79, and (ii) a human MHC encoded by the HLA-B*07:02 allele.
  • the peptide:MHC complex comprises the PRAME epitope sequence having a sequence according to SEQ ID NO: 79.
  • the peptide:MHC complex comprises the PRAME epitope sequence having at least 7 consecutive amino acids of a sequence according to SEQ ID NOs: 80 or 108, and (ii) a human MHC encoded by the HLA-A*24:02 allele.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140.
  • the linker sequence encodes a cleavable linker.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 18, 19, 20, 21, 29, 30, 31, 32, 43, 47, 49, 54, 56, 58, 45, 68, 69, 70, 114, 116, 118, 120, 122, 124, 126, 130, 132, 134, 136, 138, 146, 147, 148, 149, 150, 151, 152, and 153.
  • the sequence selected from the group consisting of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140 via a linker sequence
  • the sequence selected from the group consisting of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140 is operably linked to a sequence selected from the group consisting of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137 via a linker sequence.
  • the linker sequence is a cleavable linker.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 18, 19, 20, 21, 29, 30, 31, 32, 43, 47, 49, 54, 56, 58, 45, 68, 69, 70, 114, 116, 118, 120, 122, 124, 126, 130, 132, 134, 136, 138, 146, 147, 148, 149, 150, 151, 152, and 153.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises at least two copies of a string of sequences, and wherein each string of sequences comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of the string of sequences. [0229] In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises five copies of the string of sequences. [0230] In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide further comprises a sequence encoding a Sec sequence at the 5’ end of the string of sequences. [0231] In some embodiments, the sequence encoding the Sec sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 16, 27, 36, and 144.
  • the sequence encoding the Sec sequence is operably linked to the string of sequences via a linker sequence.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 17, 22, 28, 33, 37, 39, 42, 50, 53, 61, 66, 71, 141, 145, and 154.
  • the recombinant nucleic acid encoding the PRAME polypeptide further comprises a sequence encoding an MITD sequence at the 3’ end of the string of sequences.
  • a recombinant nucleic acid having at least 60% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 7, 15, 24, 26, 35, 41, 52, 63, 64, and 65.
  • a recombinant polypeptide having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 7, 15, 24, 26, 35, 41, 52, 63, 64, and 65.
  • a pharmaceutical composition comprising the recombinant nucleic acid of any one of the preceding embodiments or the polypeptide of any one of the preceding embodiments, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier comprising the recombinant nucleic acid of any one of the preceding embodiments, the polypeptide of any one of the preceding embodiments, or the pharmaceutical composition of any of the preceding embodiments for use in therapy.
  • FIG.1A is an exemplary mRNA construct encoding full length PRAME with linker sequences, flanked by a secretory (Sec) and MHC class I Trafficking Domain (MITD).
  • FIG.1A is an exemplary mRNA construct encoding full length PRAME with linker sequences, flanked by a secretory (Sec) and MHC class I Trafficking Domain (MITD).
  • 1B depicts exemplary mRNA constructs encoding minimal PRAME epitopes presented by HLA-A02:01 (A02), HLA-B07:02 (B07), and HLA-A24:02 (A24) in different orientations (referred to as 1xV1, 1xV2, or 1xV3).
  • Constructs 3xV2 and 5xV2 encode three- and 5-times repeats of the minimal epitopes in the V2 orientation, respectively.
  • Epitope sequences are each flanked by a cleavage linker.
  • Constructs also include GSS linker sequences and a Sec and MITD domain.
  • FIG.2 shows results of MITD protein expression, measured by mass spectrometry, of mRNA constructs expressed in A375 tumor cells. Wild type (WT) and codon optimized (Opt) constructs were compared. The solid line depicts the number of bases in the mRNA construct.
  • FIGs.3A-3C depict results of epitope protein expression, measured by targeted mass spectrometry, by indicated mRNA constructs expressed in A375 tumor cells engineered to express the HLA allele that presents the epitope as indicated.
  • FIG.3A depicts expression of epitope SLLQHLIGL (SEQ ID NO: 78) presented by HLA-A02:01.
  • FIG.3B depicts expression of epitope LYVDSLFFL (SEQ ID NO: 80) presented by HLA-A24:02.
  • FIG.3C depicts expression of epitope SPSVSQLSVL (SEQ ID NO: 79) presented by HLA-B07:02. Normalized abundance is given, normalized to the most abundant mRNA construct. [0250] FIGs.
  • FIGs. 5A-5C depict flow cytometry data verifying the generation of murine tumor cell lines engineered to express PRAME.
  • FIG.5A depicts representative flow cytometry data for LLC1 lung carcinoma cells expressing PRAME and HLA-A02:01.
  • FIG.5B depicts representative flow cytometry data for 4T1 breast cancer cells expressing PRAME and HLA- A02:01.
  • FIG.5C depicts representative flow cytometry measuring transduction efficiency of murine T cells to express the PRAME TCR.
  • FIGs.5D-5E depict results quantifying IFN- coculture of murine tumor cell lines engineered to express human PRAME and chimeric HLA-A02:01/H2-kb molecule with murine T cells from an HLA-A02:01/H2-kb transgenic mouse engineered to express the PRAME-specific TCR.
  • Various effector to target ratios were used.
  • the left bar graph illustrates results obtained by incubating target cells with the PRAME-TCR T cells and the right bar graph illustrates results obtained by incubating target cells with control non-specific splenocytes.
  • FIG.5D depicts results obtained using LLC1 lung carcinoma cells.
  • FIG.5E depicts results obtained using 4T1 breast cancer cells.
  • An antigen is a foreign substance to the body that induces an immune response.
  • a “neoantigen” refers to a class of tumor antigens which arise from tumor-specific changes in proteins.
  • Neoantigens encompass, but are not limited to, tumor antigens which arise from, for example, a substitution in a protein sequence, a frame shift mutation, a fusion polypeptide, an in-frame deletion, an insertion, and expression of an endogenous retroviral polypeptide.
  • a “neoepitope” refers to an epitope that is not present in a reference, such as a non- diseased cell, e.g., a non-cancerous cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell.
  • a “mutation” refers to a change of or a difference in a nucleic acid sequence (e.g., a nucleotide substitution, addition or deletion) compared to a reference nucleic acid.
  • a “somatic mutation” can occur in any of the cells of the body except the germ cells (sperm and egg) and are not passed on to children.
  • a mutation is a non-synonymous mutation.
  • a “non- synonymous mutation” refers to a mutation, for (e.g., a nucleotide substitution), which does result in an amino acid change such as an amino acid substitution in the translation product.
  • a “frameshift” occurs when a mutation disrupts the normal phase of a gene’s codon periodicity (also known as “reading frame”), resulting in translation of a non-native protein sequence. It is possible for different mutations in a gene to achieve the same altered reading frame.
  • Antigen processing refers to the degradation of a polypeptide or antigen into procession products, which are fragments of said polypeptide or antigen (e.g., the degradation of a polypeptide into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, for example, antigen presenting cells, to specific T cells.
  • An “antigen presenting cell” refers to a cell which presents peptide fragments of protein antigens in association with MHC molecules on its cell surface.
  • the term includes professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).
  • the APC can be a cancer cell.
  • affinity refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or II HLA, or a peptide-HLA complex and a T cell receptor (TCR)).
  • HLA human leukocyte antigen
  • TCR T cell receptor
  • K D refers to the dissociation constant between two members of a binding pair and has units of molarity.
  • K A refers to the affinity constant between two members of a binding pair is the inverse of the dissociation constant. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units.
  • K off refers to the off-rate constant of two members of a binding pair, (e.g., the off-rate constant of an HLA-binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR).
  • binding data results may be expressed in terms of an “IC 50 .” Affinity may also be expressed as the inhibitory concentration 50 (IC 50 ), or the concentration at which 50% of a first member of a binding pair (e.g., a peptide) is displaced. Likewise, ln(IC 50 ) refers to the natural log of the IC 50 .
  • an IC 50 may be the concentration of a tested peptide in a binding assay at which 50% inhibition of binding of a labeled reference peptide is observed.
  • these WSGR Docket No.: 50401-787.601 values can approximate K D values.
  • Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94/20127 and WO 94/03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J.
  • binding can be expressed relative to binding by a reference standard peptide. Binding can also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol.2:443 (1990); Hill et al., J. Immunol.147:189 (1991); del Guercio et al., J.
  • Synthetic epitopes can comprise artificial amino acid residues “amino acid mimetics,” such as D isomers of natural occurring L amino acid residues or non- natural amino acid residues such as cyclohexylalanine.
  • a derived or prepared epitope can be an analog of a native epitope.
  • the term “derived from” refers to the origin or source, and may include naturally occurring, recombinant, unpurified, purified or differentiated molecules or cells.
  • an expanded or induced antigen specific T cell may be derived from a T cell.
  • an expanded or induced antigen specific T cell may be derived from an antigen specific T cell in a biological sample.
  • a matured APC may be derived from a non-matured APC (e.g., an immature APC).
  • an APC may be derived from a monocyte (e.g., a CD14 + monocyte).
  • a dendritic cell may be derived from a monocyte (e.g., a CD14 + monocyte).
  • an APC may be derived from a bone marrow cell.
  • an “epitope” is the collective features of a molecule (e.g., a peptide’s charge and primary, secondary and tertiary structure) that together form a site recognized by another molecule (e.g., an immunoglobulin, T cell receptor, HLA molecule, or chimeric antigen receptor).
  • an epitope can be a set of amino acid residues involved in recognition WSGR Docket No.: 50401-787.601 by a particular immunoglobulin; a Major Histocompatibility Complex (MHC) receptor; or in the context of T cells, those residues recognized by a T cell receptor protein and/or a chimeric antigen receptor.
  • MHC Major Histocompatibility Complex
  • Epitopes can be prepared by isolation from a natural source, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues, amino acid mimetics, (such as D isomers of naturally-occurring L amino acid residues or non-naturally-occurring amino acid residues). Throughout this disclosure, epitopes may be referred to in some cases as peptides or peptide epitopes. In certain embodiments, there is a limitation on the length of a peptide of the present disclosure.
  • the embodiment that is length-limited occurs when the protein or peptide comprising an epitope described herein comprises a region (i.e., a contiguous series of amino acid residues) having 100% sequence identity with a native sequence.
  • a region i.e., a contiguous series of amino acid residues
  • the region with 100% sequence identity to a native sequence generally has a length of: less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues.
  • an “epitope” described herein is comprised by a peptide having a region with less than 51 amino acid residues that has 100% sequence identity to a native peptide sequence, in any increment down to 5 amino acid residues; for example 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.
  • a “T cell epitope” refers to a peptide sequence bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex.
  • a peptide-MHC complex can be recognized and bound by a TCR of a T cell (e.g., a cytotoxic T-lymphocyte or a T-helper cell).
  • a “T cell” includes CD4+ T cells and CD8+ T cells.
  • the term T cell also includes both T helper 1 type T cells and T helper 2 type T cells.
  • T cells may be generated by the method described in the application, for a clinical application.
  • T cells or adoptive T cells referred to here, such as for a clinical application are cells isolated from a biological source, manipulated and cultured ex vivo and prepared into a drug candidate for a specific therapy such as a cancer, e.g., melanoma.
  • a drug product is selected from a number of drug candidates.
  • a drug product is a T cell, more specifically, a population of T cells, or more specifically a population of T cells with heterogeneous characteristics and subtypes.
  • a drug product, as disclosed herein may have a population of T cells comprising CD8+ T cells, CD4+ T cells, with cells at least above a certain exhibiting antigen specificity, a certain percentage of each exhibiting a memory phenotype, among others.
  • Immune cells refers to a cell that plays a role in the immune response.
  • Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • an “immunogenic” peptide or an “immunogenic” epitope or an “immunogenic” peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated or humoral response, for example, a cytotoxic T lymphocyte (CTL) response, a helper T lymphocyte (HTL) response and/or a B lymphocyte response.
  • CTL cytotoxic T lymphocyte
  • HTL helper T lymphocyte
  • B lymphocyte response e.g., a B lymphocyte response.
  • a “protective immune response” or “therapeutic immune response” refers to a CTL and/or an HTL response to an antigen derived from a pathogenic antigen (e.g., a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects or progression.
  • the immune response can also include an antibody response which has been facilitated by the stimulation of helper T cells.
  • a “T cell receptor” (“TCR”) refers to a molecule, whether natural or partly or wholly synthetically produced, found on the surface of T lymphocytes (T cells) that recognizes an antigen bound to a major histocompatibility complex (MHC) molecule.
  • TCR antigen-presenting cells
  • a “chimeric antigen receptor” or “CAR” refers to an antigen binding protein in that includes an immunoglobulin antigen binding domain (e.g., an immunoglobulin WSGR Docket No.: 50401-787.601 variable domain) and a T cell receptor (TCR) constant domain.
  • an immunoglobulin antigen binding domain e.g., an immunoglobulin WSGR Docket No.: 50401-787.601 variable domain
  • TCR T cell receptor
  • a “constant domain” of a TCR polypeptide includes a membrane-proximal TCR constant domain, a TCR transmembrane domain and/or a TCR cytoplasmic domain, or fragments thereof.
  • a CAR is a monomer that includes a polypeptide comprising an embodiments, the CAR is a dimer that includes a first polypeptide comprising an domain and a second polypeptide comprising an immunoglobulin heavy or light chain variable
  • MHC Major Histocompatibility Complex
  • MHC can include any class of MHC molecule, such as MHC class I and MHC class II molecules, and relate to a complex of genes which occurs in all vertebrates.
  • HLA human leukocyte antigen
  • HLA human Leukocyte antigen
  • MHC Major Histocompatibility Complex
  • the major histocompatibility complex in the genome comprises the genetic region whose gene products expressed on the cell surface are important for binding and presenting endogenous and/or foreign antigens and thus for regulating immunological processes.
  • MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions. MHC proteins or molecules bind peptides and present them for recognition by T-cell receptors.
  • the proteins encoded by the MHC can be expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T-cell.
  • MHC binding peptides can result from the proteolytic cleavage of protein antigens and represent potential lymphocyte epitopes. (e.g., T cell epitope and B cell epitope). MHCs can transport the peptides to the cell surface and present them there to specific cells, such as cytotoxic T-lymphocytes, T-helper cells, or B cells.
  • the MHC region can be divided into three - - microglobulin (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T-cells.
  • MHC class II proteins c - -chains and they can WSGR Docket No.: 50401-787.601 present antigen fragments to T-helper cells.
  • a “receptor” refers to a biological molecule or a molecule grouping capable of binding a ligand.
  • a receptor may serve, to transmit information in a cell, a cell formation or an organism.
  • a receptor comprises at least one receptor unit, for example, where each receptor unit may consist of a protein molecule.
  • a receptor has a structure which complements that of a ligand and may complex the ligand as a binding partner.
  • a receptor is to be understood as meaning in particular proteins of MHC classes I and II capable of forming a receptor/ligand complex with a ligand, in particular a peptide or peptide fragment of suitable length.
  • a “ligand” refers to a molecule which has a structure complementary to that of a receptor and is capable of forming a complex with this receptor.
  • Naturally occurring refers to the fact that an object can be found in nature.
  • a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • the terms “peptide” and “peptide epitope” are used interchangeably with “oligopeptide” in the present specification to designate a series of residues connected one to -amino and carboxyl groups of adjacent amino acid residues.
  • a “synthetic peptide” refers to a peptide that is obtained from a non- natural source, e.g., is man-made.
  • Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in their pattern of the primary and secondary anchor residues.
  • an MHC class I motif identifies a peptide of 7, 89, 10, 11, 12 or 13 amino acid residues in length.
  • an MHC class II motif identifies a peptide of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 amino acid residues in length.
  • a “cross-reactive binding” peptide refers to a peptide that binds to more than one member of a class of a binding pair members (e.g., a peptide bound by both a class I HLA molecule and a class II HLA molecule).
  • the L- form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol
  • the D-form for those amino acid residues having D-forms is represented by a lower case single letter or a lower case three letter symbol.
  • Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or “G”.
  • the amino acid sequences of peptides set forth herein are generally designated using the standard single letter symbol.
  • a “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • “Pharmaceutically acceptable” refers to a generally non-toxic, inert, and/or physiologically compatible composition or component of a composition.
  • a “pharmaceutical excipient” or “excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like.
  • a “pharmaceutical excipient” is an excipient which is pharmaceutically acceptable.
  • the term “vaccine” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, for example, a cellular or humoral immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell.
  • a vaccine may be used for the prevention or treatment of a disease.
  • individualized cancer vaccine or “personalized cancer vaccine” “personal cancer vaccine” concerns a particular cancer patient and means that a cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.
  • polynucleotide and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA, for example, mRNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • the polynucleotide and nucleic acid can be in vitro transcribed mRNA.
  • the polynucleotide that is administered using the methods of the present disclosure is mRNA.
  • isolated or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides described herein do not contain some or all of the materials normally associated with the peptides in their in situ environment.
  • an “isolated” epitope can be an epitope that does not include the whole sequence of the protein from which the epitope was derived.
  • a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • substantially pure refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof.
  • the terms “effective amount” or “therapeutically effective amount” or “therapeutic effect” refer to an amount of a therapeutic effective to “treat” a disease or disorder in a subject or mammal.
  • the therapeutically effective amount of a drug has a therapeutic effect and as such can prevent the development of a disease or disorder; slow down the development of a disease or disorder; slow down the progression of a disease or disorder; relieve to some extent one or more of the symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
  • the terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • the term “depleted” when used to describe a cell sample refers to a cell sample in which a subpopulation of cells has been removed or depleted.
  • an immune cell sample depleted of CD25 expressing cells refers to an immune cell sample in which CD25 expressing cells have been removed or depleted.
  • one or more binding agents can be used to remove or deplete one or more cells or cell types from a sample.
  • CD14 + cells can be depleted or removed from a PBMC sample, such as by using an antibody that binds to CD14.
  • the “stimulation” refers to a response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event.
  • stimulation of a T cell can refer to binding of a TCR of a T cell to a peptide-MHC complex.
  • stimulation of a T cell can refer to a step within protocol 1 or protocol 2 in which PBMCs are cultured together with peptide loaded APCs.
  • enriched refers to a composition or fraction wherein an object species has been partially purified such that the concentration of the object species is substantially higher than the naturally occurring level of the species in a finished product without enrichment.
  • induced cell refers to a cell that has been treated with an inducing compound, cell, or population of cells that affects the cell’s protein expression, gene expression, differentiation status, shape, morphology, viability, and the like.
  • a “reference” can be used to correlate and/or compare the results obtained in the methods of the present disclosure from a diseased specimen.
  • a “reference” may be obtained on the basis of one or more normal specimens, in particular specimens which are not affected by a disease, either obtained from an individual or one or more different individuals (e.g., healthy individuals), such as individuals of the same species.
  • a “reference” can be determined empirically by testing a sufficiently large number of normal specimens.
  • a tumor unless otherwise mentioned, is a cancerous tumor, and the terms cancer and tumor are used interchangeably throughout the document.
  • T cells e.g., ex vivo activated T cells, or T-cell receptor engineered T cells (TCR-T cells)
  • TCR-T cells T-cell receptor engineered T cells
  • the cells can circulate until they encounter their cognate epitope, typically on a tumor cell.
  • T cells can infiltrate the tumor and survive the harsh tumor microenvironment.
  • infused T cell numbers and frequency may decline relatively rapidly after infusion.
  • the dose of infused TCR-T cells may be important in clinical response across several studies.
  • One strategy to support the engraftment, expansion and persistence of infused T cells after infusion can include administering a vaccine that encodes the target antigen.
  • the vaccine design can differ and may be a determinant of the success of this approach.
  • the vaccine may encode for the full-length protein to be presented on the surface of cells.
  • the vaccine can alternatively be designed around the minimal epitope target of the T cells.
  • the vaccine strings designed to lead to optimal cleavage of the target epitopes can lead to more (e.g., 10-100 times more) of the desired epitope being presented on major histocompatibility complex (MHC) molecules encoded by HLA alleles than when the full- length PRAME protein is used.
  • MHC major histocompatibility complex
  • the vaccine may encode targets epitopes presented by MHC molecules encoded by HLA-A*02:01, HLA-B*07:02, and HLA-A*24:02.
  • MHC molecules encoded by HLA-A*02:01, HLA-B*07:02, and HLA-A*24:02.
  • vaccine constructs to WSGR Docket No.: 50401-787.601 present at least the target epitope on each of these HLA alleles. There are multiple considerations, including the order of the epitopes, the number of times each epitope is encoded, the codon optimization approach, and the mRNA format.
  • PRAME vaccines comprising PRAME polypeptides or recombinant nucleic acids encoding PRAME polypeptides that, when expressed, can result in presentation of one or more epitopes of PRAME by one or more MHC molecules encoded by one or more HLA alleles.
  • a recombinant nucleic acid encoding a PRAME polypeptide comprising a multiepitopic polypeptide.
  • the PRAME polypeptide does not comprise a full-length PRAME polypeptide.
  • the PRAME polypeptide comprises a first PRAME amino acid sequence comprising a first PRAME epitope sequence.
  • the PRAME polypeptide comprises a second PRAME amino acid sequence comprising a second PRAME epitope sequence.
  • the first PRAME epitope sequence and the second PRAME epitope sequence are different.
  • the first PRAME epitope sequence and the second PRAME epitope sequence are linked via a linker.
  • presentation of the first and/or second PRAME epitope sequence as a peptide:MHC complex by the antigen presenting cells (APCs) of the subject administered the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide is higher than the presentation of the first and/or second PRAME epitope sequence as the peptide:MHC complex by the APCs of a subject administered the full-length PRAME polypeptide or a recombinant nucleic acid encoding the full-length PRAME polypeptide.
  • the first PRAME amino acid sequence is the first epitope sequence.
  • the second PRAME amino acid sequence is the second epitope sequence.
  • the first PRAME amino acid sequence is the first epitope sequence and the second PRAME amino acid sequence is the second epitope sequence.
  • the first PRAME amino acid WSGR Docket No.: 50401-787.601 sequence consists of the first epitope sequence.
  • the second PRAME amino acid sequence consists of the second epitope sequence.
  • the first PRAME amino acid sequence consists of the first epitope sequence and the second PRAME amino acid sequence consists of the second epitope sequence.
  • each different PRAME epitope sequence is separated by a linker.
  • each different PRAME epitope sequence is a minimal epitope sequence.
  • the first PRAME epitope sequence consists of from 5 to 18, from 6 to 17, from 5 to 16, from 7 to 12, or from 8 to 10 consecutive amino acids from the full length PRAME polypeptide.
  • the second PRAME epitope sequence consists of from 5 to 18, from 6 to 17, from 5 to 16, from 7 to 12, or from 8 to 10 consecutive amino acids from the full length PRAME polypeptide.
  • the first PRAME epitope sequence consists of from 8 to 30, from 9 to 29, from 10 to 28, from 11 to 27, from 12 to 26, from 13 to 25, from 14 to 24, or from 15 to 23 consecutive amino acids from the full length PRAME polypeptide.
  • the second PRAME epitope sequence consists of from 8 to 30, from 9 to 29, from 10 to 28, from 11 to 27, from 12 to 26, from 13 to 25, from 14 to 24, or from 15 to 23 consecutive amino acids from the full length PRAME polypeptide.
  • the first PRAME amino acid sequence and the second PRAME amino acid sequence is separated by a linker.
  • the first PRAME amino acid sequence comprises the first PRAME epitope sequence and one, two, three, four, five or more residues flanking the N-terminus or C-terminus of the first PRAME epitope sequence from the full length PRAME polypeptide.
  • the second PRAME amino acid sequence comprises the second PRAME epitope sequence and one, two, three, four, five or more residues flanking the N-terminus or C-terminus of the second PRAME epitope sequence from the full-length PRAME polypeptide.
  • the multiepitopic polypeptide does not comprise more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive amino acids from the full-length PRAME polypeptide. In some embodiments, the multiepitopic polypeptide does not comprise more than 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive amino acids from the full-length PRAME polypeptide.
  • a multiepitopic polypeptide comprising PRAME polypeptide or a recombinant nucleic acid encoding a PRAME polypeptide.
  • the recombinant nucleic acid encoding the PRAME polypeptide can be an RNA sequence, such as a messenger RNA (mRNA) sequence.
  • the RNA sequence encoding the PRAME polypeptide can comprise the WSGR Docket No.: 50401-787.601 corresponding RNA sequence of any one of the DNA sequences described herein.
  • the RNA sequence encoding the PRAME polypeptide can comprise a corresponding RNA sequence of any one of the sequences listed in Table 1.
  • the PRAME polypeptide comprises a full length PRAME polypeptide.
  • each sequence of the multiepitopic polypeptide is the same PRAME epitope sequence.
  • each sequence of the multiepitopic polypeptide is a different PRAME epitope sequence.
  • the recombinant nucleic acid encoding the PRAME polypeptide further comprises a sequence encoding a secretory (Sec) sequence.
  • the Sec sequence is at the N terminus of the multiepitopic polypeptide.
  • the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 74.
  • the Sec sequence comprises a sequence of SEQ ID NO: 74.
  • the Sec sequence is operably linked to the multiepitopic polypeptide via a linker.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 85.
  • the linker comprises a sequence of SEQ ID NO: 85.
  • the recombinant nucleic acid encoding the PRAME polypeptide further comprises a sequence encoding an MHC I Trafficking Domain (MITD) sequence.
  • the sequence encoding an MITD sequence is at the C terminus of the multiepitopic polypeptide.
  • the multiepitopic polypeptide is operably linked to the MITD domain.
  • the multiepitopic polypeptide is operably linked to the MITD domain via a linker sequence.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 85. In some embodiments, the linker comprises a sequence of SEQ ID NO: 85.
  • the multiepitopic polypeptide comprises a PRAME polypeptide. In some embodiments, the sequence of the multiepitopic polypeptide is the same PRAME epitope sequence. In some embodiments, the sequence of the multiepitopic polypeptide is a different PRAME epitope sequence.
  • the multiepitopic polypeptide comprises at least 2 different PRAME epitope sequences. In some embodiments, WSGR Docket No.: 50401-787.601 the multiepitopic polypeptide comprises at least 3 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 4 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 5 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 6 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 8 different PRAME epitope sequences.
  • the multiepitopic polypeptide comprises at least 9 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 10 or more different PRAME epitope sequences. [0304] In some embodiments, the multiepitopic polypeptide comprises a first PRAME epitope sequence, operably linked to a second PRAME epitope sequence. In some embodiments, the first PRAME epitope sequence and the second PRAME epitope sequence are the same. In some embodiments, the first PRAME epitope sequence and the second PRAME epitope sequence are different. In some embodiments, the first PRAME epitope sequence and second PRAME epitope sequence are presentable by different HLA alleles.
  • the first PRAME epitope sequence and second PRAME epitope sequence are presented by different HLA alleles. In some embodiments, the first PRAME epitope sequence and second PRAME epitope sequence are predicted to bind to different HLA alleles. In some embodiments, the first PRAME epitope sequence and second PRAME epitope sequence are predicted to be presented by different HLA alleles. [0305] In some embodiments, a first PRAME epitope sequence binds to a first HLA allele with a K D less than 500nM. In some embodiments, a first PRAME epitope sequence binds to a first HLA allele with a K D less than 400nM.
  • a first PRAME epitope sequence binds to a first HLA allele with a K D less than 50nM. In some embodiments, a first PRAME epitope sequence binds to a first HLA allele with a K D less than 1nM. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele. In some embodiments, a first PRAME epitope sequence is predicted to bind to a second HLA allele. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 200nM.
  • a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 800nM. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 900nM. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 1000nM. In some embodiments, a first PRAME epitope sequence binds to a first HLA allele with a K D of less than 1nM and binds to second HLA allele with a K D of more than 200nM.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 97.
  • the linker comprises a sequence of SEQ ID NO: 86.
  • the linker comprises a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 88.
  • the linker comprises a sequence of SEQ ID NO: 89.
  • the linker comprises a sequence of SEQ ID NO: 90.
  • the linker comprises a sequence of SEQ ID NO: 91.
  • the second PRAME epitope sequence is presentable by an HLA-B allele. In some embodiments, the second PRAME epitope sequence is presentable by HLA-B*07:02 allele. In some embodiments, the second PRAME epitope sequence is presented by HLA-B*07:02 allele. In some embodiments, the second PRAME epitope WSGR Docket No.: 50401-787.601 sequence binds to HLA-B*07:02 allele. In some embodiments, the second PRAME epitope sequence is predicted to bind to HLA-B*07:02 allele. In some embodiments, the second PRAME epitope sequence is predicted to be presented by HLA-B*07:02.
  • the NT seq comprises a secretory (Sec) sequence and a N- terminal linker sequence.
  • the CT seq comprises a C-terminal linker and a MITD sequence.
  • the Epitope1 is a PRAME epitope presentable by HLA- A*02:01 allele.
  • the Epitope1 is a PRAME epitope presentable by HLA- B*07:02 allele.
  • the Epitope1 is a PRAME epitope presentable by HLA- A*24:02 allele.
  • the Epitope2 is a PRAME epitope presentable by HLA- A*02:01 allele.
  • the cleavable linker1, cleavable linker2, or cleavable linker3 comprise a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 86, 89, 90, 91, 92, 93, 94, 95, 96 and 97. In some cases, the cleavable linker1, cleavable linker2, or cleavable linker3 comprise a sequence of any one of SEQ ID NOs: 86, 89, 90, 91, 92, 93, 94, 95, 96 and 97. [0319] In some embodiments, the PRAME polypeptide comprises a sequence with at least 60% sequence identity to SEQ ID NO: 75.
  • the PRAME polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises a sequence with at least 90% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises a sequence of SEQ ID NO: 79. [0322] In some embodiments, the PRAME polypeptide comprises a sequence with at least 60% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises a sequence of SEQ ID NO: 108. [0324] In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 75.
  • the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 75. [0325] In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 78. In some WSGR Docket No.: 50401-787.601 embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 78.
  • the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 78. [0326] In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 79.
  • the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 79. [0327] In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 80. [0328] In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 108.
  • the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 108. [0329] In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 60% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 90% sequence identity WSGR Docket No.: 50401-787.601 to SEQ ID NO: 75.
  • the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 75. [0330] In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 60% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises two copies of a sequence with at least 60% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 79.
  • the PRAME polypeptide comprises two copies of a sequence with at least 60% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 80.
  • the PRAME polypeptide comprises two copies of a sequence with at least 60% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 108.
  • the PRAME polypeptide comprises three copies of a sequence with at least 60% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 75.
  • the PRAME polypeptide comprises three copies of a sequence with at least 60% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises three copies of a sequence with at least 60% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 80.
  • the PRAME polypeptide comprises three copies of a sequence with at least 60% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME WSGR Docket No.: 50401-787.601 polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 108.
  • the PRAME polypeptide comprises four copies of a sequence with at least 60% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 75.
  • the PRAME polypeptide comprises four copies of a sequence with at least 60% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises four copies of a sequence with at least 60% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 79.
  • the PRAME polypeptide comprises five copies of a sequence with at least 60% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises five copies of a sequence of SEQ ID NO: 80.
  • each copy of the multiepitopic polypeptide comprises, from N to C terminus, a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 80, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 79, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 78, operably linked to a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 108, operably linked to a sequence with at least 80% sequence identity to the sequence of SEQ ID NO: 79.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 80, operably linked to the sequence of SEQ ID NO: 79.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 80 via a linker, and the sequence of SEQ ID NO: 80 operably linked to the sequence of SEQ ID NO: 79 via a linker.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 108, and the sequence of SEQ ID NO: 108 operably linked to the sequence of SEQ ID NO: 79 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 91.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the linker comprises a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence of SEQ ID NO: 89. In some embodiments, the linker comprises a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence of SEQ ID NO: 91. In some embodiments, the linker comprises a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence of SEQ ID NO: 93. In some embodiments, the linker comprises a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence of SEQ ID NO: 95. In some embodiments, the linker comprises a sequence of SEQ ID NO: 96.
  • the linker comprises a sequence of SEQ ID NO: 97.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 80, operably linked to the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78. In some cases, the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 108, operably linked to the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 operably linked to
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 108, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78
  • the linker is a cleavable linker.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 86.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity toa sequence of SEQ ID NO: 87.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity toa sequence of SEQ ID NO: 89.
  • the linker comprises a having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity toa sequence of SEQ ID NO: 91.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity toa sequence of SEQ ID NO: 93.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity toa sequence of SEQ ID NO: 95.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity toa sequence of SEQ ID NO: 97. In some embodiments, the linker comprises a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a WSGR Docket No.: 50401-787.601 sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence of SEQ ID NO: 89. In some embodiments, the linker comprises a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence of SEQ ID NO: 91. In some embodiments, the linker comprises a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence of SEQ ID NO: 93. In some embodiments, the linker comprises a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence of SEQ ID NO: 95. In some embodiments, the linker comprises a sequence of SEQ ID NO: 96.
  • the linker comprises a sequence of SEQ ID NO: 97.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 108, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 80, operably linked to the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78. In some cases, the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 108, operably linked to the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, WSGR Docket No.: 50401-787.601 at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 via a linker, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 9
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 108, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 via a linker, and the sequence of SEQ ID NO: 79 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78 via a linker.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 80 via a linker, and the sequence of SEQ ID NO: 80 is operably linked via a linker to the sequence of SEQ ID NO: 79 via a linker.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 108 via a linker, and the sequence of SEQ ID NO: 108 operably linked to the sequence of SEQ ID NO: 79 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 86.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 89.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence WSGR Docket No.: 50401-787.601 of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 91.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 93.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 95.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the linker comprises a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence of SEQ ID NO: 89. In some embodiments, the linker comprises a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence of SEQ ID NO: 91. In some embodiments, the linker comprises a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence of SEQ ID NO: 93. In some embodiments, the linker comprises a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence of SEQ ID NO: 95. In some embodiments, the linker comprises a sequence of SEQ ID NO: 96.
  • the linker comprises a sequence of SEQ ID NO: 97.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78, operably linked to a sequence with at least 60%, at least 65%, at WSGR Docket No.: 50401-787.601 least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 108.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78 via a linker, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78 operably to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO:
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 79, operably linked via a linker to the sequence of SEQ ID NO: 78, operably linked via a linker to the sequence of SEQ ID NO: 80.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 79, operably linked via a linker to the sequence of SEQ ID NO: 78, operably linked via a linker to the sequence of SEQ ID NO: 108.
  • the linker is a cleavable linker.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 89.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 93.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 95.
  • the WSGR Docket No.: 50401-787.601 linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 96.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 97.
  • the linker comprises a sequence of SEQ ID NO: 86.
  • the linker comprises a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 97.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 108, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 80, operably linked to the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide WSGR Docket No.: 50401-787.601 comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 108, operably linked to the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 via a linker, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO:
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, WSGR Docket No.: 50401-787.601 or 100% sequence identity to a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 91.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 93.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the linker comprises a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 96. In some embodiments, the linker comprises a sequence of SEQ ID NO: 97. [0385] In some embodiments, the PRAME polypeptide further comprises a secretory domain (Sec) sequence. In some embodiments, the Sec sequence is at the N terminus of the multiepitopic polypeptide. In some embodiments, the Sec comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 74.
  • the PRAME polypeptide comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 82. In some embodiments, the PRAME polypeptide comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 83.
  • the PRAME polypeptide comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 84.
  • the PRAME polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 77.
  • the PRAME polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 81.
  • the PRAME polypeptide comprises a sequence WSGR Docket No.: 50401-787.601 having at least 70% sequence identity to a sequence of SEQ ID NO: 82.
  • the PRAME polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 83. In some embodiments, the PRAME polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 84. [0389] In some embodiments, the PRAME polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 77. In some embodiments, the PRAME polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 81. In some embodiments, the PRAME polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 82.
  • the PRAME polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 83. In some embodiments, the PRAME polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 84. [0391] In some embodiments, the PRAME polypeptide comprises a sequence of SEQ ID NO: 77. In some embodiments, the PRAME polypeptide comprises a sequence of SEQ ID NO: 81. In some embodiments, the PRAME polypeptide comprises a sequence of SEQ ID NO: 82. In some embodiments, the PRAME polypeptide comprises a sequence of SEQ ID NO: 83. In some embodiments, the PRAME polypeptide comprises a sequence of SEQ ID NO: 84.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a WSGR Docket No.: 50401-787.601 sequence of SEQ ID NO: 4.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 4.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 38.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 38.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 38.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more sequences with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, 44, 46, 48, 55, 57, 59, 67, 113, 115, 117, 119, 121, 123, 125, 127, 131, 133, 135, 137, 139, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 9. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 11.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 48.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 55. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 57.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 59. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 67.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more sequences with at least 90% sequence identity to any one sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, 44, 46, 48, 55, 57, 59, 67, 113, 115, 117, 119, 121, 123, 125, 127, 131, 133, 135, 137, 139, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 9.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 11. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 13. In some WSGR Docket No.: 50401-787.601 embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 44.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 55. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 57.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more sequences selected from the group consisting of SEQ ID NOs: 9, 11, 13, 44, 46, 48, 55, 57, 59, 67, 113, 115, 117, 119, 121, 123, 125, 127, 131, 133, 135, 137, 139, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 9.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 11.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 13. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 44. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 55.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 57. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 59. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises a sequence of SEQ ID NO: 67.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of one or more sequences with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, 44, 46, 48, 55, 57, 59, 67, 113, 115, 117, 119, 121, 123, 125, 127, 131, 133, 135, 137, 139, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 9. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 11.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 13.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 44.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 48.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 55. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 57.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at WSGR Docket No.: 50401-787.601 least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 59.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 67.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of one or more sequences with at least 90% sequence identity to any one sequence selected from the group consisting of SEQ ID NOs: 9, 11, 13, 44, 46, 48, 55, 57, 59, 67, 113, 115, 117, 119, 121, 123, 125, 127, 131, 133, 135, 137, 139, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 9.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 11. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 13. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 44.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 46. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 55.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 57. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 59. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 67.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of one or more sequences selected from the group WSGR Docket No.: 50401-787.601 consisting of SEQ ID NOs: 9, 11, 13, 44, 46, 48, 55, 57, 59, 67, 113, 115, 117, 119, 121, 123, 125, 127, 131, 133, 135, 137, 139, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 9.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 55. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 57. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 59.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 11. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 13. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 44. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 46.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 55. In some embodiments, the WSGR Docket No.: 50401-787.601 recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 57. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 59.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 67. [0402] In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of one or more sequences selected from the group consisting of SEQ ID NOs: 9, 11, 13, 44, 46, 48, 55, 57, 59, 67, 113, 115, 117, 119, 121, 123, 125, 127, 131, 133, 135, 137, 139, and 140. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 9.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 11. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 13. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 44. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 46.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 55. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 57. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 59.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 11. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 13. In some embodiments, the recombinant nucleic acid encoding WSGR Docket No.: 50401-787.601 the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 44. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 46.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 55. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 57. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 59.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 67. [0404] In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises five copies of one or more sequences selected from the group consisting of SEQ ID NOs: 9, 11, 13, 44, 46, 48, 55, 57, 59, 67, 113, 115, 117, 119, 121, 123, 125, 127, 131, 133, 135, 137, 139, and 140. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises five copies of a sequence of SEQ ID NO: 9.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 9. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 11. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 13. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 44. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 46.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 48. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 55. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 57. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of a sequence of SEQ ID NO: 59.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, and operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, operably linked to a sequence of any one of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, and operably linked to a sequence of any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 12. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 14.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 21.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 29. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 30.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 43. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 45.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 47. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 49.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 54. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 56.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 58. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 60.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 68. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 69.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 70.
  • the linker sequence comprises a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence of SEQ ID NO: 10.
  • the linker sequence comprises a sequence of SEQ ID NO: 12.
  • the linker sequence WSGR Docket No.: 50401-787.601 comprises a sequence of SEQ ID NO: 14.
  • the linker sequence comprises a sequence of SEQ ID NO: 18.
  • the linker sequence comprises a sequence of SEQ ID NO: 19. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 20. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 21. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 22. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 28. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 29. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 30. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 31. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 32.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, operably linked to a sequence a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, and operably linked to a sequence a sequence having at least 80% sequence identity to any one of SEQ ID NO: 9, 44, 59, 115, 121, 127, 135, and 140.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 19.
  • the linker sequence comprises a sequence of SEQ ID NO: 43. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 45. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 47. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 49. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 54. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 56. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 58. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 60. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 68.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of any one of SEQ ID NOs: 114, 116, 118, 120, 122, 124, 126, 130, 132, 134, 136, 138, 146, 147, 148, 149, 150, 151, 152, and 153.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, operably linked to a sequence a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, via a linker sequence and the sequence a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any WSGR Docket No.: 50401-787.601 one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, via a linker sequence and the sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NO: 13, 48, 55, 117, 123, 131, and 137, via a linker sequence.
  • the linker sequence encodes a cleavable linker.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 10.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a WSGR Docket No.: 50401-787.601 sequence of SEQ ID NO: 21.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 29. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 30.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 31. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 32.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 43. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 45.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 47. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 49.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 68. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 69.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 70.
  • the linker sequence comprises a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence of SEQ ID NO: 10.
  • the linker sequence comprises a sequence of SEQ ID NO: 12.
  • the linker sequence comprises a sequence of SEQ ID NO: 14.
  • the linker sequence comprises a sequence of SEQ ID NO: 18.
  • the linker sequence comprises a sequence of SEQ ID NO: 19.
  • the linker sequence comprises a sequence of SEQ ID NO: 69. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 70. [0429] In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of any one of SEQ ID NOs: 114, 116, 118, 120, 122, 124, 126, 130, 132, 134, 136, 138, 146, 147, 148, 149, 150, 151, 152, and 153.
  • each string of sequences comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, and operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NO: 9, 44, 59, 115, 121, 127, 135, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises five copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises WSGR Docket No.: 50401-787.601 seven copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises eight copies of the string of sequences.
  • the recombinant nucleic acid encoding the PRAME polypeptide further comprises a sequence encoding a secretory (Sec) sequence.
  • the Sec sequence is at the 5’ end of the string of sequences.
  • the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 2.
  • the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 27.
  • the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 36. In some embodiments the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 144. In some embodiments the Sec sequence comprises a sequence of SEQ ID NO: 2. In some embodiments the Sec sequence comprises a sequence of SEQ ID NO: 16. In some embodiments the Sec sequence comprises of SEQ ID NO: 27.
  • the Sec sequence comprises a sequence of SEQ ID NO: 36. In some embodiments the Sec sequence comprises a sequence of SEQ ID NO: 144.
  • the recombinant nucleic acid encoding the PRAME polypeptide further comprises a sequence encoding an MHC I Trafficking Domain (MITD) sequence. In some embodiments, the sequence encoding an MITD sequence is at the 3’ end of the string of sequences. In some embodiments the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 6.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 23. In some embodiments the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a WSGR Docket No.: 50401-787.601 sequence of SEQ ID NO: 34.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 40. In some embodiments the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 51.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 62. In some embodiments the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 72.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 128. In some embodiments the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 142.
  • the MITD sequence comprises of SEQ ID NO: 34. In some embodiments the MITD sequence comprises of SEQ ID NO: 40. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 51. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 62. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 72. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 128. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 142. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 143. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 155.
  • the string of sequences is operably linked to the MITD domain. In some embodiments, the string of sequences is operably linked to the MITD domain via a linker WSGR Docket No.: 50401-787.601 sequence. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 3.
  • the linker comprises a sequence of SEQ ID NO: 22. In some embodiments, the linker comprises a sequence of SEQ ID NO: 28. In some embodiments, the linker comprises a sequence of SEQ ID NO: 33. In some embodiments, the linker comprises a sequence of SEQ ID NO: 37. In some embodiments, the linker comprises a sequence of SEQ ID NO: 39. In some embodiments, the linker comprises a sequence of SEQ ID NO: 42. In some embodiments, the linker comprises a sequence of SEQ ID NO: 50. In some embodiments, the linker comprises a sequence of SEQ ID NO: 53. In some embodiments, the linker comprises a sequence of SEQ ID NO: 61.
  • the linker comprises a sequence of SEQ ID NO: 66. In some embodiments, the linker comprises a sequence of SEQ ID NO: 71. In some embodiments, the linker comprises a sequence of SEQ ID NO: 141. In some embodiments, the linker comprises a sequence of SEQ ID NO: 145. In some embodiments, the linker comprises a sequence of SEQ ID NO: 154. [0433] Provided herein is a recombinant nucleic acid comprising a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 1.
  • the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 35. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 7.
  • the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 7. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 41. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 52. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 24.
  • a recombinant polypeptide comprising a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 73.
  • the recombinant polypeptide comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 77.
  • the recombinant polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 77. In some embodiments, the recombinant polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 81. In some embodiments, the recombinant polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 82. In some embodiments, the recombinant polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 83. In some embodiments, the recombinant polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 84.
  • the recombinant polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 73. In some embodiments, the recombinant polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 77. In some embodiments, the recombinant polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 81. In some embodiments, the recombinant polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 82.
  • the recombinant WSGR Docket No.: 50401-787.601 polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 83. In some embodiments, the recombinant polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 84. [0440] In some embodiments, the recombinant polypeptide comprises a sequence of SEQ ID NO: 73. In some embodiments, the recombinant polypeptide comprises a sequence of SEQ ID NO: 77. In some embodiments, the recombinant polypeptide comprises a sequence of SEQ ID NO: 81.
  • the recombinant polypeptide comprises a sequence of SEQ ID NO: 82. In some embodiments, the recombinant polypeptide comprises a sequence of SEQ ID NO: 83. In some embodiments, the recombinant polypeptide comprises a sequence of SEQ ID NO: 84. Table 1.
  • the peptide:MHC complex comprises a PRAME epitope sequence having at least 5 consecutive amino acids of a sequence according to SEQ ID NO: 78 and a human MHC encoded by the HLA-A*02:01 allele. In some embodiments, the peptide:MHC complex comprises a PRAME epitope sequence having at least 7 consecutive amino acids of a sequence according to SEQ ID NO: 78 and a human MHC encoded by the HLA-A*02:01 allele. In some embodiments, the peptide:MHC complex comprises a PRAME epitope sequence having at least 5 consecutive amino acids of a sequence according to SEQ ID NO: 79 and a human MHC encoded by the HLA-B*07:02 allele.
  • the peptide:MHC complex comprises a PRAME epitope sequence having at least 7 consecutive amino acids of a sequence according to SEQ ID NO: 80 and a human MHC encoded by the HLA-A*24:02 allele. In some embodiments, the peptide:MHC complex comprises a PRAME epitope sequence having at least 5 consecutive amino acids of a sequence according to SEQ ID NO: 108 and a human MHC encoded by the HLA-A*24:02 allele. In some embodiments, the peptide:MHC complex comprises a PRAME epitope sequence having at least 7 consecutive amino acids of a sequence according to SEQ ID NO: 108 and a human MHC encoded by the HLA-A*24:02 allele.
  • the peptide:MHC complex comprises the PRAME epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 78. In some embodiments the peptide:MHC complex comprises the PRAME epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 79. In some embodiments the peptide:MHC complex comprises the PRAME epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 80. In some embodiments the peptide:MHC complex comprises the PRAME epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 108.
  • cell division is increased.
  • cytotoxicity of the cell against target cell is increased compared to an otherwise identical cell not having a TCR specific for the peptide:MHC complex.
  • cytotoxicity of the cell against target cell is increased by 2% or more compared to an otherwise identical cell not having a TCR specific for the peptide:MHC complex.
  • cytotoxicity of the cell against target cell is increased by 5% or more compared to an otherwise identical cell not having a TCR specific for the peptide:MHC complex. In some embodiments, upon binding of the TCR to the peptide:MHC complex, cytotoxicity of the cell against target cell is increased by 10% or more compared to an otherwise identical cell not having a TCR specific for the peptide:MHC complex. In some embodiments, upon binding of the TCR to the peptide:MHC complex, cytotoxicity of the cell against target cell is increased by 15% or more compared to an otherwise identical cell not having a TCR specific for the peptide:MHC complex.
  • cytotoxicity of the cell against target cell is increased by 150% or more compared to an otherwise identical cell not having a TCR specific for the peptide:MHC complex.
  • cytotoxicity of the cell against target cell is increased by 200% or more compared to an otherwise identical cell not having a TCR specific for the peptide:MHC complex.
  • the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 101 and a complementarity determining region 2 (CDR2) having an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to an amino acid sequence set forth in SEQ ID NO: 102.
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 101 and a complementarity determining region 2 (CDR2) having an amino acid set forth in SEQ ID NO: 102.
  • the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3.
  • the CDR1 comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at WSGR Docket No.: 50401-787.601 least 90%, at least 95%, or 100% sequence identity to a sequence set forth in SEQ ID NO: 98
  • the CDR2 comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to identity toa sequence set forth in SEQ ID NO: 99
  • the CDR3 comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence set forth in SEQ ID NO: 100.
  • the CDR1 comprises an amino acid sequence set forth in SEQ ID NO: 98
  • the CDR2 comprises an amino acid sequence set forth in SEQ ID NO: 99
  • the CDR3 comprises an amino acid sequence set forth in SEQ ID NO: 100.
  • the TCR alpha chain construct comprises a variable region comprising a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to identity to an amino acid sequence set forth in SEQ ID NO: 104.
  • the TCR alpha chain construct comprises a variable region comprising a sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 104.
  • the TCR alpha chain construct comprises a variable region comprising an amino acid sequence set forth in SEQ ID NO: 104.
  • the TCR comprises a beta chain having an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 107 and an alpha chain having an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 105.
  • the TCR comprises a beta chain having an amino acid sequence that is at least 80% identical to SEQ ID NO: 107 and an alpha chain having an amino acid sequence that is at least 80% identical to SEQ ID NO: 105. In some embodiments, the TCR comprises a beta chain having an amino acid sequence set forth in SEQ ID NO: 107 and an alpha chain having an amino acid sequence set forth in SEQ ID NO: 105.
  • Methods of Treatment [0445] The methods of the disclosure can be used to treat any type of cancer known in the art.
  • Non-limiting examples of cancers to be treated by the methods of the present disclosure can include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), cutaneous melanoma, synovial sarcoma, myxoid and round cell liposarcoma, osteosarcoma, WSGR Docket No.: 50401-787.601 and neuroblastoma ,esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies.
  • melanoma e.g., metastatic mal
  • a cancer to be treated by the methods of treatment of the present disclosure is selected from the group consisting of carcinoma, squamous carcinoma, adenocarcinoma, sarcomata, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, squamous cell carcinoma of the anogenital region, melanoma and renal cell carcinoma.
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas.
  • Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom’s macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer (e.g., metastatic, hormone refractory prostate cancer), pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological
  • cancers include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
  • the cancer whose phenotype is determined by the method of the present disclosure is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
  • the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma.
  • the APCs of the subject express more of the PRAME epitope sequence compared to the APCs of a subject administered the WSGR Docket No.: 50401-787.601 full-length PRAME polypeptide or a recombinant nucleic acid encoding the full-length PRAME polypeptide.
  • the therapy comprises a cell comprising the multiepitopic polypeptide or the recombinant nucleic acid encoding the multiepitopic polypeptide.
  • the multiepitopic polypeptide comprises a first PRAME amino acid sequence comprising a first PRAME epitope sequence and a second PRAME amino acid sequence comprising a second PRAME epitope sequence.
  • APCs having (e.g., transfected or otherwise delivered with) the multiepitopic polypeptide comprising a PRAME polypeptide comprising different PRAME epitopes (or the recombinant nucleic acid encoding the PRAME polypeptide) can lead to increased cytokine release by the PRAME specific T cells of at least 1.1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times more compared to APCs having the full-length PRAME polypeptide or a recombinant nucleic acid encoding the full-length PRAME polypeptide.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises the second PRAME epitope sequence and a human MHC encoded by an HLA allele.
  • Also provided herein is a method of treating a subject with a disease or condition.
  • the disease or condition can comprise cancer.
  • the method comprises administering to the subject a PRAME polypeptide.
  • the method comprises administering to the subject a recombinant nucleic acid encoding the PRAME polypeptide.
  • the method comprises administering to the subject a cell a cell comprising the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide.
  • the PRAME polypeptide comprises a PRAME epitope sequence.
  • the subject has been previously administered a T-cell receptor (TCR).
  • TCR T-cell receptor
  • the subject has been previously administered a recombinant nucleic acid encoding the TCR.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises the PRAME epitope sequence.
  • the peptide:MHC complex comprises a human MHC encoded by an HLA allele.
  • the peptide:MHC complex comprises (i) the PRAME epitope sequence and (ii) a human MHC encoded by an HLA allele.
  • the disease or condition can comprise a cancer.
  • the method comprises administering to the subject a T-cell receptor (TCR).
  • the method comprises administering to the subject a recombinant nucleic acid encoding the TCR.
  • the method can comprise administering to the subject an immune cell comprising the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises a PRAME epitope sequence.
  • the peptide:MHC complex comprises a human MHC encoded by an HLA allele.
  • the peptide:MHC complex comprises (i) a PRAME epitope sequence and (ii) a human MHC encoded by an HLA allele.
  • WSGR Docket No.: 50401-787.601 In some embodiments, the subject has been previously administered a PRAME polypeptide. In some embodiments, the subject has been previously administered a recombinant nucleic acid encoding the PRAME polypeptide.
  • the PRAME polypeptide comprises the PRAME epitope sequence.
  • the disease or condition can comprise a cancer.
  • the method comprises administering to the subject a PRAME polypeptide.
  • the method comprises administering to the subject a recombinant nucleic acid encoding the PRAME polypeptide.
  • the PRAME polypeptide comprises a PRAME epitope sequence.
  • the method comprises administering the subject a TCR or an immune cell comprising the TCR.
  • the method comprises administering to the subject a recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises a PRAME epitope sequence.
  • the peptide:MHC complex comprises a human MHC encoded by an HLA allele.
  • the peptide:MHC complex comprises (i) a PRAME epitope sequence and (ii) a human MHC encoded by an HLA allele.
  • the method comprises administering to the subject the PRAME polypeptide concurrently with the TCR or an immune cell comprising the TCR. In some embodiments, the method comprises administering to the subject the PRAME polypeptide concurrently with the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some embodiments, the method comprises administering to the subject the recombinant nucleic acid encoding the PRAME polypeptide concurrently with the TCR or an immune cell comprising the TCR.
  • the method comprises administering to the subject the recombinant nucleic acid encoding the PRAME polypeptide concurrently with the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some embodiments, the method comprises administering to the subject the PRAME polypeptide prior to the TCR or an immune cell comprising the TCR. In some embodiments, the method comprises administering to the subject the PRAME polypeptide prior to the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the method comprises administering to the subject the PRAME polypeptide subsequent to the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some embodiments, the method comprises administering to the subject the recombinant nucleic acid encoding the PRAME polypeptide subsequent to the TCR or an immune cell comprising the TCR. In some embodiments, the method comprises administering to the subject the recombinant nucleic acid encoding the PRAME polypeptide subsequent to the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the TCR described herein can be a soluble TCR.
  • the TCR described herein can be a membrane-bound TCR.
  • the TCR described herein can be expressed a surface of an immune cell.
  • the method comprises administering a cell comprising the PRAME polypeptide or nucleic acid encoding the PRAME polypeptide concurrently with administering to the subject a TCR or a recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the PRAME peptide sequence, and (ii) a human MHC encoded by an HLA allele.
  • the method comprises administering a cell comprising the PRAME polypeptide or nucleic acid encoding the PRAME polypeptide subsequent to administering to the subject a TCR or a recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the PRAME peptide sequence, and (ii) a human MHC encoded by an HLA allele.
  • the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide can be administered at least 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, 1 day, 2 days, 5 days, 10 days, 20 days, 30 days, 1 month, 2 months, 3 WSGR Docket No.: 50401-787.601 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more after the subject has been administered the TCR or the recombinant nucleic acid encoding the TCR.
  • the immune cell comprising the TCR or the recombinant nucleic acid encoding the TCR can be administered at least 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, 15 hours, 20 hours, 1 day, 2 days, 5 days, 10 days, 20 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, or more after the subject has been administered the PRAME polypeptide or the recombinant nucleic acid encoding the PRAME polypeptide.
  • the PRAME polypeptide is administered at least 1 hour after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 2 hours after the subject has been administered the TCR or an immune cell comprising the TCR or an immune cell comprising the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 4 hours after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 8 hours after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the PRAME polypeptide is administered at least 12 hours after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 1 day after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 2 days after the subject has been administered the TCR or WSGR Docket No.: 50401-787.601 an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 5 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 10 days after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the PRAME polypeptide is administered at least 20 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 30 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 1 month after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 2 months after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 3 months after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the PRAME polypeptide is administered at least 6 months after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 1 year after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 2 years after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the PRAME polypeptide is administered at least 3 years or more after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 1 hour after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 2 hours after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 4 hours after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the recombinant nucleic acid encoding WSGR Docket No.: 50401-787.601 the PRAME polypeptide is administered at least 2 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 5 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 10 days after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 20 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 30 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 1 month after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 2 months after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 3 months after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 6 months after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 1 year after the subject has been administered the TCR or an immune cell comprising the TCR. In some cases, the Recombinant nucleic acid encoding the PRAME polypeptide is administered at least 2 years after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 3 years or more after the subject has been administered the TCR or an immune cell comprising the TCR. [0470] In some cases, the PRAME polypeptide is administered at least 1 hour after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 2 hours after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the PRAME polypeptide is administered at least 4 hours after the subject has been administered the recombinant nucleic acid encoding WSGR Docket No.: 50401-787.601 the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 8 hours after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the PRAME polypeptide is administered at least 12 hours after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 1 day after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 2 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the PRAME polypeptide is administered at least 5 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 10 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 20 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the PRAME polypeptide is administered at least 30 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 1 month after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 2 months after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the PRAME polypeptide is administered at least 3 months after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 6 months after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the PRAME polypeptide is administered at least 1 year after the subject has been administered the WSGR Docket No.: 50401-787.601 recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the PRAME polypeptide is administered at least 2 years after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the PRAME polypeptide is administered at least 3 years or more after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 1 hour after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 2 hours after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 4 hours after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 8 hours after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 12 hours after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 1 day after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 2 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 5 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 10 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding WSGR Docket No.: 50401-787.601 the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 20 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 30 days after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 1 month after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 2 months after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 3 months after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 6 months after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 1 year after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR. In some cases, the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 2 years after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the recombinant nucleic acid encoding the PRAME polypeptide is administered at least 3 years or more after the subject has been administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the TCR or an immune cell comprising the TCR is administered at least 1 hour after the subject has been administered the PRAME polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 2 hours after the subject has been administered the PRAME polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 4 hours after the subject has been administered the PRAME polypeptide.
  • the TCR or an immune cell comprising WSGR Docket No.: 50401-787.601 the TCR is administered at least 8 hours after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 12 hours after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 1 day after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 2 days after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 5 days after the subject has been administered the PRAME polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 10 days after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 20 days after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 30 days after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 1 month after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 2 months after the subject has been administered the PRAME polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 3 months after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 6 months after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 1 year after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 2 years after the subject has been administered the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 3 years or more after the subject has been administered the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 1 hour after the subject has been administered the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 2 hours after the subject has been administered the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 4 hours after the subject has been administered the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 10 days after the subject has been administered the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 20 days after the subject has been administered the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 30 days after the subject has been administered the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 1 month after the subject has been administered the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 2 months after the subject has been administered the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 3 months after the subject has been administered the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 6 months after the subject has been administered the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 1 year after the subject has been WSGR Docket No.: 50401-787.601 administered the PRAME polypeptide.
  • the Recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 2 years after the subject has been administered the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 3 years or more after the subject has been administered the PRAME polypeptide. [0474] In some cases, the TCR or an immune cell comprising the TCR is administered at least 1 hour after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 2 hours after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 4 hours after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 8 hours after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 12 hours after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 1 day after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 2 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 5 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 10 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 20 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 30 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 1 month after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 2 months after the subject has been administered WSGR Docket No.: 50401-787.601 the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 3 months after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the TCR or an immune cell comprising the TCR is administered at least 6 months after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 12 hours after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 1 day after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 2 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an WSGR Docket No.: 50401-787.601 immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 5 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 10 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 20 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 30 days after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 1 month after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 2 months after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 3 months after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 6 months after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 1 year after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 2 years after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide. In some cases, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 3 years or more after the subject has been administered the recombinant nucleic acid encoding the PRAME polypeptide.
  • the cell is an immune cell.
  • the recombinant nucleic acid encoding the TCR is expressed by a cell.
  • the cell is an immune cell.
  • the TCR is a soluble TCR.
  • the method comprises administering two or more different TCRs.
  • the method comprises administering two or more recombinant nucleic acids encoding the two or more different TCRs.
  • the two or more TCRs comprise a first TCR and a second TCR.
  • the two or more different TCRs are expressed on the surface of two different immune cells.
  • the first TCR and the second TCR bind to different peptide:MHC complexes, each peptide:MHC complex comprising (i) an epitope sequence and (ii) a human MHC encoded by an HLA allele.
  • the two or more different TCRs are administered separately or co-administered in a same mixture.
  • recombinant nucleic acids encoding the two or more different TCRs are administered separately or co-administered in a same mixture.
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 79 and an MHC encoded by an HLA-B*07:02 allele and the second TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 80 or SEQ ID NO: 108 and an MHC encoded by an HLA-A*24:02 allele.
  • the PRAME polypeptide does not comprise a full-length PRAME protein sequence.
  • the PRAME polypeptide comprises a PRAME epitope sequence.
  • the PRAME epitope sequence comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 78. In some embodiments, the PRAME epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 78. In some embodiments, the PRAME epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of WSGR Docket No.: 50401-787.601 SEQ ID NO 78. In some embodiments, the PRAME epitope sequence comprises a sequence of SEQ ID NO: 78.
  • the HLA allele is an HLA-A allele. In some cases, the HLA allele is an HLA-A*02:01 allele.
  • the PRAME epitope sequence comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 79. In some embodiments, the PRAME epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 79. In some embodiments, the PRAME epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO 79.
  • the PRAME epitope sequence comprises a sequence of SEQ ID NO: 79.
  • the HLA allele is an HLA-B allele.
  • the HLA allele is an HLA-B*07:02 allele.
  • the PRAME epitope sequence comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 80.
  • the PRAME epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 80.
  • the PRAME epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO 80. In some embodiments, the PRAME epitope sequence comprises a sequence of SEQ ID NO: 80. In some embodiments, the PRAME epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NO: 108. In some embodiments, the PRAME epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 108. In some embodiments, the PRAME epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO 108. In some embodiments, the PRAME epitope sequence comprises a sequence of SEQ ID NO: 108.
  • the HLA allele is an HLA-A allele. In some embodiments, the HLA allele is an HLA-A*24:02 allele.
  • the PRAME polypeptide comprises a multiepitopic polypeptide. In some embodiments, the multiepitopic polypeptide does not comprise a full- length PRAME polypeptide. In some embodiments, the sequence of the multiepitopic polypeptide is the same PRAME epitope sequence. In some embodiments, the multiepitopic polypeptide comprises two or more different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 2 different PRAME epitope sequences.
  • the multiepitopic polypeptide comprises at least 3 different WSGR Docket No.: 50401-787.601 PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 4 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 5 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 6 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 8 different PRAME epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 9 different PRAME epitope sequences.
  • the multiepitopic polypeptide comprises at least 10 or more different PRAME epitope sequences. In some embodiments, the different PRAME epitope sequences are separated. In some embodiments, the different PRAME epitope sequences are separated by linker sequences.
  • antigen presenting cells (APCs) of subject administered the PRAME polypeptide present a PRAME epitope sequence as a peptide:MHC complex. In some cases, antigen presenting cells of subject administered the nucleic acid encoding the PRAME polypeptide comprising the multiepitopic polypeptide present a PRAME epitope sequence as a peptide:MHC complex.
  • the APCs present more of a PRAME epitope as a peptide:MHC complex compared to the APCs of a subject administered a full length PRAME polypeptide or recombinant nucleic acid encoding the full-length PRAME polypeptide.
  • T cells of the subject administered the multiepitopic polypeptide or a recombinant nucleic acid encoding the multiepitopic polypeptide exhibit increased expansion compared to T cells of a subject administered a full-length PRAME polypeptide or a recombinant nucleic acid encoding the full-length PRAME polypeptide.
  • the multiepitopic polypeptide comprises a first PRAME amino acid sequence comprising a first PRAME epitope sequence. In some embodiments, the multiepitopic polypeptide comprises a second PRAME amino acid sequence comprising a second PRAME epitope sequence. In some embodiments, the first PRAME epitope sequence and the second PRAME epitope sequence are different. In some embodiments, the first PRAME epitope sequence and the second PRAME epitope sequence is separated by a linker. [0484] In some embodiments, the first amino acid sequence is the first epitope sequence. In some embodiments the second amino acid sequence is the second epitope sequence.
  • the first amino acid sequence is the first epitope sequence
  • the second amino acid sequence is the second epitope sequence.
  • the first amino acid sequence consists of the first epitope sequence.
  • the second amino acid sequence consists of the second epitope sequence.
  • the first amino WSGR Docket No.: 50401-787.601 acid sequence consists of the first epitope sequence
  • the second amino acid sequence consists of the second epitope sequence.
  • the first PRAME epitope sequence consists of from 5 to 18, from 6 to 17, from 5 to 16, from 7 to 12, or from 8 to 10 consecutive amino acids from the full length PRAME polypeptide.
  • a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 300nM. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 500nM. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 600nM. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 700nM. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 800nM.
  • a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 900nM. In some embodiments, a first PRAME epitope sequence binds to a second HLA allele with a K D of more than 1000nM. In some embodiments, a first PRAME epitope sequence binds to a first HLA allele with a K D of less than 1nM and binds to second HLA allele with a K D of more than 200nM. In some embodiments, a first PRAME epitope sequence binds to a first HLA allele with a K D of less than 50nM and binds to second HLA allele with a K D of more than 300nM.
  • a first PRAME epitope sequence binds to a first HLA allele with a K D of less than 100nM and binds to second HLA allele with a K D of more than 500nM. In some embodiments, a first PRAME epitope sequence binds to a first HLA allele with a K D of less than 200 nM and binds to second HLA allele with a K D of more than 600nM. [0491] In some aspects, the first PRAME epitope sequence is operably linked to the second PRAME epitope sequence. In some aspects, the first PRAME epitope sequence is operably linked to the second PRAME epitope sequence via a linker.
  • the linker WSGR Docket No.: 50401-787.601 comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 86.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 91.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 93.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 95.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the linker comprises a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence of SEQ ID NO: 89. In some embodiments, the linker comprises a sequence of SEQ ID NO: 90. In some WSGR Docket No.: 50401-787.601 embodiments, the linker comprises a sequence of SEQ ID NO: 91. In some embodiments, the linker comprises a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence of SEQ ID NO: 93. In some embodiments, the linker comprises a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence of SEQ ID NO: 95.
  • the first PRAME epitope sequence binds to HLA-A*02:01 allele. In some embodiments, the first PRAME epitope sequence is predicted to bind to HLA-A*02:01 allele. In some embodiments, the first PRAME epitope sequence is predicted to be presented by HLA-A*02:01. [0494] In some embodiments, the first PRAME epitope sequence is presentable by an HLA- A allele. In some embodiments, the first PRAME epitope sequence is presentable by HLA- A*24:02 allele. In some embodiments, the first PRAME epitope sequence is presented by HLA-A*24:02 allele.
  • the first PRAME epitope sequence binds to HLA-B*07:02 allele. In some embodiments, the first PRAME epitope sequence is predicted to bind to HLA-B*07:02 allele. In some embodiments, the first PRAME epitope sequence is predicted to be presented by HLA-B*07:02. [0496] In some embodiments, the second PRAME epitope sequence is presentable by an HLA-A allele. In some embodiments, the second PRAME epitope sequence is presentable by HLA-A*02:01 allele. In some embodiments, the second PRAME epitope sequence is presented by HLA-A*02:01 allele.
  • the second PRAME epitope WSGR Docket No.: 50401-787.601 sequence binds to HLA-A*02:01 allele. In some embodiments, the second PRAME epitope sequence is predicted to bind to HLA-A*02:01 allele. In some embodiments, the second PRAME epitope sequence is predicted to be presented by HLA-A*02:01. [0497] In some embodiments, the second PRAME epitope sequence is presentable by an HLA-A allele. In some embodiments, the second PRAME epitope sequence is presentable by HLA-A*24:02 allele. In some embodiments, the second PRAME epitope sequence is presented by HLA-A*24:02 allele.
  • the second PRAME epitope sequence binds to HLA-B*07:02 allele. In some embodiments, the second PRAME epitope sequence is predicted to bind to HLA-B*07:02 allele. In some embodiments, the second PRAME epitope sequence is predicted to be presented by HLA-B*07:02. [0499] In some embodiments, the third PRAME epitope sequence is presentable by an HLA- A allele. In some embodiments, the third PRAME epitope sequence is presentable by HLA- A*02:01 allele. In some embodiments, the third PRAME epitope sequence is presented by HLA-A*02:01 allele.
  • the third PRAME epitope sequence binds to HLA-B*07:02 allele. In some embodiments, the third PRAME epitope sequence is predicted to bind to HLA-B*07:02 allele. In some embodiments, the third PRAME epitope sequence is predicted to be presented by HLA-B*07:02. [0502] In some cases, the subject does not express an HLA that binds to each PRAME epitope of the multiepitopic polypeptide. In some cases, the subject only expresses HLA alleles that bind to a subset of PRAME epitope sequences of the multiepitopic polypeptide.
  • the PRAME polypeptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80.
  • the PRAME WSGR Docket No.: 50401-787.601 polypeptide comprises a sequence with at least 70% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises a sequence with at least 90% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 75. [0509] In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 78.
  • the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 79.
  • the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises one or more copies of a sequence of SEQ ID NO: 80. [0512] In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 108.
  • the PRAME polypeptide comprises two copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 79.
  • the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 79. [0516] In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 80. [0517] In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least WSGR Docket No.: 50401-787.601 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 108.
  • the PRAME polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises two copies of a sequence of SEQ ID NO: 108. [0518] In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 75.
  • the PRAME polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 75.
  • the PRAME polypeptide comprises three copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 78.
  • the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 79. WSGR Docket No.: 50401-787.601 [0521] In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 80. [0522] In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 108.
  • the PRAME polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises three copies of a sequence of SEQ ID NO: 108.
  • the PRAME polypeptide comprises four copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 75.
  • the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 75. [0524] In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 78.
  • the PRAME polypeptide comprises four copies of a sequence with at least 90% sequence identity WSGR Docket No.: 50401-787.601 to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 78. [0525] In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 79.
  • the PRAME polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 79. [0526] In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 80.
  • the PRAME polypeptide comprises four copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 108.
  • the PRAME polypeptide comprises four copies of a sequence of SEQ ID NO: 108. [0528] In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises five copies of WSGR Docket No.: 50401-787.601 a sequence with at least 80% sequence identity to SEQ ID NO: 75.
  • the PRAME polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises five copies of a sequence of SEQ ID NO: 75. [0529] In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 78.
  • the PRAME polypeptide comprises five copies of a sequence with at least 80% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 78. In some embodiments, the PRAME polypeptide comprises five copies of a sequence of SEQ ID NO: 78. [0530] In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 79.
  • the PRAME polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 80% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 79. In some embodiments, the PRAME polypeptide comprises five copies of a sequence of SEQ ID NO: 79.
  • the PRAME polypeptide comprises five copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 80% sequence identity to SEQ ID NO: 80. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 80.
  • the PRAME polypeptide comprises five copies of a sequence of SEQ ID NO: 80. [0532] In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 108. In some embodiments, the WSGR Docket No.: 50401-787.601 PRAME polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises five copies of a sequence with at least 80% sequence identity to SEQ ID NO: 108.
  • the PRAME polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 108. In some embodiments, the PRAME polypeptide comprises five copies of a sequence of SEQ ID NO: 108. [0533] In some embodiments, the PRAME polypeptide comprises six or more copies of a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 75. In some embodiments, the PRAME polypeptide comprises six or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 75.
  • the linker is a cleavable linker.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 86.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 93.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the linker comprises a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises WSGR Docket No.: 50401-787.601 a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence of SEQ ID NO: 89. In some embodiments, the linker comprises a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence of SEQ ID NO: 91. In some embodiments, the linker comprises a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence of SEQ ID NO: 93. In some embodiments, the linker comprises a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence of SEQ ID NO: 95. In some embodiments, the linker comprises a sequence of SEQ ID NO: 96.
  • the linker comprises a sequence of SEQ ID NO: 97.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 80, operably linked to the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78. In some cases, the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 108, operably linked to the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, WSGR Docket No.: 50401-787.601 at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 108, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 80 via a linker, and the sequence of SEQ ID NO: 80 operably linked to the sequence of SEQ ID NO: 79 via a linker.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 108 via a linker, and the sequence of SEQ ID NO: 108 operably linked to the sequence of SEQ ID NO: 79 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 89.
  • the WSGR Docket No.: 50401-787.601 linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 90.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 91.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 93.
  • the linker comprises a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence of SEQ ID NO: 89. In some embodiments, the linker comprises a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence of SEQ ID NO: 91. In some embodiments, the linker comprises a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence of SEQ ID NO: 93. In some embodiments, the linker comprises a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence of SEQ ID NO: 95. In some embodiments, the linker comprises a sequence of SEQ ID NO: 96.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 via a linker, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO:
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 108, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79 via a linker, and the sequence of SEQ ID NO: 79 operably linked to a sequence WSGR Docket No.: 50401-787.601 with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78 via a linker.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 80 via a linker, and the sequence of SEQ ID NO: 80 is operably linked via a linker to the sequence of SEQ ID NO: 79 via a linker.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 108 via a linker, and the sequence of SEQ ID NO: 108 operably linked to the sequence of SEQ ID NO: 79 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 86.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 89.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 91.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 93.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least WSGR Docket No.: 50401-787.601 95%, or 100% sequence identity to a sequence of SEQ ID NO: 95.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the linker comprises a sequence of SEQ ID NO: 86. In some embodiments, the linker comprises a sequence of SEQ ID NO: 87.
  • the linker comprises a sequence of SEQ ID NO: 88. In some embodiments, the linker comprises a sequence of SEQ ID NO: 89. In some embodiments, the linker comprises a sequence of SEQ ID NO: 90. In some embodiments, the linker comprises a sequence of SEQ ID NO: 91. In some embodiments, the linker comprises a sequence of SEQ ID NO: 92. In some embodiments, the linker comprises a sequence of SEQ ID NO: 93. In some embodiments, the linker comprises a sequence of SEQ ID NO: 94. In some embodiments, the linker comprises a sequence of SEQ ID NO: 95. In some embodiments, the linker comprises a sequence of SEQ ID NO: 96.
  • the linker comprises a sequence of SEQ ID NO: 97.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 80.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 108.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78, WSGR Docket No.: 50401-787.601 operably linked to the sequence of SEQ ID NO: 80.
  • the PRAME polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 79, operably linked to the sequence of SEQ ID NO: 78, operably linked to the sequence of SEQ ID NO: 108.
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78 via a linker, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78 operably to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO:
  • the PRAME polypeptide comprises, from N terminus to C terminus, a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 79, operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78 via a linker, and the sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 78 operably linked to a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence of SEQ ID NO:
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 56. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 58.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 70.
  • the linker sequence comprises a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence of SEQ ID NO: 10.
  • the linker sequence comprises a sequence of SEQ ID NO: 12.
  • the linker sequence comprises a sequence of SEQ ID NO: 14.
  • the linker sequence comprises a sequence of SEQ ID NO: 18.
  • the linker sequence comprises a sequence of SEQ ID NO: 19.
  • the linker sequence comprises a sequence of SEQ ID NO: 70.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, WSGR Docket No.: 50401-787.601 or 100% sequence identity to a sequence of any one of SEQ ID NOs: 114, 116, 118, 120, 122, 124, 126, 130, 132, 134, 136, 138, 146, 147, 148, 149, 150, 151, 152, and 153.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, and operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NO: 13, 13, 48, 55, 117, 123, 131, and 137.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, operably linked to a sequence of any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, and operably linked to a sequence of any one of SEQ ID NO: 13, 13, 48, 55, 117, 123, 131, and 137.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, via a linker sequence and the sequence having at least 90% sequence identity to any one of SEQ ID NOs: 9, 44, 59, 115, 121, 127, 135, and 140, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NO: 13, 13, 48, 55, 117, 123, 131, and 137, via a linker sequence.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 12. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 14.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 19.
  • the linker sequence comprises a sequence having at least 60%, at least WSGR Docket No.: 50401-787.601 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 20.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 21.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 22. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 28.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 29. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 30.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 31. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 32.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 43. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 45.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 47. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 49.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 58. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 60.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 68. In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 69.
  • the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 70.
  • the linker sequence comprises a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence of SEQ ID NO: 10.
  • the linker sequence comprises a sequence of SEQ ID NO: 12.
  • the linker sequence comprises a sequence of SEQ ID NO: 14.
  • the linker sequence comprises a sequence of SEQ ID NO: 18.
  • the linker sequence comprises a sequence of SEQ ID NO: 19.
  • the linker sequence comprises a sequence of SEQ ID NO: 20. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 21. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 22. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 28. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 29. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 30. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 31. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 32. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 43.
  • the linker sequence comprises a sequence of SEQ ID NO: 45. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 47. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 49. In some embodiments, the linker sequence WSGR Docket No.: 50401-787.601 comprises a sequence of SEQ ID NO: 54. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 56. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 58. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 60. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 68.
  • the linker sequence comprises a sequence of SEQ ID NO: 69. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 70. [0607] In some embodiments, the linker sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of any one of SEQ ID NOs: 114, 116, 118, 120, 122, 124, 126, 130, 132, 134, 136, 138, 146, 147, 148, 149, 150, 151, 152, and 153.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises at least two copies of a string of sequences.
  • each string of sequences comprises, from 5’ end to 3’ end, a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, operably linked to a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, and operably linked to a sequence a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at
  • each string of sequences comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, and operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NO: 9, 44, 59, 115, 121, 127, 135, and 140.
  • each string of sequences comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 13, 48, 55, 117, 123, 131, and 137, operably linked to a sequence of any one of SEQ ID NOs: 11, 46, 57, 67, 113, 119, 125, 133, and 139, and operably linked to a sequence of any one of SEQ ID NO: 9, 44, 59, 115, 121, 127, 135, and 140.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises three copies of the string of sequences.
  • the recombinant nucleic acid encoding the PRAME polypeptide comprises four copies of the string of sequences. In some embodiments, WSGR Docket No.: 50401-787.601 the recombinant nucleic acid encoding the PRAME polypeptide comprises five copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises six copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises seven copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide comprises eight copies of the string of sequences.
  • the recombinant nucleic acid encoding the PRAME polypeptide further comprises a sequence encoding a secretory (Sec) sequence.
  • the Sec sequence is at the 5’ end of the string of sequences.
  • the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 2.
  • the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 27.
  • the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 36. In some embodiments the Sec sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 144. In some embodiments the Sec sequence comprises a sequence of SEQ ID NO: 2. In some embodiments the Sec sequence comprises a sequence of SEQ ID NO: 16. In some embodiments the Sec sequence comprises of SEQ ID NO: 27.
  • the Sec sequence comprises a sequence of SEQ ID NO: 36.
  • the string of sequences is operably linked to the Sec sequence.
  • the Sec sequence comprises a sequence of SEQ ID NO: 144.
  • the string of sequences is operably linked to the Sec sequence via a linker sequence.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 3.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 5.
  • the linker WSGR Docket No.: 50401-787.601 comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 17.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 33. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 37.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 39. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 42.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 50. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 53.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 61. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 66.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 71. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 141.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least WSGR Docket No.: 50401-787.601 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 145.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 154.
  • the linker comprises a sequence of SEQ ID NO: 3.
  • the linker comprises a sequence of SEQ ID NO: 5.
  • the linker comprises a sequence of SEQ ID NO: 17. In some embodiments, the linker comprises a sequence of SEQ ID NO: 22. In some embodiments, the linker comprises a sequence of SEQ ID NO: 28. In some embodiments, the linker comprises a sequence of SEQ ID NO: 33. In some embodiments, the linker comprises a sequence of SEQ ID NO: 37. In some embodiments, the linker comprises a sequence of SEQ ID NO: 39. In some embodiments, the linker comprises a sequence of SEQ ID NO: 42. In some embodiments, the linker comprises a sequence of SEQ ID NO: 50. In some embodiments, the linker comprises a sequence of SEQ ID NO: 53.
  • the linker comprises a sequence of SEQ ID NO: 61. In some embodiments, the linker comprises a sequence of SEQ ID NO: 66. In some embodiments, the linker comprises a sequence of SEQ ID NO: 71. In some embodiments, the linker comprises a sequence of SEQ ID NO: 141. In some embodiments, the linker comprises a sequence of SEQ ID NO: 145. In some embodiments, the linker comprises a sequence of SEQ ID NO: 154. [0610] In some embodiments, the recombinant nucleic acid encoding the PRAME polypeptide further comprises a sequence encoding an MHC I Trafficking Domain (MITD) sequence.
  • MIMD MHC I Trafficking Domain
  • the sequence encoding an MITD sequence is at the 3’ end of the string of sequences.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 6.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 23.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 34. In some embodiments the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 40.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or WSGR Docket No.: 50401-787.601 100% sequence identity to a sequence of SEQ ID NO: 51.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 62.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 72. In some embodiments the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 128.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 142. In some embodiments the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 143.
  • the MITD sequence comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 155.
  • the MITD sequence comprises a sequence of SEQ ID NO: 6.
  • the MITD sequence comprises a sequence of SEQ ID NO: 23.
  • the MITD sequence comprises of SEQ ID NO: 34.
  • the MITD sequence comprises of SEQ ID NO: 40.
  • the MITD sequence comprises a sequence of SEQ ID NO: 51.
  • the MITD sequence comprises a sequence of SEQ ID NO: 62.
  • the MITD sequence comprises a sequence of SEQ ID NO: 72. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 128. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 142. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 143. In some embodiments the MITD sequence comprises a sequence of SEQ ID NO: 155. In some embodiments, the string of sequences is operably linked to the MITD sequence. In some embodiments, the string of sequences is operably linked to the MITD sequence via a linker sequence.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 3.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of WSGR Docket No.: 50401-787.601 SEQ ID NO: 5.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 22.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 28. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 33.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 37. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 39.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 42. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 50.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 53. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 61.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 66. In some embodiments, the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 71.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 141.
  • the linker comprises a sequence having at least 60%, at least WSGR Docket No.: 50401-787.601 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 145.
  • the linker comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 154.
  • the linker comprises a sequence of SEQ ID NO: 3.
  • the linker comprises a sequence of SEQ ID NO: 5.
  • the linker comprises a sequence of SEQ ID NO: 17.
  • the linker comprises a sequence of SEQ ID NO: 22.
  • the linker comprises a sequence of SEQ ID NO: 28.
  • the linker comprises a sequence of SEQ ID NO: 33.
  • the linker comprises a sequence of SEQ ID NO: 37. In some embodiments, the linker comprises a sequence of SEQ ID NO: 39. In some embodiments, the linker comprises a sequence of SEQ ID NO: 42. In some embodiments, the linker comprises a sequence of SEQ ID NO: 50. In some embodiments, the linker comprises a sequence of SEQ ID NO: 53. In some embodiments, the linker comprises a sequence of SEQ ID NO: 61. In some embodiments, the linker comprises a sequence of SEQ ID NO: 66. In some embodiments, the linker comprises a sequence of SEQ ID NO: 71. In some embodiments, the linker comprises a sequence of SEQ ID NO: 141.
  • the linker comprises a sequence of SEQ ID NO: 145. In some embodiments, the linker comprises a sequence of SEQ ID NO: 154. [0611] In some embodiments, the recombinant nucleic acid is codon optimized. In some embodiments, the recombinant nucleic acid is not codon optimized. In some embodiments, the recombinant nucleic acid is DNA. In some embodiments, the recombinant nucleic acid is RNA. In some embodiments the recombinant nucleic acid is mRNA.
  • the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 1.
  • the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 35.
  • the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 52.
  • the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 64. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 26.
  • the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 65.
  • the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 1.
  • the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 35.
  • the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 7.
  • the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 41. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 52. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 24.63. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 64.
  • the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 7. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 41. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 52. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 24.
  • the recombinant nucleic acid comprises a sequence of SEQ ID NO: 64. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 26. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 65.
  • WSGR Docket No.: 50401-787.601 the subject expresses more of the PRAME epitope when administered with the codon optimized recombinant nucleic acid than administered with a wild-type recombinant nucleic acid.
  • the TCR comprises a beta chain construct. In some embodiments, the TCR comprises an alpha chain construct.
  • the TCR comprises a beta chain construct and an alpha chain construct.
  • the TCR beta chain construct comprises a complementarity determining region 3 (CDR3).
  • the TCR beta chain construct comprises a CDR3 having an amino acid sequence with at least 60%, 70%, 80%, or 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 103.
  • the TCR beta chain construct comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 103.
  • the TCR beta chain construct comprises a variable region.
  • the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 60%, 70%, 80%, or 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 106. In some embodiments, the TCR beta chain construct comprises a CDR1. In some embodiments, the TCR beta chain construct comprises a CDR1 having an amino acid sequence with at least 60%, 70%, 80%, or 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the TCR beta chain construct comprises a CDR1 having an amino acid set forth in SEQ ID NO: 101. In some embodiments, the TCR beta chain construct comprises a CDR2.
  • the TCR beta chain construct comprises a CDR2 having an amino acid sequence with at least 60%, 70%, 80%, or 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 102. In some embodiments, the TCR beta chain construct comprises a CDR2 having an amino acid sequence set forth in SEQ ID NO: 102. [0618] In some embodiments, the TCR comprises an alpha chain construct. In some embodiments, the alpha chain construct comprises a CDR1. In some embodiments, the alpha chain construct comprises a CDR2. In some embodiments, the alpha chain construct comprises a CDR3.
  • the alpha chain construct comprises a CDR1 having an amino acid sequence with at least 60%, 70%, 80%, or 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the alpha chain construct comprises a CDR1 having an amino acid sequence set forth in SEQ ID NO: 98. In some embodiments, the alpha chain construct comprises a CDR2 having an amino acid sequence with at least 60%, 70%, 80%, or 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 99. In some embodiments, the alpha chain construct comprises a WSGR Docket No.: 50401-787.601 CDR2 having an amino acid sequence set forth in SEQ ID NO: 99.
  • the TCR comprises a beta chain having an amino acid sequence with at least 60%, 70%, 80%, or 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, the TCR comprises a beta chain having an amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence with at least 60%, 70%, 80%, or 90% sequence identity to an amino acid sequence set forth in SEQ ID NO: 105. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence set forth in SEQ ID NO: 105. [0619] Provided herein is a method of treating a subject with a disease or condition. In some embodiments, the disease or condition is a cancer.
  • the method comprises administering to the subject a therapy.
  • the therapy comprises a multiepitopic polypeptide.
  • the therapy comprises a recombinant nucleic acid encoding the multiepitopic polypeptide.
  • the therapy comprises a cell comprising the multiepitopic polypeptide.
  • the therapy comprises a cell comprising the recombinant nucleic acid encoding the multiepitopic polypeptide.
  • the multiepitopic polypeptide comprises a first amino acid sequence comprising a first epitope sequence from a cancer protein.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises the first epitope sequence and a human MHC encoded by an HLA allele.
  • the peptide:MHC complex comprises the second epitope sequence and a human MHC encoded by an HLA allele.
  • Provided herein is a method of treating a subject with a disease or condition.
  • the disease or condition is a cancer.
  • the method comprises administering to the subject a therapy.
  • the therapy comprises a TCR.
  • the therapy comprises a recombinant nucleic acid encoding the TCR.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises an epitope sequence form a cancer protein and a human MHC encoded by an HLA allele.
  • the subject has been previously treated with a multiepitopic polypeptide.
  • the subject has been previously treated with a recombinant nucleic acid encoding the multiepitopic polypeptide.
  • the subject has been previously treated with a cell comprising a multiepitopic polypeptide.
  • the subject has been previously treated with a cell comprising a recombinant nucleic acid encoding the multiepitopic polypeptide.
  • the first epitope sequence or the second epitope sequence are linked by a linker.
  • the multiepitopic polypeptide does not comprise the full-length cancer protein.
  • the method comprises administering to the subject a second therapy.
  • the second therapy comprises a TCR.
  • the second therapy comprises a recombinant nucleic acid encoding the TCR.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises the first epitope sequence and a human MHC encoded by an HLA allele.
  • the cancer protein is PRAME.
  • the TCR recognizes an epitope derived from the cancer protein.
  • the method comprises administering two or more different TCRs.
  • the method comprises administering two or more recombinant nucleic acids encoding the two or more different TCRs.
  • the two or more TCRs comprise a first TCR and a second TCR.
  • the two or more different TCRs are expressed on the surface of two different immune cells.
  • the first TCR and the second TCR bind to different peptide:MHC complexes, each peptide:MHC complex comprising (i) an epitope sequence and (ii) a human MHC encoded by an HLA allele.
  • the two or more different TCRs are administered separately or co-administered in a same mixture.
  • recombinant nucleic acids encoding the two or more different TCRs are administered separately or co-administered in a same mixture.
  • the first TCR binds WSGR Docket No.: 50401-787.601 to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 78 and an MHC encoded by an HLA-A*02:01 allele and the second TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 79 and an MHC encoded by an HLA- B*07:02 allele.
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 79 and an MHC encoded by an HLA-B*07:02 allele and the second TCR binds to a peptide:MHC complex comprising an epitope sequence of SEQ ID NO: 80 or SEQ ID NO: 108 and an MHC encoded by an HLA-A*24:02 allele.
  • the APCs of the subject administered the multiepitopic polypeptide or a recombinant nucleic acid encoding the multiepitopic polypeptide present more of an epitope sequence derived from the cancer protein as a peptide:MHC complex compared to the APCs of a subject administered a full-length cancer protein.
  • the T cells of the subject administered the multiepitopic polypeptide or a recombinant nucleic acid encoding the multiepitopic polypeptide exhibit increased expansion compared to T cells of a subject administered a full-length cancer protein or a recombinant nucleic acid encoding the full-length cancer protein.
  • the cell is an APC.
  • Generating antigen specific (e.g., PRAME-specific) T cells by controlled ex vivo induction or expansion of T cells can provide highly specific and beneficial T cell and T-cell receptors (TCRs).
  • TCRs T cell manufacturing methods, TCRs, therapeutic T cell compositions which can be used for treating cancer.
  • the TCRs can be used for validating a whether its cognate epitope sequence is presented by a cell of interest and can be used for validating whether a candidate WSGR Docket No.: 50401-787.601 antigen is suitable for use as a part of a vaccine design.
  • the first step is to expand and induce antigen specific T cells with a favorable phenotype and function.
  • the present disclosure provides compositions and methods for manufacturing of T cells which can be used for antigen specific T cell therapy (e.g., personal or personalized T cell therapies for treating cancer).
  • the T cell compositions provided herein can be personal antigen specific T cell therapies.
  • the process includes on one hand, identification of the cancer-specific antigens, leading to the production of antigenic peptides or mRNAs encoding such antigenic peptides; and on the other hand, preparing activated, antigen specific cells for immunotherapy and identification of functional TCRs that can be used in TCR recognition assays as described herein.
  • the NEO-STIM process can comprise methods of preparing activated, antigen-specific T cells.
  • Provided herein are methods for stimulating T cells.
  • the methods provided herein can be used to stimulate antigen specific T cells.
  • the methods provided herein can be used to induce or activate T cells.
  • the methods provided herein can be used to expand activated T cells.
  • the methods provided herein can be used to induce na ⁇ ve T cells.
  • the methods provided herein can be used to expand antigen specific CD8 + T cells.
  • the methods provided herein can be used to expand antigen specific CD4 + T cells.
  • the APCs and/or T cells are derived from a biological sample which is peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the subject is administered FLT3L prior to obtaining the biological sample for preparing the APCs and/or T cells.
  • the APCs and/or T cells are derived from a biological sample, for example, from healthy human donors.
  • antigen-presenting cells are first loaded with antigenic peptides ex vivo and used to prepare antigen activated T cells.
  • compositions provided herein comprise T cells that are stimulated by APCs, such as APCs pre-loaded with antigen peptides.
  • the compositions can comprise a population of immune cells comprising T cells from a sample (e.g., a biological sample), wherein the T cells comprise APC-stimulated T cells.
  • mRNA encoding one or more antigenic peptides are introduced into APCs for expression of the antigenic peptides. Such APCs are used for stimulating or activating T cells.
  • the biological sample comprises greater than about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% antigen naive cells of the total cell count in the biological sample that is derived from peripheral blood or leukapheresis.
  • a composition comprises a population of immune cells that has been incubated with one or more cytokines, growth factors or ligands, such as a ligand that binds to a cell surface receptor of an APC or a T cell.
  • cytokines, growth factors and ligands include, for example, GM-CSF, IL-4, IL-7, FLT3L, TNF- - -15, PGE1, IL-6, IFN- - -rna40, and poly(I:C).
  • a composition comprises a population of immune cells that has been incubated with one or more APCs or APC preparations.
  • a composition can comprise a population of immune cells that has been incubated with one or more cytokine, growth factor and/or ligand stimulated APCs or cytokine, growth factor and/or ligand stimulated APC preparations.
  • a composition can comprise a population of immune cells that has been incubated with one or more cytokine stimulated APCs or cytokine stimulated APC preparations.
  • a composition can comprise a population of immune cells that have been incubated with one or more growth factor stimulated APCs or growth factor stimulated APC preparations.
  • a composition can comprise a population of immune cells that has been incubated with one or more ligand stimulated APCs or ligand stimulated APC preparations.
  • the APC is an autologous APC, an allogenic APC, or an artificial APC. In some embodiments, the APC is an artificial APC.
  • WSGR Docket No.: 50401-787.601 Immune cells can be characterized by cell surface molecules. In some embodiments the immune cells are preferably selected based on the cell surface markers, for example, from the biological sample, by using antibodies that can bind to the cell surface receptors. In some embodiments some cells are negatively selected to enrich one or more cell types that do not express the cell surface molecule that they are negatively selected for.
  • antigen-presenting cells are prepared from the biological sample by selecting from APCs or precursor cells that can be cultured in presence of antigenic peptides to generate antigen-loaded APCs, which are used for activating T cells. Some of the related cell surface markers for selecting and/or enriching for a set of cells is described below.
  • CD1 cluster of differentiation 1 is a family of glycoproteins expressed on the surface of various human antigen-presenting cells. They are related to the class I MHC molecules, and are involved in the presentation of lipid antigens to T cells.
  • CD11b or Integrin alpha M is one protein subunit that forms heterodimeric integrin alpha-M beta- M 2) molecule, also known as macrophage-1 antigen (Mac-1) or complement receptor 3(CR3). ITGAM is also known as CR3A, and cluster of differentiation M 2 is the common integrin 2 subunit known as M 2 2 subfamily (or leukocyte) integrins. M 2 is expressed on the surface of many leukocytes involved in the innate immune system, including monocytes, granulocytes, macrophages, and natural killer cells.
  • Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. This protein combines with the beta 2 chain (ITGB2) to form a leukocyte- specific integrin referred to as inactivated-C3b (iC3b) receptor 4 (CR4).
  • IGB2 inactivated-C3b
  • iC3b inactivated-C3b receptor 4
  • the alpha X beta 2 complex seems to overlap the properties of the alpha M beta 2 integrin in the adherence of neutrophils and monocytes to stimulated endothelium cells, and in the phagocytosis of complement coated particles.
  • CD11c is a type I transmembrane protein found at high levels on WSGR Docket No.: 50401-787.601 most human dendritic cells, but also on monocytes, macrophages, neutrophils, and some B cells that induces cellular activation and helps trigger neutrophil respiratory burst; expressed in hairy cell leukemias, acute nonlymphocytic leukemias, and some B-cell chronic lymphocytic leukemias.
  • CD14 is a surface antigen that is preferentially expressed on monocytes/macrophages. It cooperates with other proteins to mediate the innate immune response to bacterial lipopolysaccharide. Alternative splicing results in multiple transcript variants encoding the same protein.
  • CD14 also recognizes other pathogen-associated molecular patterns such as lipoteichoic acid.
  • CD25 is expressed by conventional T cells after stimulation, and it has been shown that in human peripheral blood, only the CD4 + CD25 hi T cells are 'suppressors'.
  • the APC comprises a dendritic cell (DC).
  • the APC is derived from a CD14 + monocyte.
  • the APCs can be obtained from skin, spleen, bone marrow, thymus, lymph nodes, peripheral blood, or cord blood.
  • the CD14 + monocyte is from a biological sample from a subject comprising PBMCs.
  • a CD14 + monocyte can be isolated from, enriched from, or purified from a biological sample from a subject comprising PBMCs.
  • the CD14 + monocyte is stimulated with one or more cytokines or growth factors.
  • the one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, TNF- - -6, IL-7, IL-15, IFN- , IFN- -rna40, poly I:C, or a combination thereof.
  • the CD14 + monocyte is from a second biological sample comprising PBMCs.
  • an isolated population of APCs can be enriched or substantially enriched.
  • the isolated population of APCs is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous.
  • the isolated population of APCs is at least 60%, at least 75%, or at least 90% homogeneous.
  • APCs, such as APCs can include, for example, APCs derived in culture from monocytic dendritic precursors as well as WSGR Docket No.: 50401-787.601 endogenously-derived APCs present in tissues such as, for example, peripheral blood, cord blood, skin, spleen, bone marrow, thymus, and lymph nodes.
  • APCs and cell populations substantially enriched for APCs can be isolated by methods also provided by the present disclosure.
  • the methods generally include obtaining a population of cells that includes APC precursors, differentiation of the APC precursors into immature or mature APCs, and can also include the isolation of APCs from the population of differentiated immature or mature APCs.
  • APC precursor cells can be obtained by methods known in the art.
  • APC precursors can be isolated, for example, by density gradient separation, fluorescence activated cell sorting (FACS), immunological cell separation techniques such as panning, complement lysis, rosetting, magnetic cell separation techniques, nylon wool separation, and combinations of such methods.
  • FACS fluorescence activated cell sorting
  • Methods for immuno-selecting APCs include, for example, using antibodies to cell surface markers associated with APC precursors, such as anti-CD34 and/or anti-CD14 antibodies coupled to a substrate.
  • Enriched populations of APC precursors can also be obtained. Methods for obtaining such enriched precursor populations are known in the art. For example, enriched populations of APC precursors can be isolated from a tissue source by selective removal of cells that adhere to a substrate. Using a tissue source such as, e.g., bone marrow or peripheral blood, adherent monocytes can be removed from cell preparations using a commercially-treated plastic substrate (e.g., beads or magnetic beads) to obtain a population enriched for nonadherent APC precursors.
  • tissue source such as, e.g., bone marrow or peripheral blood
  • adherent monocytes can be removed from cell preparations using a commercially-treated plastic substrate (e.g., beads or magnetic beads) to obtain a population enriched for nonad
  • Monocyte APC precursors can also be obtained from a tissue source by using an APC precursor-adhering substrate.
  • peripheral blood leukocytes isolated by, e.g., leukapheresis are contacted with a monocytic APC precursor-adhering substrate having a high surface area to volume ratio and the adherent monocytic APC precursors are separated.
  • the substrate coupled can be a particulate or fibrous substrate having a high surface-to-volume ratio, such as, for example, microbeads, microcarrier beads, pellets, granules, powder, capillary tubes, microvillous membrane, and the like.
  • the particulate or fibrous substrate can be glass, polystyrene, plastic, glass-coated polystyrene microbeads, and the like.
  • the APC precursors can also be cultured in vitro for differentiation and/or expansion. Methods for differentiation/expansion of APC precursors are known in the art. Generally, expansion can be achieved by culturing the precursors in the presence of at least one cytokine that induces APC (e.g., dendritic cell) differentiation/proliferation.
  • APC e.g., dendritic cell
  • these cytokines WSGR Docket No.: 50401-787.601 are granulocyte colony stimulating factor (G-CSF) or granulocyte/macrophage colony stimulating factor (GM-CSF).
  • G-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte/macrophage colony stimulating factor
  • agents can be used to inhibit proliferation and/or maturation of non-APC cell types in the culture, thereby further enriching the population of APC precursors.
  • agents include cytokines such as, e.g., IL-13, IL-4, or IL- 15, and the like.
  • the isolated populations of APC precursors are cultured and differentiated to obtain immature or mature APCs.
  • Suitable tissue culture media include, for example, AIM-V®, RPMI 1640, DMEM, X-VIVO, and the like.
  • the tissue culture media is typically supplemented with amino acids, vitamins, divalent cations, and cytokines to promote differentiation of the precursors toward the APC phenotype.
  • the differentiation-promoting cytokines are GM-CSF and/or IL-4.
  • cultures of APC precursors during expansion, differentiation, and maturation to the APC phenotype can include plasma to promote the development of APCs.
  • a typical plasma concentration is about 5%.
  • APC precursors are isolated by adherence to a substrate
  • plasma can be included in the culture media during the adherence step to promote the CD14 + phenotype early in culture.
  • a typical plasma concentration during adherence is about 1% or more.
  • the monocytic APC precursors can be cultured for any suitable time.
  • suitable culture times for the differentiation of precursors to immature APCs can be about 1 to about 10 days, e.g., about 4 to about 7 days.
  • immature APCs can be monitored by methods known to those skilled in the art, such as by the presence or absence of cell surface markers (e.g., CD11c+, CD83low, CD86 /low, HLA- DR+ ).
  • Immature APCs can also be cultured in appropriate tissue culture medium to maintain the immature APCs in a state for further differentiation or antigen uptake, processing and presentation.
  • immature APCs can be maintained in the presence of GM-CSF and IL-4.
  • APC precursors can be isolated prior to differentiation.
  • the isolated population can be enriched or substantially enriched for APC precursors.
  • APC precursors are isolated with a CD14 specific probe.
  • CD14 expressing cells are detected by FACS using a CD14 specific probe either directly conjugated to a fluorescent molecule (e.g., FITC or PE) or with an unlabeled antibody specific for CD14 and a labeled second antibody specific for the first antibody.
  • CD14+ cells can also be separated from CD14low and CD14 cells by FACS sorting. Gating for CD14high positivity can be determined in reference to CD14 staining on, e.g., PBMC- WSGR Docket No.: 50401-787.601 derived monocytes.
  • the CD14 specific binding agent is, for example, an anti-CD14 antibody (e.g., monoclonal or antigen binding fragments thereof).
  • an anti-CD14 antibody e.g., monoclonal or antigen binding fragments thereof.
  • a number of anti-CD14 antibodies suitable for use in the present disclosure are well known to the skilled artisan and many can be purchased commercially. Differentiation into immature APCs (CD14 negative) can take place following isolation.
  • a CD14 specific probe is coupled to a substrate and the CD14+ cells are isolated by affinity selection. A population of cells that includes CD14 + cells is exposed to the coupled substrate and the CD14 + cells are allowed to specifically adhere.
  • Non- adhering CD14 cells are then washed from the substrate, and the adherent cells are then eluted to obtain an isolated cell population substantially enriched in APC precursors.
  • the CD14 specific probe can be, for example, an anti-CD14 antibody.
  • the substrate can be, for example, commercially available tissue culture plates or beads (e.g., glass or magnetic beads). Methods for affinity isolation of cell populations using substrate-coupled antibodies specific for surface markers are generally known.
  • immature APCs can optionally be exposed to a predetermined antigen. Suitable predetermined antigens can include any antigen for which T-cell modulation is desired.
  • Antigens can include, for example, bacterial cells, viruses, partially purified or purified bacterial, viral, fungal, protozoan, or helminth antigens, recombinant cells expressing an antigen on its surface, and any other antigen. Any of the antigens can also be presented as a peptide or recombinantly produced protein or portion thereof. Following contact with antigen, the cells can be cultured for any suitable time to allow antigen uptake and processing, to expand the population of antigen-specific APCs, and the like. [0661] For example, in some embodiments, the immature APCs can be cultured following antigen uptake to promote maturation of the immature APCs into mature APCs that present antigen in the context of MHC molecules.
  • APC maturation can be performed, for example, by culture in the presence of known maturation factors, such as cytokines (e.g., TNF- - r CD40 ligand), bacterial products (e.g., LPS or BCG), and the like.
  • cytokines e.g., TNF- - r CD40 ligand
  • bacterial products e.g., LPS or BCG
  • the maturation of immature APCs to mature APCs can be monitored by methods known in the art, such as, for example by measuring the presence or absence of cell surface markers (e.g., upregulation of CD83, CD86, and MHC molecules) or testing for the expression of mature APC specific mRNA or proteins using, for example, an oligonucleotide array.
  • cell surface markers e.g., upregulation of CD83, CD86, and MHC molecules
  • the immature APCs can be cultured in an appropriate tissue culture medium to expand the cell population and/or maintain the immature APCs in state for further WSGR Docket No.: 50401-787.601 differentiation or antigen uptake.
  • immature APCs can be maintained and/or expanded in the presence of GM-CSF and IL-4.
  • the immature APCs can be cultured in the presence of anti-inflammatory molecules such as, for example, anti-inflammatory cytokines (e.g., IL-10 and TGF- [0663]
  • the isolated population of APCs is enriched for mature APCs.
  • the isolated population of mature APCs can be obtained by culturing a differentiated population of immature APCs in the presence of maturation factors as described above (e.g., bacterial products, and/or proinflammatory cytokines), thereby inducing maturation. Immature APCs can be isolated by removing CD14+ cells. [0664] According to yet another aspect of the present disclosure, APCs can be preserved, e.g., by cryopreservation either before exposure or following exposure to a suitable antigen.
  • maturation factors as described above (e.g., bacterial products, and/or proinflammatory cytokines)
  • Cryopreservation agents which can be used include dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i- erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, and inorganic salts.
  • a controlled slow cooling rate can be critical. Different cryoprotective agents and different cell types may have different optimal cooling rates.
  • the heat of fusion phase where water turns to ice typically can be minimal.
  • the cooling procedure can be carried out by use of, e.g., a programmable freezing device or a methanol bath procedure.
  • Programmable freezing apparatuses allow determination of optimal cooling rates and facilitate standard reproducible cooling.
  • Programmable controlled- rate freezers such as Cryomed or Planar permit tuning of the freezing regimen to the desired cooling rate curve.
  • APCs can be rapidly transferred to a long-term cryogenic storage vessel.
  • samples can be cryogenically stored in liquid nitrogen cryopreservation, and long term storage of hematopoietic stem cells, particularly from bone marrow or peripheral blood, is largely applicable to the APCs of the present disclosure.
  • the expanded population of cells comprises from at least 5x105 to at least 5x10 11 total cells. In some embodiments, the expanded population of cells comprises at least 1x10 6 total cells. In some embodiments, the expanded population of cells comprises at least 5x10 6 total cells. In some embodiments, the expanded population of cells comprises at least 1x10 7 total cells. In some embodiments, the expanded population of cells comprises at least 5x10 7 total cells. In some embodiments, the expanded population of cells comprises at least 1x10 8 total cells. In some embodiments, the expanded population of cells comprises at least 5x10 8 total cells. In some embodiments, the expanded population of cells comprises at least 1x10 9 total cells. In some embodiments, the expanded population of cells comprises at least 5x10 9 total cells.
  • the expanded population of cells comprises at least 1x10 10 total cells. In some embodiments, the expanded population of cells comprises at least 5x10 10 total cells. In some embodiments, the expanded population of cells comprises at least 1x10 11 total cells. In some embodiments, the expanded population of cells comprises at least 5x1011 total cells. In some embodiments, the expanded population of cells comprises from 1x10 8 to 1x10 11 total cells. In some embodiments, the expanded population of cells comprises from 0,75x10 8 to 1.25x10 10 total cells. In some embodiments the expanded population of cells comprises from 5x10 8 to 1x10 9 total cells. In some embodiments, the expanded population of cells comprises 5x10 8 to 1x10 9 total cells.
  • the expanded population of cells comprises 5x10 8 to 2x10 9 total cells.
  • the subject is pretreated with FLT3L at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week before isolation of PBMC or leukapheresis.
  • the subject is pretreated with FLT3L at least about 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks before isolation of PBMC or leukapheresis.
  • the cell population is enriched for CD11c+ cells.
  • the antigen loaded APC comprises dendritic cells (DCs). In some embodiments, the antigen loaded APC comprises plasmacytoid dendritic cells (pDCs). In some embodiments, the antigen loaded APC comprises CD1c+ DCs. In some embodiments, the antigen loaded APC comprises CD141+ DCs. In some embodiments, the cell population comprises macrophages. In some embodiments, the method further comprises reducing or depleting CD19+ cells from the cell population for activating or enriching antigen activated T cells. In some embodiments, the method further comprises reducing or depleting both CD11b+ and CD19+ cells from the cell population for activating or enriching antigen activated T cells.
  • the method further comprises reducing or depletingCD14+ cells from the cell population for preparing and enriching antigen activated T cells. In some embodiments, the method further comprises reducing or depleting CD25+ cells from the cell population for preparing and enriching antigen activated T cells. In some embodiments, the method further comprises reducing or depleting one or more of CD19+, CD14+, CD25+ or CD11b+ cells from the cell population for activating or enriching antigen activated T cells. In some embodiments, depleting comprises depleting CD14+ cells directly from a washed peripheral blood mononuclear cell (PBMC) sample from a human subject.
  • PBMC peripheral blood mononuclear cell
  • depleting comprises depleting CD25+ cells directly from a washed peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, depleting comprises depleting CD14+ cells and CD25+ cells directly from a washed peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, depleting comprises depleting CD14+ cells directly from a cryopreserved peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, depleting comprises depleting CD25+ cells directly from a cryopreserved peripheral blood mononuclear cell (PBMC) sample from a human subject.
  • PBMC washed peripheral blood mononuclear cell
  • depleting comprises depleting CD14+ cells and CD25+ cells directly from a cryopreserved peripheral blood mononuclear cell (PBMC) sample from a human subject.
  • PBMC peripheral blood mononuclear cell
  • the depleted population of cells is incubated for a first time period in the presence of FLT3L and a polypeptide comprising at least two different epitope sequences.
  • the depleted population of cells is incubated for a first time period in the presence of FLT3L and a polynucleic acid encoding a polypeptide comprising at least two different epitope sequences.
  • each of the at least two different epitope sequences is from the same protein encoded by the genome of a cancer cell.
  • each of the at least two different epitope sequences is from a different protein encoded by the genome of a cancer cell.
  • WSGR Docket No.: 50401-787.601 the same protein encoded by the genome of a cancer cell is PRAME.
  • a first epitope sequence of the at least two different epitope sequences are connected to a second epitope sequence of the at least 2 different epitope sequences via a linker sequence.
  • the at least two different epitope sequences are expressed as a single polypeptide chain.
  • the polypeptide comprises at least 2 to at least 15 or more different epitope sequences from two or more different proteins encoded by the genome of a cancer cell.
  • the method is performed in less than 30 days. In some embodiments, the method is performed in less than 28 days. In some embodiments, the method is performed in less than 25 days. In some embodiments, the method is performed in less than 20 days, in some embodiments. the method is performed in less than 15 days. In some embodiments, the method is performed in less than 10 days. In some embodiments, the method is performed in less than 5 days.
  • CD8+ antigen-specific T cells are expanded. In some embodiments CD4+ antigen-specific T cells are expanded.
  • the fraction of CD8+ antigen-specific T cells of the total number of T cells in the expanded population of cells is at least two-fold higher than the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the CD14 and/or CD25 depleted population of immune cell.
  • the fraction of CD8+ antigen-specific T cells of the total number of T cells in the expanded population of cells is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9- fold, at least 10-fold higher than the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the CD14 and/or CD25 depleted population of immune cell.
  • the fraction of CD4+ antigen-specific T cells of the total number of T cells in the expanded population of cells is at least two-fold higher than the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the CD14 and/or CD25 depleted population of immune cell.
  • the fraction of CD4+ antigen-specific T WSGR Docket No.: 50401-787.601 cells of the total number of T cells in the expanded population of cells is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold higher than the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the CD14 and/or CD25 depleted population of immune cell.
  • At least 10% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells. In some embodiments, at least 20% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells. In some embodiments, at least 30% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells. In some embodiments, at least 40% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells.
  • At least 50% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells. In some embodiments, at least 60% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells. In some embodiments, at least 70% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells. In some embodiments, at least 80% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells.
  • At least 90% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from na ⁇ ve CD8+ T cells.
  • at least 0.1% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells.
  • at least 0.5% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells.
  • at least 1% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells.
  • At least 5% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ WSGR Docket No.: 50401-787.601 T cells. In some embodiments, at least 10% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells. In some embodiments, at least 20% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells.
  • At least 30% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells. In some embodiments, at least 40% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells. In some embodiments, at least 50% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells. In some embodiments, at least 60% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells.
  • depleting CD14+ cells and/or CD25+ cells from the population of immune cells comprising a first population of APCs and T cells comprises contacting the population of immune cells comprising a first population of APCs and T cells with a CD14 binding agent and/or a CD25 binding agent.
  • the stimulant for activating the cells comprises FL3TL.
  • the agent promoting cell growth and maintenance ex vivo comprises a growth factor, a cytokine, an amino acid, a supplement or a combination thereof.
  • the antigen loaded APCs can stimulate T cells for 2, 3, 4, 5, 6, or 7 days.
  • the antigenic peptides used to prepare antigen loaded APCs are long peptides comprising at least 20 amino acids, or at least 30 amino acids or at least 40 amino acids or at least 50 amino acids, or any number of amino acids in between.
  • the antigenic peptides used to prepare antigen loaded APCs comprise the amino acids flanking on either side of the epitope that facilitate endogenous processing of the antigenic peptide for increased rate of presentation to a T cell.
  • a longer immunogenic peptide can be designed in several ways.
  • the coding sequence for each antigenic peptide is 24-120 nucleotides long.
  • the mRNA is 50-10,000 nucleotides long. In some embodiments, the mRNA is 100- 10,000 nucleotides long. In some embodiments, the mRNA is 200-10,000 nucleotides long. In some embodiments, the mRNA is 50-5,000 nucleotides long. In some embodiments, the mRNA is 100-5,000 nucleotides long. In some embodiments, the mRNA is 100-1,000 nucleotides long. In some embodiments, the mRNA is 300-800 nucleotides long.

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Abstract

La présente divulgation concerne une thérapie à base de compositions de vaccin comprenant des polypeptides multiépitopiques, des acides nucléiques recombinants codant pour des polypeptides multiépitopiques et leurs méthodes d'utilisation. La présente divulgation concerne également des récepteurs de lymphocytes T (TCR) qui peuvent reconnaître un ou plusieurs épitopes des polypeptides multiépitopiques. La thérapie selon la présente divulgation peut comprendre (i) un TCR, un acide nucléique recombinant codant pour le TCR, ou des cellules comprenant le TCR ou l'acide nucléique recombinant codant pour le TCR, et (ii) une composition de vaccin divulguée. Les méthodes et les compositions de la divulgation peuvent être utilisées pour le traitement du cancer.
PCT/US2025/016645 2024-02-21 2025-02-20 Compositions de vaccin et méthodes d'utilisation Pending WO2025179050A2 (fr)

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