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WO2025227066A2 - Compositions de vaccins ras et méthodes d'utilisation - Google Patents

Compositions de vaccins ras et méthodes d'utilisation

Info

Publication number
WO2025227066A2
WO2025227066A2 PCT/US2025/026433 US2025026433W WO2025227066A2 WO 2025227066 A2 WO2025227066 A2 WO 2025227066A2 US 2025026433 W US2025026433 W US 2025026433W WO 2025227066 A2 WO2025227066 A2 WO 2025227066A2
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
hla
seq
ras
nucleic acid
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/US2025/026433
Other languages
English (en)
Other versions
WO2025227066A3 (fr
Inventor
Kendra FOLEY
Vikram JUNEJA
Shirisha MEDA
Christina M. ARIETA
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 WO2025227066A2 publication Critical patent/WO2025227066A2/fr
Publication of WO2025227066A3 publication Critical patent/WO2025227066A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61K39/001154—Enzymes
    • A61K39/001164—GTPases, e.g. Ras or Rho
    • 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
    • A61K2039/70—Multivalent vaccine

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 RAS polypeptide and comprises a first RAS amino acid sequence comprising a first RAS epitope sequence and a second RAS amino acid sequence comprising a second RAS epitope sequence, wherein the first RAS epitope sequence and the second RAS epitope sequence are different, and wherein presentation of the first and/or second RAS epitope sequence as a peptide:MHC complex by antigen presenting cells (APCs) of the subject administered
  • APCs antigen presenting cells
  • Also provided herein is 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 RAS polypeptide and comprises a first RAS amino acid sequence comprising a first RAS epitope sequence and a second RAS amino acid sequence comprising a second RAS epitope sequence, wherein the first RAS epitope sequence and the second RAS epitope sequence are different, and wherein the first RAS amino acid sequence and the second RAS amino acid sequence is separated by a linker.
  • the full-length RAS polypeptide is a full-length KRAS, NRAS or HRAS polypeptide, wherein the first RAS amino acid sequence is a first KRAS, NRAS or HRAS WSGR Docket No.50401-795.601 amino acid sequence, and wherein the second RAS amino acid sequence is a second KRAS, NRAS or HRAS amino acid sequence.
  • the method of any one of the preceding embodiments further comprises administering the subject a T-cell receptor (TCR), a recombinant nucleic acid encoding the TCR, or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR.
  • TCR T-cell receptor
  • the first RAS epitope sequence and the second RAS epitope sequence is separated by a linker.
  • the first RAS amino acid sequence is the first epitope sequence and/or the second RAS amino acid sequence is the second epitope sequence.
  • the first RAS amino acid sequence comprises a first RAS mutation
  • the second RAS amino acid sequence comprises a second RAS mutation different from the first RAS mutation.
  • the first RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the second RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the first RAS amino acid sequence is processed into one or more epitopes comprising the first RAS mutation within a cell.
  • the first RAS mutation is G12V, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 58-62 and 65-72.
  • the first RAS mutation is G12D, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 28, 75-79.
  • the first RAS mutation is G12C, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096.
  • the first RAS epitope sequence and/or the second RAS epitope sequence consists of from 7 to 12 consecutive amino acids from the full-length RAS polypeptide.
  • the first RAS epitope sequence and/or the second RAS epitope sequence consists of from 13 to 25 consecutive amino acids from the full-length RAS polypeptide.
  • the first RAS amino acid sequence and the second RAS amino acid sequence is separated by a linker.
  • the first RAS amino acid sequence comprises the first RAS epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the first RAS epitope sequence from the full-length RAS polypeptide.
  • the second RAS amino acid sequence comprises the second RAS epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the second RAS epitope sequence from the full-length RAS polypeptide.
  • the multiepitopic polypeptide does not comprise more than 12 or more consecutive amino acids from the full-length RAS polypeptide, or the multiepitopic polypeptide does not comprise more than 25 or more consecutive amino acids from the full-length RAS polypeptide.
  • the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the first or the second RAS epitope sequence, and (ii) a human MHC encoded by an HLA allele.
  • the multiepitopic polypeptide is a RAS polypeptide comprising the first RAS epitope sequence and the second RAS epitope sequence.
  • the linker is a cleavable linker.
  • a method of treating a subject with a disease or condition comprising administering to the subject (a) a RAS polypeptide, (b) a recombinant nucleic acid encoding the RAS polypeptide, or (c) a cell comprising the RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide, wherein the RAS polypeptide comprises a RAS epitope sequence, wherein the subject has been previously administered a T-cell receptor (TCR), a recombinant nucleic acid encoding the TCR, or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the RAS epitope sequence, and (ii) a T-cell receptor (TCR), a recombinant
  • 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), a recombinant nucleic acid encoding the TCR, or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) a RAS epitope sequence, and (ii) a human MHC encoded by an HLA allele, wherein the subject has been previously administered (a) a RAS polypeptide, (b) a recombinant nucleic acid encoding the RAS polypeptide, or (c) a cell comprising the RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide, wherein the RAS polypeptide comprises the RAS epitope sequence.
  • TCR T-cell receptor
  • Also provided herein is a method of treating a subject with a disease or condition comprising, administering to the subject (i) a RAS polypeptide, (ii) a recombinant nucleic acid encoding the RAS polypeptide, or (iii) a cell comprising the RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide, wherein the RAS polypeptide comprises a RAS epitope sequence; and administering to the subject a TCR, a recombinant nucleic acid encoding the TCR, or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR, wherein the TCR recognizes WSGR Docket No.50401-795.601 and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the RAS peptide sequence, and (ii) a human MHC encoded by an HLA allele.
  • administering in (a) is performed concurrently with administering in (b). [0029] In some embodiments, administering in (a) is prior to administering in (b). [0030] In some embodiments, administering in (a) is subsequent to administering in (b). [0031] In some embodiments, the RAS polypeptide or the recombinant nucleic acid encoding the RAS 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 RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide.
  • the cell is an immune cell.
  • 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 of any one of the preceding embodiments further comprises administering two or more different TCRs or recombinant nucleic acids encoding the two or more different TCRs, or cells comprising the two or more different TCRs or the 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 having a G12V mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA- C*01:02, HLA-C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-A*03:02, and HLA-A*03:05
  • the second TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12D WSGR Docket No.50401-795.601 mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA- B*07:02, HLA-C*08:02, HLA-C*03:04, HLA
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12V mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA- C*01:02, HLA-C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-A*03:02, and HLA-A*03:05
  • the second TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12C mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA- DRB1*11:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12D mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*08:02, HLA-C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA- A*11:01, and HLA-A*68:01.
  • the second TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12C mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA- C*03:03.
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 58-62 and 65-72 and an MHC encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA- A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA- DRB1*07:01, HLA-A*03:02, and HLA-A*03:05 and the second TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 28, 75- 79 and an MHC encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*08:02, H
  • the RAS polypeptide does not comprise a full-length RAS protein sequence.
  • the RAS epitope sequence comprises a sequence of SEQ ID NOs: 58 or 59.
  • the HLA allele is selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*03:02, HLA-A*03:05, HLA-B*40:01, and HLA-A*68:01.
  • the RAS epitope sequence comprises a sequence of SEQ ID NO: 60.
  • the HLA allele is HLA-C*01:02.
  • the RAS epitope sequence comprises a sequence of SEQ ID NOs: 61 or 62.
  • the HLA allele is HLA-C*03:03, or HLA-C*03:04.
  • the RAS epitope sequence comprises a sequence of SEQ ID NOs: 58 or 59.
  • the MHC allele is HLA-A*11:01.
  • the RAS epitope sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 65-71.
  • the MHC allele is HLA-DRB1*07:01.
  • the RAS epitope sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 58 or 72.
  • the MHC allele is selected from the group consisting of HLA- A*03:01, HLA-A*03:02, and HLA-A*03:05.
  • the RAS epitope sequence comprises a sequence of SEQ ID NO: 75.
  • the MHC allele is selected from the group consisting of HLA- B*07:02, HLA-C*08:02, HLA-C*03:04, and HLA-C*05:01.
  • the RAS epitope sequence comprises a sequence of SEQ ID NO: 76.
  • the MHC allele is selected from the group consisting of HLA-A*-3:01, HLA-A*11:01, and HLA-A*68:01.
  • the RAS epitope sequence comprises a sequence of SEQ ID NO: 77.
  • the MHC allele is HLA-A*11:01.
  • the RAS epitope sequence comprises a sequence of SEQ ID NOs: 78 or 79.
  • the MHC allele is HLA-C*08:02.
  • the RAS epitope sequence comprises a sequence of SEQ ID NO: 29.
  • the MHC allele is HLA-DRB1*11:01.
  • the RAS epitope sequence comprises a sequence of SEQ ID NO: 81 or 1096.
  • the MHC allele is selected from the group consisting of HLA-A*3:01, HLA-A*11:01, and HLA-A*68:01.
  • the RAS epitope sequence comprises a sequence of SEQ ID NO: 82.
  • the MHC allele is HLA-C*03:03.
  • the RAS polypeptide is a multiepitopic polypeptide, and wherein the multiepitopic polypeptide does not comprise a full-length RAS polypeptide and comprises two or more different RAS epitope sequences.
  • the multiepitopic polypeptide comprises at least 3, 4, 5, or more different RAS epitope sequences.
  • the two or more different RAS epitope sequences are separated by linker sequences.
  • antigen presenting cells (APCs) of the subject administered the RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide comprising the multiepitopic polypeptide present more of a RAS epitope sequence as a peptide:MHC complex compared to the APCs of a subject administered a full-length RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS 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 RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • the multiepitopic polypeptide comprises a first RAS amino acid sequence comprising a first RAS epitope sequence and a second RAS amino acid sequence comprising a second RAS epitope sequence, and wherein the first RAS epitope sequence and the second RAS epitope sequence are different or wherein the first RAS amino acid sequence and the second RAS amino acid sequence comprise different RAS mutations.
  • the first RAS epitope sequence and the second RAS epitope sequence is separated by a linker.
  • WSGR Docket No.50401-795.601 [0078]
  • 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 RAS amino acid sequence comprises a first RAS mutation
  • the second RAS amino acid sequence comprises a second RAS mutation different from the first RAS mutation.
  • the first RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the second RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the first RAS amino acid sequence is processed into one or more epitopes comprising the first RAS mutation within a cell.
  • the first RAS mutation is G12V, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 27, 58-62 and 65-72.
  • the first RAS mutation is G12D, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 28, 75-79.
  • the first RAS mutation is G12C, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096.
  • the first RAS epitope sequence and/or the second RAS epitope sequence consists of from 7 to 12 consecutive amino acids from the full-length RAS polypeptide.
  • the first RAS epitope sequence and/or the second RAS epitope sequence consists of from 13 to 25 consecutive amino acids from the full-length RAS polypeptide.
  • the first RAS amino acid sequence and the second RAS amino acid sequence is separated by a linker.
  • the first amino acid sequence comprises the first RAS epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the first RAS epitope sequence from the full-length RAS polypeptide.
  • the second amino acid sequence comprises the second RAS epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the second RAS epitope sequence from the full-length RAS polypeptide.
  • the multiepitopic polypeptide does not comprise more than 12 or more consecutive amino acids from the full-length RAS polypeptide, or the multiepitopic polypeptide does not comprise more than 25 or more consecutive amino acids from the full-length RAS polypeptide.
  • WSGR Docket No.50401-795.601 the first RAS epitope sequence and the second RAS 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 RAS 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 amino acid sequence is operably linked to the second amino acid sequence via a linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the multiepitopic polypeptide comprises a first amino acid sequence comprising a first RAS epitope sequence, operably linked to a second amino acid sequence comprising a second RAS epitope sequence, operably linked to a third amino acid sequence comprising a third RAS epitope sequence.
  • the first RAS epitope sequence, the second RAS epitope sequence, and/or the third RAS epitope sequence comprises a RAS mutation selected from the group consisting of G12V, G12D, and G12C, and wherein the first RAS epitope sequence, the second RAS epitope sequence, and the third RAS epitope sequence comprise different RAS mutations.
  • the first RAS epitope sequence comprises a G12V mutation.
  • the second RAS epitope sequence comprises a G12D mutation.
  • the third RAS epitope sequence comprises a G12C mutation.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA- A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA- DRB1*07:01, HLA-A*03:02, and HLA-A*03:05, is presented by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-
  • the subject does not express an HLA allele that recognizes each RAS epitope sequence of the multiepitopic polypeptide.
  • the subject only expresses HLA alleles that recognize a subset of RAS epitope sequences of the multiepitopic polypeptide.
  • the RAS polypeptide comprises one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises one or more copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises two, three, four, or five copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. [0108] In some embodiments, the RAS polypeptide comprises two, three, four, or five copies of each sequence of SEQ ID NOs: 27-29. WSGR Docket No.50401-795.601 [0109] In some embodiments, the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 27, operably linked to a sequence of SEQ ID NO: 29, and operably linked to a sequence of SEQ ID NO: 28.
  • the sequence of SEQ ID NO: 27 is operably linked to the sequence of SEQ ID NOs: 29 via a linker, and the sequence of SEQ ID NO: 29 is operably linked to the sequence of SEQ ID NO: 28 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NOs: 29, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 27.
  • the sequence of SEQ ID NO: 29 is operably linked to the sequence of SEQ ID NO: 28 via a linker, and the sequence of SEQ ID NO: 28 is operably linked to the sequence of SEQ ID NO: 27 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 28, operably linked to a sequence of SEQ ID NO: 27, and operably linked to a sequence of SEQ ID NO: 29.
  • the sequence of SEQ ID NO: 28 is operably linked to the sequence of SEQ ID NO: 27 via a linker
  • the sequence of SEQ ID NO: 27 is operably linked to a sequence of SEQ ID NO: 29 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 27, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 29.
  • the sequence of SEQ ID NO: 27 is operably linked to the sequence of SEQ ID NO: 28 via a linker, and the sequence of SEQ ID NO: 28 is operably linked to a sequence of SEQ ID NO: 29 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 28, operably linked to a sequence of SEQ ID NO: 29, and operably linked to a sequence of SEQ ID NO: 27.
  • the sequence of SEQ ID NO: 28 is operably linked to the sequence of SEQ ID NO: 29 via a linker
  • the sequence of SEQ ID NO: 29 is operably linked to a sequence of SEQ ID NO: 27 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 29, operably linked to a sequence of SEQ ID NO: 27, and operably linked to a sequence of SEQ ID NO: 28.
  • the sequence of SEQ ID NO: 29 is operably linked to the sequence of SEQ ID NO: 27 via a linker
  • the sequence of SEQ ID NO: 27 is operably linked to a sequence of SEQ ID NO: 28 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises at least two copies of a multiepitopic polypeptide, and wherein each copy comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 29, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises three copies of the multiepitopic polypeptide.
  • the RAS polypeptide comprises five copies of the multiepitopic polypeptide.
  • the RAS polypeptide further comprises a secretory domain (Sec) sequence at the N terminus of the multiepitopic polypeptide.
  • the Sec sequence comprises a sequence of SEQ ID NO: 32.
  • the Sec sequence is operably linked to the multiepitopic polypeptide via a linker.
  • the linker comprises a sequence of SEQ ID NO: 30 or 112.
  • the RAS polypeptide further comprises an MHC class I trafficking domain (MITD) sequence at the C terminus of the multiepitopic polypeptide.
  • the MITD sequence comprises a sequence of SEQ ID NO: 33.
  • the multiepitopic polypeptide is operably linked to the MITD sequence via a linker.
  • the linker comprises a sequence of SEQ ID NO: 30 or 112.
  • WSGR Docket No.50401-795.601 [0144]
  • the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 25.
  • the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 26.
  • the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 113.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. [0148] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14- 23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two, three, four, or five copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two, three, four, or five copies of each sequence of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 14- 16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109.
  • the sequence selected from the group consisting of SEQ ID NOs: 14- 16, 84, 92, 96, 100, 104, and 108 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 via a linker sequence
  • the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109 via a linker sequence.
  • the linker sequence encodes a cleavable linker.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 5, 6, 8-13.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 20- 23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence selected from the group consisting of WSGR Docket No.50401-795.601 SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108.
  • the sequence selected from the group consisting of SEQ ID NOs: 20- 23, 88, 94, 98, 102, 106, and 110 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109 via a linker sequence
  • the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108 via a linker sequence.
  • the linker sequence encodes a cleavable linker.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 5, 6, 8-13.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 17- 19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110.
  • the sequence selected from the group consisting of SEQ ID NOs: 17- 19, 86, 93, 97, 101, 105, and 109 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108 via a linker sequence
  • the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108 is operably linked to a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 via a linker sequence.
  • the linker sequence is a cleavable linker.
  • the linker sequence comprises a sequence of selected from the group consisting of SEQ ID NOs: 5, 6, 8-13.
  • the recombinant nucleic acid encoding the RAS 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: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 17- 19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of the string of sequences. [0165] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of the string of sequences. WSGR Docket No.50401-795.601 [0166] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide further comprises a sequence encoding a secretory (Sec) sequence at the 5’end of the string of sequences. [0167] In some embodiments, the sequence encoding the Sec sequence comprises a sequence of SEQ ID NO: 3.
  • 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: 4, 7, 83, and 89.
  • the recombinant nucleic acid encoding the RAS polypeptide further comprises a sequence encoding an MITD sequence at the 3’ end of the string of sequences.
  • the sequence encoding the MITD domain comprises a sequence of SEQ ID NO: 24 or 90.
  • the string of sequences is operably linked to the sequence encoding the MITD sequence via a linker sequence.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 7, 83, and 89.
  • the recombinant nucleic acid is codon-optimized.
  • the recombinant nucleic acid is an RNA.
  • the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 91, 95, 99, 103, 107, 111.
  • the subject expresses more of the RAS epitope when administered with the codon-optimized recombinant nucleic acid than administered with wild-type recombinant nucleic acid.
  • the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence set forth in SEQ ID NO: 57.
  • the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 53.
  • the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 55 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 56.
  • CDR1 complementarity determining region 1
  • CDR2 complementarity determining region 2
  • the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 46, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 47, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 48.
  • the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44.
  • the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 51, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 49 or SEQ ID NO: 51, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 42, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 40 or SEQ ID NO: 42.
  • the TCR is selected from the TCRs presented in Tables 8A-8D. [0185] In some embodiments, binding of the TCR to the peptide:MHC complex results in production of a cytokine by the immune cell. [0186] In some embodiments, the cytokine is IFN- ⁇ , TNF- ⁇ , IL-2, IL-18, or any combination thereof.
  • a recombinant nucleic acid encoding a RAS polypeptide comprising a multiepitopic polypeptide, wherein the multiepitopic polypeptide does not comprise a full-length RAS polypeptide and comprises a first RAS amino acid sequence comprising a first RAS epitope sequence and a second RAS amino acid sequence comprising a second RAS epitope sequence, wherein the first RAS epitope sequence and the second RAS epitope sequence are different, and wherein the first RAS amino acid sequence and the second RAS amino acid sequence are linked via a linker.
  • presentation of the first and/or second RAS epitope sequence as a peptide:MHC complex by antigen presenting cells (APCs) of the subject administered the RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide is higher than the presentation of the first and/or second RAS epitope sequence as the peptide:MHC complex by the APCs of a subject administered the full-length RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • the first RAS amino acid sequence is the first epitope sequence and/or the second RAS amino acid sequence is the second epitope sequence.
  • the first RAS amino acid sequence consists of the first epitope sequence, and/or the second RAS amino acid sequence consists of the second epitope sequence.
  • the first RAS amino acid sequence comprises a first RAS mutation
  • the second RAS amino acid sequence comprises a second RAS mutation different from the first RAS mutation.
  • WSGR Docket No.50401-795.601 [0192]
  • the first RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the second RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the first RAS amino acid sequence is processed into one or more epitopes comprising the first RAS mutation within a cell.
  • the first RAS mutation is G12V, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 27, 58-62 and 65-72.
  • the first RAS mutation is G12D, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 28, 75-79.
  • the first RAS mutation is G12C, and wherein the one or more epitopes are selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096.
  • the first RAS epitope sequence and/or the second RAS epitope sequence consists of from 7 to 12 consecutive amino acids from the full-length RAS polypeptide.
  • the first RAS epitope sequence and/or the second RAS epitope sequence consists of from 13 to 25 consecutive amino acids from the full-length RAS polypeptide.
  • the first RAS amino acid sequence and the second RAS amino acid sequence is separated by a linker.
  • the first RAS amino acid sequence comprises the first RAS epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the first RAS epitope sequence from the full-length RAS polypeptide.
  • the second RAS amino acid sequence comprises the second RAS epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the second RAS epitope sequence from the full-length RAS polypeptide.
  • the multiepitopic polypeptide does not comprise more than 12 or more consecutive amino acids from the full-length RAS polypeptide, or the multiepitopic polypeptide does not comprise more than 25 or more consecutive amino acids from the full-length RAS polypeptide.
  • the RAS polypeptide further comprises a Secretory (Sec) sequence at the N terminus of the multiepitopic polypeptide.
  • the Sec sequence comprises a sequence of SEQ ID NO: 32.
  • the Sec sequence is operably linked to the multiepitopic polypeptide via a linker.
  • the linker comprises a sequence of SEQ ID NO: 30 or 112.
  • the RAS polypeptide further comprises an MHC class I trafficking signal (MITD) sequence at the C terminus of the multiepitopic polypeptide.
  • MITD MHC class I trafficking signal
  • the MITD sequence comprises a sequence of SEQ ID NO: 33.
  • the multiepitopic polypeptide is operably linked to the MITD sequence via a linker.
  • the linker comprises a sequence of SEQ ID NO: 30 or 112.
  • the multiepitopic polypeptide comprises at least 3, 4, 5, or more different RAS epitope sequences.
  • the first RAS epitope sequence and the second RAS 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.
  • the first RAS 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 linker comprises a sequence of SEQ ID NO: 31.
  • the multiepitopic polypeptide comprises a first amino acid sequence comprising a first RAS epitope sequence, operably linked to a second amino acid sequence comprising a second RAS epitope sequence, operably linked to a third amino acid sequence comprising a third RAS epitope sequence.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA- A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA- DRB1*07:01, HLA-A*03:02, and HLA-A*03:05, is presented by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-
  • the multiepitopic polypeptide comprises the formula NT seq – ([Antigen1]-cleavable linker1-[Antigen2])x-CT seq, where x is an integer from 1 to 10.
  • the multiepitopic polypeptide comprises the formula NT seq – ([Antigen1]-cleavable linker1-[Antigen2]-cleavable linker2-[Antigen3])x-CT seq, where x is an integer from 1 to 10.
  • 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 Antigen1, Antigen2, or Antigen3 is comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-29.
  • the cleavable linker1 or cleavable linker2 is selected from the group consisting of SEQ ID NOs: 30 and 31.
  • the N-terminal linker sequence or the C-terminal linker sequence is selected from the group consisting of SEQ ID NOs: 30 and 31.
  • the RAS polypeptide comprises one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. [0226] In some embodiments, the RAS polypeptide comprises one or more copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. [0227] In some embodiments, the RAS polypeptide comprises two, three, four, or five copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. [0228] In some embodiments, the RAS polypeptide comprises two, three, four, or five copies of each sequence of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 27, operably linked to a sequence of SEQ ID NO: 29, and operably linked to a sequence of SEQ ID NO: 28.
  • the sequence of SEQ ID NO: 27 is operably linked to the sequence of SEQ ID NO: 29 via a linker
  • the sequence of SEQ ID NO: 29 is operably linked to the sequence of SEQ ID NO: 28 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 29, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 27.
  • the sequence of SEQ ID NO: 29 is operably linked to the sequence of SEQ ID NO: 28 via a linker
  • the sequence of SEQ ID NO: 28 is operably linked to the sequence of SEQ ID NO: 27 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 28, operably linked to a sequence of SEQ ID NO: 27, and operably linked to a sequence of SEQ ID NO: 29.
  • the sequence of SEQ ID NO: 28 is operably linked to the sequence of SEQ ID NO: 27 via a linker
  • the sequence of SEQ ID NO: 27 is operably linked to a sequence of SEQ ID NO: 29 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the RAS polypeptide comprises at least two copies of a multiepitopic polypeptide, and wherein each copy comprises, from N terminus to C terminus, a sequence of SEQ ID WSGR Docket No.50401-795.601 NO: 29, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises three copies of the multiepitopic polypeptide.
  • the RAS polypeptide comprises five copies of the multiepitopic polypeptide.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. [0245] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of one or more sequences selected from the group consisting of SEQ ID NO:s 14- 23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two, three, four, or five copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two, three, four, or five copies of each sequence of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 14- 16, 84, 92, 96, 100, 104, and 108 , operably linked to a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109.
  • sequence selected from the group consisting of SEQ ID NOs: 14- 16, 84, 92, 96, 100, 104, and 108 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 via a linker sequence
  • sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109 via a linker sequence.
  • the linker sequence encodes a cleavable linker.
  • the linker sequence comprises a sequence of selected from the group consisting of SEQ ID NOs: 5, 6, 8-13.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 20- 23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence selected from the group consisting of WSGR Docket No.50401-795.601 SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108.
  • the sequence selected from the group consisting of SEQ ID NOs: 20- 23, 88, 94, 98, 102, 106, and 110 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109 via a linker sequence
  • the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108 via a linker sequence.
  • the linker sequence encodes a cleavable linker.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 5, 6, 8-13.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 17- 19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110.
  • the sequence selected from the group consisting of SEQ ID NOs: 17- 19, 86, 93, 97, 101, 105, and 109 is operably linked to the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108 via a linker sequence
  • the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108 is operably linked to a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 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: 5, 6, 8-13.
  • the recombinant nucleic acid encoding the RAS 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: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 17- 19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of the string of sequences. [0262] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of the string of sequences. WSGR Docket No.50401-795.601 [0263] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide further comprises a sequence encoding a Sec sequence at the 5’ end of the string of sequences. [0264] In some embodiments, the sequence encoding the Sec sequence comprises a sequence of SEQ ID NO: 3.
  • 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: 4, 7, 83, and 89.
  • the recombinant nucleic acid encoding the RAS polypeptide further comprises a sequence encoding an MITD sequence at the 3’ end of the string of sequences.
  • the sequence encoding the MITD sequence comprises a sequence of SEQ ID NO: 24 or 90.
  • the string of sequences is operably linked to the sequence encoding the MITD sequence via a linker sequence.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 7, 83, and 89.
  • a recombinant nucleic acid having at least 60% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 91, 95, 99, 103, 107, 111.
  • a recombinant nucleic acid having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 91, 95, 99, 103, 107, 111.
  • a polypeptide encoded by the recombinant nucleic acid of any one of the preceding embodiments is also provided herein.
  • 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.
  • nucleic acid of any one of the preceding embodiments polypeptide of any one of the preceding embodiments, or the pharmaceutical composition of any one of the preceding embodiments for use in therapy.
  • FIGs.1-2 depict an exemplary vaccine construct containing three different antigenic KRAS protein mutants.
  • FIG.1 is a cartoon depicting the protein domains and linkers encoded by the construct.
  • FIG.2 shows the fully annotated sequence of the exemplary construct.
  • FIGs.3A-3D depict results from a T cell activation experiment measuring IFN- ⁇ secretion by T cells incubated with monocyte-derived dendritic cells (moDCs) transfected with the construct shown in FIG.1, a construct containing three repeats of the antigenic KRAS mutants, an irrelevant construct, or with no moDCs.
  • FIG.3A depicts results using T cells expressing a TCR recognizing KRAS G12V mutant presented by HLA-DRB1:07:01, left and right panels are from different T cell donors.
  • FIG.3B depicts results using T cells expressing a TCR recognizing KRAS G12V mutant presented by HLA-A11:01, left and right panels are from different T cell donors.
  • FIG.3C depicts results using T cells expressing a TCR recognizing KRAS G12D mutant presented by HLA-A11:01, left and right panels are from different T cell donors.
  • FIG.3D depicts results using T cells expressing a TCR recognizing KRAS G12D mutant presented by HLA-C08:02, left and right panels are from different T cell donors.
  • FIG.4 depicts results from a T cell activation experiment measuring IFN- ⁇ secretion by T cells incubated with monocyte-derived dendritic cells (moDCs) transfected with the constructs described in Table 6. Left and right panels depict results obtained using two different T cell donors.
  • moDCs monocyte-derived dendritic cells
  • Neoantigens encompass, but are not limited to, tumor antigens which arise from, for example, a WSGR Docket No.50401-795.601 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. These alterations can (but do not always) cause cancer or other diseases.
  • 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
  • KD refers to the dissociation constant between two members of a binding pair and has units of molarity.
  • KA 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.
  • Koff 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 WSGR Docket No.50401-795.601 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. Given the conditions in which the assays are run (e.g., limiting HLA protein concentrations and/or labeled reference peptide concentrations), these 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. Immunol.154:247 (1995); and Sette, et al., Mol.
  • 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. Immunol.154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo 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 WSGR Docket No.50401-795.601 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 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.
  • 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).
  • epitopes may be referred to in some cases as peptides or peptide epitopes.
  • 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).
  • 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 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 candidate may be designated a drug product.
  • 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 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.
  • An “immune cell” 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.
  • Immunogenic peptides described herein are capable of binding to an HLA molecule and thereafter induce a cell-mediated or humoral response (e.g., a CTL (cytotoxic) response, or a HTL response) to the peptide.
  • 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.
  • MHC major histocompatibility complex
  • TCR The ability of a T cells to recognize an antigen associated with various diseases (e.g., cancers) or infectious organisms is conferred by its TCR, which is made up of both an alpha ( ⁇ ) chain and a beta ( ⁇ ) chain or a gamma ( ⁇ ) and a delta ( ⁇ ) chain.
  • the proteins which make up these chains are encoded by DNA, which employs a unique mechanism for generating the tremendous diversity of the TCR.
  • This multi-subunit immune recognition receptor associates with the CD3 complex and binds peptides presented by the MHC class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of a TCR to a peptide on an APC is a central event in T cell activation.
  • a “chimeric antigen receptor” or “CAR” refers to an antigen binding protein in that includes an immunoglobulin antigen binding domain (e.g., an immunoglobulin variable WSGR Docket No.50401-795.601 domain) and a T cell receptor (TCR) constant domain.
  • 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 immunoglobulin heavy chain variable domain linked to a TCR ⁇ constant domain.
  • the CAR is a dimer that includes a first polypeptide comprising an immunoglobulin heavy or light chain variable domain linked to a TCR ⁇ or TCR ⁇ constant domain and a second polypeptide comprising an immunoglobulin heavy or light chain variable domain (e.g., a ⁇ or ⁇ variable domain) linked to a TCR ⁇ or TCR ⁇ constant domain.
  • a first polypeptide comprising an immunoglobulin heavy or light chain variable domain linked to a TCR ⁇ or TCR ⁇ constant domain
  • a second polypeptide comprising an immunoglobulin heavy or light chain variable domain (e.g., a ⁇ or ⁇ variable domain) linked to a TCR ⁇ or TCR ⁇ constant domain.
  • MHC Major Histocompatibility Complex
  • 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. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex.
  • HLA human leukocyte antigen
  • a “Human Leukocyte Antigen” or “HLA” refers to a human Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., Immunology, 8TH Ed., Lange Publishing, Los Altos, Calif. (1994).
  • 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 subgroups, class I, class II, and class III.
  • MHC class I proteins can contain an ⁇ -chain and ⁇ 2- microglobulin (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T-cells.
  • MHC class II proteins can contain ⁇ - and ⁇ -chains and they can present antigen fragments to T-helper cells.
  • MHC class III region can encode for other immune components, such as complement components and cytokines.
  • the MHC can be both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).
  • WSGR Docket No.50401-795.601 [0301]
  • 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. The information is transmitted in particular by conformational changes of the receptor following complexation of the ligand on the surface of a cell.
  • 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.
  • a ligand is to be understood as meaning a peptide or peptide fragment which has a suitable length and suitable binding motifs in its amino acid sequence, so that the peptide or peptide fragment is capable of forming a complex with MHC proteins such as MHC class I or MHC class II proteins.
  • a “receptor/ligand complex” is also to be understood as meaning a “receptor/peptide complex” or “receptor/peptide fragment complex”, including a peptide- or peptide fragment- presenting MHC molecule such as MHC class I or MHC class II molecules.
  • a “native” or a “wild type” sequence refers to a sequence found in nature.
  • the term “naturally occurring” as used herein refers to the fact that an object can be found in nature. For example, 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.
  • peptide and peptide epitope are used interchangeably with “oligopeptide” in the present specification to designate a series of residues connected one to the other, typically by peptide bonds between the ⁇ -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. Such peptides can be produced using such methods as chemical synthesis or recombinant DNA technology.
  • “Synthetic peptides” include “fusion proteins.”
  • the term “motif” refers to a pattern of residues in an amino acid sequence of defined length, for example, a peptide of less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, for example, from about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for a class I HLA motif and from about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for a class II HLA motif, which is recognized by a particular HLA molecule.
  • 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, WSGR Docket No.50401-795.601 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 term “residue” refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or that is encoded by a nucleic acid (DNA or RNA). The nomenclature used to describe peptides or proteins follows the conventional practice.
  • amino group is presented to the left (the amino- or N-terminus) and the carboxyl group to the right (the carboxy- or C-terminus) of each amino acid residue.
  • amino acid residue positions are referred to in a peptide epitope, they are numbered in an amino to carboxyl direction with the first position being the residue located at the amino terminal end of the epitope, or the peptide or protein of which it can be a part.
  • the amino- and carboxyl-terminal groups although not specifically shown, are in the form they can assume at physiologic pH values, unless otherwise specified.
  • each residue is generally represented by standard three letter or single letter designations.
  • 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.
  • 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.
  • 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 naturally-occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such a polynucleotide could be part of a vector, and/or such a polynucleotide or peptide could be part of a composition, and still be “isolated” in that such vector or composition is not part of its natural environment.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules produced synthetically.
  • 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.
  • the terms “identical” or percent “identity” in the context of two or more 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.
  • two nucleic acids or polypeptides described herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as an amino acid sequence of a peptide or a coding region of a nucleotide sequence.
  • subject refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • 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 WSGR Docket No.50401-795.601 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.
  • a cell sample e.g., a peripheral blood mononuclear cell (PBMC) sample
  • PBMC peripheral blood mononuclear cell
  • 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. While a tumor is a cancer of solid tissue, several of the compositions and methods described herein are in principle applicable to cancers of the blood, leukemia.
  • 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 WSGR Docket No.50401-795.601 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 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 RAS protein is used.
  • MHC major histocompatibility complex
  • the full-length RAS polypeptide can be a full-length KRAS, NRAS or HRAS polypeptide.
  • the vaccine may encode targets epitopes presented by MHC molecules encoded by HLA- A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA- C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-DRB1*11:01, HLA-A*03:02, HLA-B*07:02, HLA-C*08:02, HLA-C*03:04, HLA-C*05:01, and HLA-A*03:05.
  • vaccine constructs to present at least the target epitope on each of these HLA alleles.
  • a series of in vitro and in vivo assays have been defined to prioritize and select the best vaccine design, starting by choosing the best codon optimization approach and then selecting the best order of the epitopes. This information can be used to evaluate the approach in vivo.
  • a mouse model for this approach can utilize mice expressing human HLA and MHC.
  • HLA-engineered tumor cells can also be utilized to evaluate the approach.
  • RAS vaccines comprising RAS polypeptides or recombinant nucleic acids encoding RAS polypeptides that, when expressed, can result in presentation of one or more epitopes of RAS by one or more MHC molecules encoded by one or more HLA alleles.
  • a recombinant nucleic acid encoding a RAS polypeptide comprising a multiepitopic polypeptide.
  • the RAS polypeptide does not comprise a full- length RAS polypeptide.
  • the RAS polypeptide comprises a first RAS amino acid sequence comprising a first RAS epitope sequence.
  • the RAS polypeptide comprises a second RAS amino acid sequence comprising a second RAS epitope sequence.
  • the first RAS epitope sequence and the second RAS epitope sequence are different.
  • the first RAS epitope sequence and the second RAS epitope sequence are linked via a linker.
  • presentation of the first and/or second RAS epitope sequence as a peptide:MHC complex by the antigen presenting cells (APCs) of the subject administered the RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide is higher than the presentation of the first and/or second RAS epitope sequence as the peptide:MHC complex by the APCs of a subject administered the full-length RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • the first RAS amino acid sequence is the first epitope sequence.
  • the second RAS amino acid sequence is the second epitope sequence.
  • the first RAS amino acid sequence is the first epitope sequence and the second RAS amino acid sequence is the second epitope sequence. In some embodiments, the first RAS amino acid sequence consists of the first epitope sequence. In some embodiments, the second RAS amino acid sequence consists of the second epitope sequence. In some embodiments, the first RAS amino acid sequence consists of the first epitope sequence and the second RAS amino acid sequence consists of the second epitope sequence. In some cases, each different RAS epitope sequence is separated by a linker. [0325] In some embodiments, the first RAS amino acid sequence comprises a first RAS mutation. In some embodiments, the second RAS amino acid sequence comprises a second RAS mutation.
  • the second RAS mutation is different from the first RAS mutation. In some embodiments, the second RAS mutation is same as the first RAS mutation. In some embodiments, the first RAS amino acid sequence comprises a first RAS mutation, and the second RAS amino acid sequence comprises a second RAS mutation different from the first RAS mutation. In some embodiments, the first RAS amino acid sequence comprises a first RAS mutation, and the second RAS amino acid sequence comprises a second RAS mutation same as the first RAS mutation. [0326] In some embodiments, the first RAS mutation is selected from the group consisting of G12V, G12D, and G12C. In some embodiments, the second RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the first RAS amino acid sequence is processed into one or more epitopes comprising the first RAS mutation within a cell.
  • the first RAS mutation is G12V, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 27, 58-62 and 65-72.
  • the first RAS mutation is G12D, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 28, 75-79.
  • the first RAS mutation is G12C, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096.
  • the first RAS epitope sequence first RAS epitope sequence consists of from 5 to 18, from 6 to 17, from 5 to 16, from 7 to WSGR Docket No.50401-795.601 12, or from 8 to 10 consecutive amino acids from the full length RAS polypeptide.
  • the second RAS 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 RAS polypeptide. [0328]
  • the first RAS 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 RAS polypeptide.
  • the second RAS 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 RAS polypeptide.
  • the first RAS amino acid sequence and the second RAS amino acid sequence is separated by a linker.
  • the first RAS amino acid sequence comprises the first RAS epitope sequence and one, two, three, four, five or more residues flanking the N-terminus or C-terminus of the first RAS epitope sequence from the full length RAS polypeptide.
  • the second RAS amino acid sequence comprises the second RAS epitope sequence and one, two, three, four, five or more residues flanking the N-terminus or C-terminus of the second RAS epitope sequence from the full-length RAS 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 RAS 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 RAS polypeptide.
  • the recombinant nucleic acid encoding the RAS 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: 32.
  • the Sec sequence comprises a sequence of SEQ ID NO: 32.
  • 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: 30. In some embodiments, the linker comprises a sequence of SEQ ID NO: 30. In some embodiments, the Sec sequence is operably linked to the multiepitopic polypeptide via a linker. 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: 112.
  • the linker comprises a sequence of SEQ ID NO: 112. In some embodiments, the linker comprises a sequence having at least 60%, at least WSGR Docket No.50401-795.601 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: 31. In some embodiments, the linker comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 30. In some cases, the linker comprises a sequence of SEQ ID NO: 31.
  • the recombinant nucleic acid encoding the RAS 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 MITD sequence comprises at least 60%, at least 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 MITD sequence comprises a sequence of SEQ ID NO: 33.
  • 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: 30.
  • the linker comprises a sequence of SEQ ID NO: 30.
  • 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: 112.
  • the linker comprises a sequence of SEQ ID NO: 112.
  • the multiepitopic polypeptide comprises a RAS polypeptide.
  • the sequence of the multiepitopic polypeptide is the same RAS epitope sequence.
  • the sequence of the multiepitopic polypeptide is a different RAS epitope sequence.
  • the multiepitopic polypeptide comprises at least 2 different RAS epitope sequences.
  • the multiepitopic polypeptide comprises at least 3 different RAS epitope sequences.
  • the multiepitopic polypeptide comprises at least 4 different RAS epitope sequences.
  • the multiepitopic polypeptide comprises at least 5 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 6 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 8 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 9 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 10 or more different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide is a RAS polypeptide comprising the first RAS epitope sequence and the second RAS epitope sequence.
  • the multiepitopic polypeptide comprises a first RAS epitope sequence, operably linked to a second RAS epitope sequence.
  • the first RAS epitope WSGR Docket No.50401-795.601 sequence and the second RAS epitope sequence are the same.
  • the first RAS epitope sequence and the second RAS epitope sequence are different.
  • the first RAS epitope sequence and second RAS epitope sequence are presentable by different HLA alleles.
  • the first RAS epitope sequence and second RAS epitope sequence are presented by different HLA alleles.
  • the first RAS epitope sequence and second RAS epitope sequence are predicted to bind to different HLA alleles. In some embodiments, the first RAS epitope sequence and second RAS epitope sequence are predicted to be presented by different HLA alleles. [0335] In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 500nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 400nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 300nM.
  • a first RAS epitope sequence binds to a first HLA allele. In some embodiments, a first RAS epitope sequence is predicted to bind to a first HLA allele. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 200nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 100nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 50nM.
  • a first RAS epitope sequence binds to a first HLA allele with a KD less than 1nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele. In some embodiments, a first RAS epitope sequence is predicted to bind to a second HLA allele. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 200nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 300nM.
  • a first RAS epitope sequence binds to a second HLA allele with a KD of more than 500nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 600nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 700nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 800nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 900nM.
  • a first RAS epitope sequence binds to a second HLA allele with a KD of more than 1000nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD of less than 1nM and binds to second HLA allele with a KD of more than 200nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD of less than 50nM and binds to second HLA allele with a KD of more than 300nM.
  • a first RAS epitope sequence binds to a first HLA allele with a KD of less than 100nM and binds to second HLA allele with a KD of more than 500nM.
  • a first WSGR Docket No.50401-795.601 RAS epitope sequence binds to a first HLA allele with a KD of less than 200 nM and binds to second HLA allele with a KD of more than 600 nM.
  • the linker comprises at least 60%, at least 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 comprises a sequence of SEQ ID NO: 31. In some embodiments, the linker is a cleavable linker. 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112.
  • the multiepitopic polypeptide comprises a first RAS epitope sequence, operably linked to a second RAS epitope sequence, operably linked to a third RAS epitope sequence.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*11:01 allele. In some embodiments, the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*11:01 allele.
  • the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*11:01 allele. In some embodiments, the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*11:01 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*11:01 allele. [0339] In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*03:01 allele.
  • the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*03:01 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*03:01 allele. In some embodiments, the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*03:01 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*03:01 allele. [0340] In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*30:01 allele. In some embodiments, the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*30:01 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*30:01 allele. In some embodiments, the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*30:01 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*30:01 allele.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*68:01 allele. In some embodiments, the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*68:01 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*68:01 allele.
  • the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*68:01 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*68:01 allele. [0342] In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-B allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-B*40:01 allele. In some embodiments, the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-B*40:01 allele.
  • the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- B*40:01 allele. In some embodiments, the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-B*40:01 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-B*40:01 allele. [0343] In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C*01:02 allele.
  • the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-C*01:02 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- C*01:02 allele. In some embodiments, the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-C*01:02 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-C*01:02 allele. [0344] In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C allele.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C*03:03 allele. In some embodiments, the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-C*03:03 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- C*03:03 allele. In some embodiments, the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-C*03:03 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-C*03:03 allele.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C*03:04 allele. In some embodiments, the first RAS epitope WSGR Docket No.50401-795.601 sequence is presented by an MHC molecule encoded by an HLA-C*03:04 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- C*03:04 allele.
  • the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-C*03:04 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-C*03:04 allele. [0346] In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-DRB1 allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-DRB1*07:01 allele.
  • the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-DRB1*07:01 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA-DRB1*07:01 allele. In some embodiments, the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-DRB1*07:01 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA- DRB1*07:01 allele.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*03:02 allele. In some embodiments, the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*03:02 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*03:02 allele. In some embodiments, the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*03:02 allele.
  • the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*03:02 allele. [0348] In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*03:05 allele. In some embodiments, the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*03:05 allele. In some embodiments, the first RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*03:05 allele.
  • the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*03:05 allele. In some embodiments, the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*03:05 allele.
  • the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-B allele. In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-B*07:02 allele. In some embodiments, the second RAS epitope sequence is presented by an MHC molecule encoded by an HLA-B*07:02 allele.
  • the second RAS epitope sequence binds to an MHC molecule encoded by an HLA- WSGR Docket No.50401-795.601 B*07:02 allele. In some embodiments, the second RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-B*07:02 allele. In some embodiments, the second RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-B*07:02 allele. [0350] In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C allele.
  • the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C*03:04 allele. In some embodiments, the second RAS epitope sequence is presented by an MHC molecule encoded by an HLA-C*03:04 allele. In some embodiments, the second RAS epitope sequence binds to an MHC molecule encoded by an HLA- C*03:04 allele. In some embodiments, the second RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-C*03:04 allele. In some embodiments, the second RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-C*03:04 allele.
  • the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C allele. In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C*05:01 allele. In some embodiments, the second RAS epitope sequence is presented by an MHC molecule encoded by an HLA-C*05:01 allele. In some embodiments, the second RAS epitope sequence binds to an MHC molecule encoded by an HLA- C*05:01 allele. In some embodiments, the second RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-C*05:01 allele.
  • the second RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-C*05:01 allele. [0352] In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*-3:01 allele. In some embodiments, the second RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*-3:01 allele. In some embodiments, the second RAS epitope sequence binds to an MHC molecule encoded by an HLA-A*- 3:01 allele.
  • the second RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*-3:01 allele. In some embodiments, the second RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*-3:01 allele. [0353] In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*-11:01 allele. In some embodiments, the second RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*-11:01 allele.
  • the second RAS epitope sequence binds to an MHC molecule encoded by an HLA-A*- 11:01 allele. In some embodiments, the second RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*-11:01 allele. In some embodiments, the second RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*-11:01 allele. WSGR Docket No.50401-795.601 [0354] In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele.
  • the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*-68:01 allele. In some embodiments, the second RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*-68:01 allele. In some embodiments, the second RAS epitope sequence binds to an MHC molecule encoded by an HLA-A*- 68:01 allele. In some embodiments, the second RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*-68:01 allele.
  • the second RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*-68:01 allele. [0355] In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C allele. In some embodiments, the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C*08:02 allele. In some embodiments, the second RAS epitope sequence is presented by an MHC molecule encoded by an HLA-C*08:02 allele. In some embodiments, the second RAS epitope sequence binds to an MHC molecule encoded by an HLA- C*08:02 allele.
  • the second RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-C*08:02 allele. In some embodiments, the second RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-C*08:02 allele. [0356] In some embodiments, the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-DRB allele. In some embodiments, the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-DRB1*11:01 allele.
  • the third RAS epitope sequence is presented by an MHC molecule encoded by an HLA-DRB1*11:01 allele. In some embodiments, the third RAS epitope sequence binds to an MHC molecule encoded by an HLA-DRB1*11:01 allele. In some embodiments, the third RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-DRB1*11:01 allele. In some embodiments, the third RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA- DRB1*11:01 allele.
  • the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*3:01 allele. In some embodiments, the third RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*3:01 allele. In some embodiments, the third RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*3:01 allele. In some embodiments, the third RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*3:01 allele.
  • the third RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*3:01 allele. [0358] In some embodiments, the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the third RAS epitope sequence is presentable by WSGR Docket No.50401-795.601 an MHC molecule encoded by an HLA-A*11:01 allele. In some embodiments, the third RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*11:01 allele.
  • the third RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*11:01 allele. In some embodiments, the third RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*11:01 allele. In some embodiments, the third RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*11:01 allele. [0359] In some embodiments, the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A allele. In some embodiments, the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-A*68:01 allele.
  • the third RAS epitope sequence is presented by an MHC molecule encoded by an HLA-A*68:01 allele. In some embodiments, the third RAS epitope sequence binds to an MHC molecule encoded by an HLA- A*68:01 allele. In some embodiments, the third RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-A*68:01 allele. In some embodiments, the third RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-A*68:01 allele. [0360] In some embodiments, the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C allele.
  • the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA-C*03:03 allele. In some embodiments, the third RAS epitope sequence is presented by an MHC molecule encoded by an HLA-C*03:03 allele. In some embodiments, the third RAS epitope sequence binds to an MHC molecule encoded by an HLA- C*03:03 allele. In some embodiments, the third RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA-C*03:03 allele. In some embodiments, the third RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA-C*03:03 allele.
  • the multiepitopic polypeptide comprises the formula NT seq – ([Antigen1]-cleavable linker1-[Antigen2])x-CT seq, where x is an integer from 1 to 50. In some embodiments, the multiepitopic polypeptide comprises the formula NT seq – ([Antigen1]-cleavable linker1-[Antigen2])x-CT seq, where x is an integer from 1 to 25. In some embodiments, the multiepitopic polypeptide comprises the formula NT seq – ([Antigen1]-cleavable linker1- [Antigen2])x-CT seq, where x is an integer from 1 to 10.
  • the multiepitopic polypeptide comprises the formula NT seq – ([Antigen1]-cleavable linker1-[Antigen2]-cleavable linker2-[Antigen3])x-CT seq, where x is an integer from 1 to 50. In some embodiments, the multiepitopic polypeptide comprises the formula NT seq – ([Antigen1]-cleavable linker1-[Antigen2]-cleavable linker2-[Antigen3])x-CT seq, where x is an integer from 1 to 25.
  • the multiepitopic polypeptide comprises the formula NT seq – ([Antigen1]-cleavable linker1-[Antigen2]-cleavable linker2-[Antigen3])x-CT seq, where x is an integer from 1 to 10.
  • NT seq comprises a secretory (Sec) sequence and a N-terminal linker sequence.
  • CT seq comprises a C-terminal linker and a MITD sequence.
  • the Antigen1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-29.
  • the Antigen2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-29.
  • the Antigen3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 27- 29.
  • the cleavable linker1 or cleavable linker2 comprise a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 30 or 31.
  • the cleavable linker1 or cleavable linker2 is selected from the group consisting of SEQ ID NOs: 30 and 31.
  • the N-terminal linker sequence is selected from the group consisting of SEQ ID NOs: 30 and 31. In some embodiments, the C-terminal linker sequence is selected from the group consisting of SEQ ID NOs: 30 and 31.
  • the RAS polypeptide comprises a sequence with at least 60% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises a sequence with at least 70% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises a sequence with at least 90% sequence identity to SEQ ID NO: 75.
  • the RAS polypeptide comprises a sequence of SEQ ID NO: 27. [0365] In some embodiments, the RAS polypeptide comprises a sequence with at least 60% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises a sequence with at least 70% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises a sequence with at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises a sequence of SEQ ID NO: 28.
  • the RAS polypeptide comprises a sequence with at least 60% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises a sequence with at least 70% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises a sequence with at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises a sequence with at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises a sequence of SEQ ID NO: 29. [0367] In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 27.
  • the RAS polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least WSGR Docket No.50401-795.601 80% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 27. [0368] In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 28.
  • the RAS polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 28. [0369] In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 29.
  • the RAS polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 29. [0370] In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 60% sequence identity to SEQ ID NO: 27.
  • the RAS polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 27. [0371] In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 60% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 28.
  • the RAS polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 28. [0372] In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 60% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 70% sequence identity to SEQ ID NO: 29.
  • the RAS polypeptide comprises two copies of a sequence with at least 80% sequence identity to SEQ WSGR Docket No.50401-795.601 ID NO: 29. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 29. [0373] In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 60% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 27.
  • the RAS polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 27. [0374] In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 60% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 28.
  • the RAS polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 28. [0375] In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 60% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 70% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 90% sequence identity to SEQ ID NO: 29.
  • the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 29. [0376] In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 60% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises four copies of a sequence with at least 60% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 80% sequence WSGR Docket No.50401-795.601 identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 28.
  • the RAS polypeptide comprises four copies of a sequence with at least 60% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 70% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 29.
  • the RAS polypeptide comprises five copies of a sequence with at least 60% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 80% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises five copies of a sequence with at least 60% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 28.
  • the RAS polypeptide comprises five copies of a sequence with at least 60% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 70% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 29.
  • the RAS polypeptide comprises six or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least WSGR Docket No.50401-795.601 80% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises six or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence of SEQ ID NO: 28.
  • the RAS polypeptide comprises six or more copies of a sequence with at least 60% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 70% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 80% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 90% sequence identity to SEQ ID NO: 29. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence of SEQ ID NO: 29.
  • the RAS 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: 27, 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: 29, 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: 28.
  • the RAS 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: 27, 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: 29 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: 29 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: 29 operably linked
  • the RAS polypeptide comprises, from N terminus to C terminus, WSGR Docket No.50401-795.601 the sequence of SEQ ID NO: 27, operably linked to the sequence of SEQ ID NO: 29 via a linker, and the sequence of SEQ ID NO: 29 operably linked to the sequence of SEQ ID NO: 28 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31. In some embodiments, the linker is a cleavable linker. 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS 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: 29, 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: 28, 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: 27.
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 29, operably linked to the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 27.
  • the RAS 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: 29, 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: 28 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:
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 29, operably linked to the sequence of SEQ ID NO: 28 via a linker, and the sequence of SEQ ID NO: 28 operably linked to the sequence of SEQ ID NO: 27 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • 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: 112.
  • the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS 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: 28, 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: 27, 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: 29.
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 27, operably linked to the sequence of SEQ ID NO: 29.
  • the RAS 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: 28, 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: 27 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:
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 27 via a linker, and the sequence of SEQ ID NO: 27 operably linked to the sequence of SEQ ID NO: 29 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • 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: 112.
  • the linker comprises a sequence of SEQ ID NO: 112.
  • WSGR Docket No.50401-795.601 [0391]
  • the RAS polypeptide comprises two copies of a multiepitopic polypeptide.
  • 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: 29, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 28, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises three copies of a multiepitopic polypeptide.
  • 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: 29, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 28, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises four copies of a multiepitopic polypeptide.
  • 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: 29, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 28, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises five copies of a multiepitopic polypeptide.
  • 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: 29, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 28, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises six copies of a multiepitopic polypeptide.
  • 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: 29, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 28, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises seven copies of a multiepitopic polypeptide.
  • 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: 29, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 28, operably linked to a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 27.
  • the recombinant nucleic acid encoding the RAS 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: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS 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: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 14.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 14.
  • the recombinant nucleic acid encoding the RAS 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: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS 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: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 16.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID WSGR Docket No.50401-795.601 NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 16.
  • the recombinant nucleic acid encoding the RAS 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: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 17.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 17.
  • the recombinant nucleic acid encoding the RAS 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: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the RAS 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: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS WSGR Docket No.50401-795.601 polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS 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: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS 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: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 21.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 21.
  • the recombinant nucleic acid encoding the RAS 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: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a WSGR Docket No.50401-795.601 sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS 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: 23. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 23. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 23.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 23. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 23. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 23.
  • the recombinant nucleic acid encoding the RAS 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. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 84. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 84.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 84. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 84. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 84.
  • the recombinant nucleic acid encoding the RAS 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: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 86.
  • the recombinant nucleic WSGR Docket No.50401-795.601 acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 86.
  • the recombinant nucleic acid encoding the RAS 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: 88. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 88. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 88.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 88. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 88. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 88.
  • the recombinant nucleic acid encoding the RAS 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: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 92.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 92.
  • the recombinant nucleic acid encoding the RAS 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: 93. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 93.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% WSGR Docket No.50401-795.601 sequence identity to a sequence of SEQ ID NO: 93. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 93. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 93.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 93. [0414] In some embodiments, the recombinant nucleic acid encoding the RAS 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: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 94.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 94.
  • the recombinant nucleic acid encoding the RAS 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: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 96.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 96.
  • the recombinant nucleic acid encoding the RAS 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: 97. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 97.
  • the WSGR Docket No.50401-795.601 recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 97.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 97. [0417] In some embodiments, the recombinant nucleic acid encoding the RAS 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: 98. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 98.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 98. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 98. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 98. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 98.
  • the recombinant nucleic acid encoding the RAS 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: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 100.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 100.
  • the recombinant nucleic acid encoding the RAS 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: 101.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence WSGR Docket No.50401-795.601 having at least 60% sequence identity to a sequence of SEQ ID NO: 101.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 101.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 101. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 101. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 101.
  • the recombinant nucleic acid encoding the RAS 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: 102. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 102. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 102.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 102. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 102. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 102.
  • the recombinant nucleic acid encoding the RAS 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: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 104.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 104.
  • the recombinant nucleic acid encoding the RAS 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: 105.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 105.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 105.
  • the recombinant nucleic acid encoding the RAS 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: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 106.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 106.
  • the recombinant nucleic acid encoding the RAS 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: 108. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 108. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 108. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 108. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at WSGR Docket No.50401-795.601 least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 109.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 109.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 109.
  • the recombinant nucleic acid encoding the RAS 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: 110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 110.
  • the recombinant nucleic acid encoding the RAS 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: 14-23, 84, 86, 88, 92-94, 96-98, 100- 102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS 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: 14. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 15.
  • the recombinant nucleic acid encoding the RAS 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 WSGR Docket No.50401-795.601 sequence of SEQ ID NO: 16.
  • the recombinant nucleic acid encoding the RAS 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: 17.
  • the recombinant nucleic acid encoding the RAS 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: 18. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 19.
  • the recombinant nucleic acid encoding the RAS 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: 20. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 21.
  • the recombinant nucleic acid encoding the RAS 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: 22. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 23.
  • the recombinant nucleic acid encoding the RAS 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: 84. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 86.
  • the recombinant nucleic acid encoding the RAS 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: 88. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 92.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 60%, at least 65%, at least 70%, WSGR Docket No.50401-795.601 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 recombinant nucleic acid encoding the RAS 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: 94.
  • the recombinant nucleic acid encoding the RAS 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: 96. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 97.
  • the recombinant nucleic acid encoding the RAS 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: 98. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 100.
  • the recombinant nucleic acid encoding the RAS 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: 101. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 102.
  • the recombinant nucleic acid encoding the RAS 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: 104. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 105.
  • the recombinant nucleic acid encoding the RAS 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: 106. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 108.
  • the recombinant nucleic acid encoding the RAS WSGR Docket No.50401-795.601 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: 109.
  • the recombinant nucleic acid encoding the RAS 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: 110.
  • the recombinant nucleic acid encoding the RAS 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: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104- 106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 14.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 17.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 23.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 84. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ WSGR Docket No.50401-795.601 ID NO: 88.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 93. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 94.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 97. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 98.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 101. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 102.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 106.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 108. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14- 23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID WSGR Docket No.50401-795.601 NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 23.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 84. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 88. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 92.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 93. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 97.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 98. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 101. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 102.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 105. In some embodiments, WSGR Docket No.50401-795.601 the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 110. [0430] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 23. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 84. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 88.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 93. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 96.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 97. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide WSGR Docket No.50401-795.601 comprises two copies of a sequence of SEQ ID NO: 98. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 101.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 102. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 106.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 108. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 23. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 84.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 88. WSGR Docket No.50401-795.601 In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 93.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 97. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 98.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 101. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 102. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 104.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 108. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 109.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 110. [0432] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 14.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid WSGR Docket No.50401-795.601 encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 23.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 84. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 88. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 92.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 93. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 97.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 98. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 101. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 102.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 110. [0433] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a WSGR Docket No.50401-795.601 sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 17.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 21.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 23. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 84. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 86.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 88. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 93. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 94.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 97. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 98. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 100.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 101. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 102. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 104. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 105.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 108. In some embodiments, the recombinant WSGR Docket No.50401-795.601 nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 109. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 23. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 84.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 86. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 88. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 92. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 93.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 94. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 96. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 97. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 98.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 100. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 101. WSGR Docket No.50401-795.601 In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 102. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 104.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 105. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 106. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 108. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 109.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 110.
  • the recombinant nucleic acid encoding the RAS 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 the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, 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 the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, and operably linked to a sequence having
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, and operably linked to a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 17-19, 86, 93, 97, 101, 105, and 109.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, and operably linked to a sequence selected from the group consisting of SEQ ID NO: 17-19, 86, 93, 97, 101, 105, and 109.
  • the recombinant nucleic acid encoding the RAS 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 the sequence selected WSGR Docket No.50401-795.601 from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, 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 the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, via a linker sequence and the sequence a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence a sequence having at least 90% sequence identity the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, via a linker sequence and the sequence having at least 90% sequence identity the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence a sequence having at least 90% sequence identity the sequence selected from the group consisting of SEQ ID NO: 17-19, 86, 93, 97, 101, 105, and 109 via a linker sequence.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, via a linker sequence and the sequence of any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 operably linked to a sequence selected from the group consisting of SEQ ID NO: 17-19, 86, 93, 97, 101, 105, and 109 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: 5.
  • 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: 6.
  • 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 WSGR Docket No.50401-795.601 identity to a sequence of SEQ ID NO: 9.
  • 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. 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: 11.
  • 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: 13. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 5. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 6. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence of SEQ ID NO: 9. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 10. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 11. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 12. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 13.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 80% sequence identity the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence having at least 80% sequence identity the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence having at least 80% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 14-16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 14-16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence selected from the group consisting of SEQ ID NOs: WSGR Docket No.50401-795.601 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence selected from the group consisting of SEQ ID NO: 14-16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS 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 the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, 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 the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, 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%,
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, via a linker sequence and the sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 14-16, 84, 92, 96, 100, 104, and 108, via a linker sequence.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, via a linker sequence and the sequence of any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence selected from the group consisting of SEQ ID NO: 14-16, 84, 92, 96, 100, 104, and 108, 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: 5.
  • 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 WSGR Docket No.50401-795.601 100% 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: 6. 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: 8. 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: 9.
  • 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. 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: 11.
  • 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: 13. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 5. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 6. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence of SEQ ID NO: 10. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 11. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 12. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 13.
  • the recombinant nucleic acid encoding the RAS 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 the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, 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 the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, 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 WSGR Docket No.50401
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, and operably linked to a sequence a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 20-23, 88, 94, 98, 102, 106, and 110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, and operably linked to a sequence selected from the group consisting of SEQ ID NO: 20-23, 88, 94, 98, 102, 106, and 110.
  • the recombinant nucleic acid encoding the RAS 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 the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, 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 the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, via a linker sequence and the 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%
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 14-19, via a linker sequence and the sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NO: 20-23, 88, 94, 98, 102, 106, and 110, via a linker sequence.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence of any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, via a linker sequence and the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence selected from the group consisting of SEQ ID NO: 20-23, 88, 94, 98, 102, 106, and 110, 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: 5.
  • 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: 6.
  • 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. 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: 9.
  • 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. 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: 11.
  • 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: 13. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 8. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 5. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 6.
  • the linker sequence comprises a sequence of SEQ ID NO: 8. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 9. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 10. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 11. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 12. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 13. WSGR Docket No.50401-795.601 [0456] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises at least two copies of a string of sequences.
  • each string of sequences comprises, 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 the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, 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 the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, 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 least 85%, at least 90%, at least 95%, or 100% sequence identity to the sequence selected from the group consisting of
  • each string of sequences comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence a sequence having at least 90% sequence identity to the sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108.
  • each string of sequences comprises, from 5’ end to 3’ end, a sequence selected from the group consisting of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence selected from the group consisting of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence selected from the group consisting of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of the string of sequences.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises seven copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises eight copies of the string of sequences.
  • the recombinant nucleic acid encoding the RAS 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: 3.
  • the Sec sequence comprises a sequence of SEQ ID NO: 3.
  • the sequence encoding the Sec sequence is operably linked to the string of sequences 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 the sequence selected from the group consisting of SEQ ID NOs: 4, 7, 83, and 89.
  • the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 7, 83, and 89.
  • the recombinant nucleic acid encoding the RAS polypeptide further comprises a sequence encoding an MHC I Trafficking Domain (MITD) sequence.
  • 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: 24 or 90.
  • the MITD sequence comprises a sequence of SEQ ID NO: 24 or 90.
  • the string of sequences is operably linked to the sequence encoding the MITD domain. In some embodiments, the string of sequences is operably linked to the sequence encoding the MITD domain 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 the sequence selected from the group consisting of SEQ ID NOs: 4, 7, 83, and 89. In some embodiments, the linker sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 7, 83, and 89.
  • 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: 2.
  • 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: 91. 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: 95.
  • 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: 99. 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%, WSGR Docket No.50401-795.601 at least 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 103.
  • 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: 107. 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: 111. [0460] In some embodiments, 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: 2. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 91. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 95. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 99. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 103.
  • the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 107. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 111. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 1.
  • the recombinant nucleic acid comprises a sequence of SEQ ID NO: 2. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 91. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 95. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 99. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 103.
  • the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 107. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 111.
  • Immune Cells e.g., T cells
  • T cell receptors can also comprise endogenous T cell receptors WSGR Docket No.50401-795.601 that have been activated. Immune cells expressing a TCR can be activated and/or expanded using methods such as NEO-STIM.
  • T cell receptors can comprise receptors encoded by recombinant nucleic acids that are specific for a RAS polypeptide or fragment thereof.
  • a recombinant nucleic acid encoding a TCR is provided herein.
  • the TCR comprises a TCR beta chain construct.
  • the TCR comprises a TCR alpha chain construct.
  • the TCR comprises a TCR beta chain construct and a TCR alpha chain construct.
  • the TCR binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises a RAS epitope sequence having at least 5 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 27, 58-62 and 65-72 and a human MHC encoded by an HLA allele selected from the group consisting of HLA- A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA- C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-A*03:02, and HLA-A*03:05.
  • the peptide:MHC complex comprises a RAS epitope sequence having at least 7 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 27, 58-62 and 65-72 and a human MHC encoded by an HLA allele selected from the group consisting of HLA- A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA- C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-A*03:02, and HLA-A*03:05.
  • the peptide:MHC complex comprises a RAS epitope sequence having at least 5 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 28, 75-79 and a human MHC encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*08:02, HLA-C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA-A*11:01, and HLA-A*68:01.
  • the peptide:MHC complex comprises a RAS epitope sequence having at least 7 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 28, 75-79 and a human MHC encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*08:02, HLA-C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA-A*11:01, and HLA-A*68:01.
  • the peptide:MHC complex comprises a RAS epitope sequence having at least 5 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 29, 81, 82, and 1096 and a human MHC encoded by an HLA allele selected from the group consisting of HLA- DRB1*11:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • the peptide:MHC complex comprises a RAS epitope sequence having at least 7 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NO: 29, 81, 82, and 1096 and a human MHC encoded by an HLA allele selected from the group consisting of HLA- DRB1*11:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least WSGR Docket No.50401-795.601 85%, at least 90%, at least 95%, or 100% sequence identity to a sequence of SEQ ID NO: 58.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 59.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 60.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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 peptide:MHC complex comprises the RAS epitope sequence having 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 peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 65. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 66. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having 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: 67.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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 peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 69.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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 peptide:MHC complex comprises the RAS epitope sequence having 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 peptide:MHC complex comprises the RAS epitope sequence having 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 peptide:MHC complex comprises the RAS epitope sequence having 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: 75.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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 WSGR Docket No.50401-795.601 identity to a sequence of SEQ ID NO: 76.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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 peptide:MHC complex comprises the RAS epitope sequence having 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: 78. In some embodiments the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 79.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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: 81.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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.
  • the peptide:MHC complex comprises the RAS epitope sequence having 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: 1096.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 58. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 59. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 60. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 61.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 62. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 65. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 66. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 67.
  • the peptide:MHC complex WSGR Docket No.50401-795.601 comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 68. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 69. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 70. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 71.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 72. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 75. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 76. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 77.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 78. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 79. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 29. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 81.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 82. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 1096. [0465] In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 58. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 59.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 60. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 61. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 62. In some embodiments the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 65. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 66.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 67. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 68. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 69. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 70. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 71.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 72. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 75. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 76. In some embodiments the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 77. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 78.
  • the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 79. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 29. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 81. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 82. In some embodiments, the peptide:MHC complex comprises the RAS epitope sequence having a sequence according to SEQ ID NO: 1096.
  • the cell comprising the recombinant nucleic acids presented herein.
  • the cell is an immune cell.
  • the cell is a myeloid lineage cell.
  • the cell is a lymphocyte.
  • the cell is a B cell.
  • the cell is a T cell.
  • the proinflammatory cytokine is IFN- ⁇ .
  • the proinflammatory cytokine is TNF- ⁇ .
  • the proinflammatory cytokine is IL-12.
  • the proinflammatory cytokine is IL-6. In some cases, the proinflammatory cytokine is IL-17. In some embodiments, the proinflammatory cytokine is a chemokine. In some cases, upon binding of the TCR to the peptide:MHC complex, cell division is increased. In some embodiments, upon binding of the TCR to the peptide:MHC complex, 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.
  • 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. In some embodiments, upon binding of the TCR to the peptide:MHC complex, cytotoxicity of the cell against target cell is increased by 25% 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 50% 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 100% 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 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. In some embodiments, upon binding of the TCR to the peptide:MHC complex, cytotoxicity of the cell against target cell is increased by 500% or more compared to an otherwise identical cell not having a TCR specific for the peptide:MHC complex.
  • a recombinant nucleic acid encoding a TCR comprising a TCR beta chain construct. In some embodiments, the recombinant nucleic acid encoding a TCR comprises a TCR alpha chain construct.
  • the beta chain construct comprises a complementarity determining region 3 (CDR3).
  • CDR3 complementarity determining region 3
  • the beta chain CDR3 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 an amino acid sequence set forth in SEQ ID NO: 57.
  • the beta chain CDR3 comprises an amino acid sequence set forth in SEQ ID NO: 57.
  • the TCR beta 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: 52 or SEQ ID NO: 53.
  • the TCR beta chain construct comprises a variable region comprising a sequence having at least 80% sequence identity to WSGR Docket No.50401-795.601 an amino acid sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 53.
  • the TCR beta chain construct comprises a variable region comprising an amino acid sequence set forth in SEQ ID NO: 52 or SEQ ID NO: 53.
  • 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: 55 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: 56.
  • 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: 55 and a complementarity determining region 2 (CDR2) having an amino acid set forth in SEQ ID NO: 56.
  • 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 least 90%, at least 95%, or 100% sequence identity to a sequence set forth in SEQ ID NO: 46
  • 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: 47
  • 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: 48.
  • the CDR1 comprises an amino acid sequence set forth in SEQ ID NO: 46
  • the CDR2 comprises an amino acid sequence set forth in SEQ ID NO: 47
  • the CDR3 comprises an amino acid sequence set forth in SEQ ID NO: 48.
  • 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: 43 or SEQ ID NO: 44.
  • 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: 43 or SEQ ID NO: 44. In some embodiments, the TCR alpha chain construct comprises a variable region comprising an amino acid sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44.
  • 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: 49 or SEQ ID NO: 51 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: 40 or SEQ ID NO: 42.
  • the TCR comprises a beta chain having an amino acid WSGR Docket No.50401-795.601 sequence that is at least 80% identical to SEQ ID NO: 49 or SEQ ID NO: 51 and an alpha chain having an amino acid sequence that is at least 80% identical to SEQ ID NO: 40 or SEQ ID NO: 42.
  • the TCR comprises a beta chain having an amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 51 and an alpha chain having an amino acid sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 42.
  • 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, 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 malignant melanoma
  • renal cancer e.g.
  • 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 that can be prevented and/or treated in accordance with present disclosure include, but are not limited to, the following: renal cancer, kidney cancer, glioblastoma multiforme, metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myclodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hod
  • 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
  • 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 epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, brenner, or undifferentiated.
  • the present disclosure is used in the treatment, diagnosis, and/or prognosis of lymphoma or its subtypes, including, but not limited to, mantle cell lymphoma. Lymphoproliferative disorders are also considered to be proliferative diseases.
  • the present disclosure provides a method of treating a subject with a disease or condition.
  • the disease or condition can comprise a cancer.
  • the method can comprise administering to the subject a therapy comprising a multiepitopic polypeptide or a recombinant nucleic acid encoding the multiepitopic polypeptide.
  • the multiepitopic polypeptide can comprise a RAS polypeptide.
  • the RAS polypeptide may not be a full-length RAS polypeptide.
  • each sequence of the multiepitopic polypeptide can be a different RAS epitope sequence.
  • the antigen presenting cells (APCs) of the subject administered the RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide can present a RAS epitope sequence as a peptide:MHC complex.
  • the APCs of the subject present more of a RAS epitope sequence compared to the APCs of a subject administered the full-length RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • the APCs of the subject express more peptide:MHC complexes comprising the RAS epitope sequence compared to the APCs of a subject administered the full-length RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • the APCs of the subject express more of the RAS epitope sequence compared to the APCs of a subject administered the full-length RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • the full-length RAS polypeptide is a full-length KRAS, NRAS or HRAS polypeptide.
  • the therapy comprises a cell comprising the multiepitopic polypeptide or the recombinant nucleic acid encoding the multiepitopic polypeptide.
  • the WSGR Docket No.50401-795.601 multiepitopic polypeptide comprises a first RAS amino acid sequence comprising a first RAS epitope sequence and a second RAS amino acid sequence comprising a second RAS epitope sequence. In some embodiments, the first RAS epitope sequence and the second RAS epitope sequence are different.
  • the presentation of the first and/or second RAS epitope sequence as a peptide:MHC complex by APCs of the subject administered the therapy is higher than the presentation of the first and/or second RAS epitope sequence as the peptide:MHC complex by the APCs of a subject administered the full-length RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • the full-length RAS polypeptide is a full-length KRAS, NRAS or HRAS polypeptide.
  • the first RAS amino acid sequence is a first KRAS, NRAS or HRAS amino acid sequence.
  • the second RAS amino acid sequence is a second KRAS, NRAS or HRAS amino acid sequence.
  • the APCs of the subject administered with the multiepitopic polypeptide having different RAS epitopes can present more (e.g., 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, 100 times, 120 times, 150 times, 200 times or more) of a RAS epitope sequence compared to the APCs of a subject administered the full-length RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • Presentation can be measured by mass spectrometry detecting RAS -specific MHC molecules following lysis and protein extraction of APCs. Presentation can also be measured by mass spectrometry detecting components of the RAS epitope sequence.
  • the APCs having (e.g., transfected or otherwise delivered with) the multiepitopic polypeptide having different RAS epitopes can present more (e.g., 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, 100 times, 120 times, 150 times, 200 times or more) of a RAS epitope sequence compared to the APCs having the full-length RAS polypeptide or a recombinant nucleic acid encoding the full
  • Presentation can be measured by mass spectrometry detecting RAS -specific MHC molecules following lysis and protein extraction of APCs. Presentation can also be measured by mass spectrometry detecting components of the RAS epitope sequence.
  • the method further comprises administering to the subject a T-cell receptor (TCR) or a cell comprising the TCR. In some embodiments, the method comprises administering to the subject a recombinant nucleic acid encoding the TCR or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR.
  • TCR T-cell receptor
  • the method further WSGR Docket No.50401-795.601 comprises administering to the subject a cell comprising a chimeric antigen receptor (CAR) or a recombinant nucleic acid encoding the CAR.
  • CAR chimeric antigen receptor
  • the multiepitopic polypeptide comprising a RAS polypeptide or the recombinant nucleic acid encoding the multiepitopic polypeptide administered in the subject can elicit specific T cell responses of the T cells expressing TCRs that can recognize RAS epitopes in complex with MHC molecules.
  • the multiepitopic polypeptide having different RAS epitopes can elicit enhanced T cell responses compared to the full-length RAS polypeptide.
  • the multiepitopic polypeptide comprising a RAS polypeptide comprising a different RAS epitopes (or the recombinant nucleic acid encoding the RAS polypeptide) administered in a subject can lead to increased cytokine release by the RAS 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 that in a subject administered with the full-length RAS polypeptide or a recombinant nucleic acid encoding the full
  • APCs having (e.g., transfected or otherwise delivered with) the multiepitopic polypeptide comprising a RAS polypeptide comprising different RAS epitopes (or the recombinant nucleic acid encoding the RAS polypeptide) can lead to increased cytokine release by the RAS 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 RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • Cytokine release can be measured using a cytometric bead assay, ELISA, or any other suitable method of quantifying levels of cytokine release.
  • the first RAS epitope sequence and the second RAS epitope sequence is separated by a linker.
  • the first RAS amino acid sequence is the first epitope sequence.
  • the second RAS amino acid sequence is the second epitope sequence.
  • the first RAS amino acid sequence is the first epitope sequence and the second RAS amino acid sequence is the second epitope sequence.
  • the first RAS amino acid sequence consists of the first epitope sequence.
  • the second the second RAS amino acid sequence consists of the second epitope sequence.
  • the first RAS amino acid sequence consists of the first epitope sequence and the second the second RAS amino acid sequence consists of the second epitope sequence. [0479] In some embodiments, each different RAS epitope sequence is separated by a linker. [0480] In some embodiments, the first RAS amino acid sequence comprises a first RAS mutation. In some embodiments, the second RAS amino acid sequence comprises a second RAS mutation. In some WSGR Docket No.50401-795.601 embodiments, the second RAS mutation is different from the first RAS mutation. In some embodiments, the second RAS mutation is same as the first RAS mutation.
  • the first RAS amino acid sequence comprises a first RAS mutation
  • the second RAS amino acid sequence comprises a second RAS mutation different from the first RAS mutation.
  • the first RAS amino acid sequence comprises a first RAS mutation
  • the second RAS amino acid sequence comprises a second RAS mutation same as the first RAS mutation.
  • the first RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the second RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the first RAS amino acid sequence is processed into one or more epitopes comprising the first RAS mutation within a cell.
  • the first RAS mutation is G12V, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 27, 58-62 and 65-72. In some embodiments, the first RAS mutation is G12D, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 28, 75-79. In some embodiments, the first RAS mutation is G12C, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096.
  • the first RAS epitope sequence first RAS 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 RAS polypeptide.
  • the second RAS 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 RAS polypeptide.
  • the first RAS 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 RAS polypeptide.
  • the second RAS 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 RAS polypeptide.
  • the first RAS amino acid sequence and the second RAS amino acid sequence is separated by a linker.
  • the first RAS amino acid sequence comprises the first RAS epitope sequence and one, two, three, four, five or more residues flanking the N-terminus or C-terminus of the first RAS epitope sequence from the full length RAS polypeptide.
  • the second RAS amino acid sequence comprises the second RAS epitope sequence and one, two, three, four, five or more residues flanking the N-terminus or C-terminus of the second RAS epitope sequence from the full-length RAS 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 RAS polypeptide.
  • WSGR Docket No.50401-795.601 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 RAS polypeptide.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises the first RAS epitope sequence and a human MHC encoded by an HLA allele.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises the second RAS 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 RAS polypeptide.
  • the method comprises administering to the subject a recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, the method comprises administering to the subject a cell comprising the RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, the RAS polypeptide comprises a RAS epitope sequence. In some embodiments, the subject has been previously administered a T-cell receptor (TCR). In some embodiments, the subject has been previously administered a recombinant nucleic acid encoding the TCR or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR.
  • TCR T-cell receptor
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises the RAS epitope sequence.
  • the peptide:MHC complex comprises a human MHC encoded by an HLA allele.
  • the peptide:MHC complex comprises (i) the RAS epitope sequence and (ii) a human MHC encoded by an HLA allele.
  • a method of treating a subject with a disease or condition can comprise a cancer.
  • the method comprises administering to the subject a T-cell receptor (TCR).
  • TCR T-cell receptor
  • the method comprises administering to the subject a recombinant nucleic acid encoding the TCR. In some embodiments, 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. In some embodiments, the TCR recognizes and binds to a peptide:MHC complex. In some embodiments, the peptide:MHC complex comprises a RAS epitope sequence. In some embodiments, the peptide:MHC complex comprises a human MHC encoded by an HLA allele.
  • the peptide:MHC complex comprises (i) a RAS epitope sequence and (ii) a human MHC encoded by an HLA allele.
  • the subject has been previously administered a RAS polypeptide.
  • the subject has been previously administered a recombinant nucleic acid encoding the RAS polypeptide.
  • the RAS polypeptide comprises the RAS epitope sequence.
  • WSGR Docket No.50401-795.601 [0489] Also provided herein is a method of treating a subject with a disease or condition.
  • the disease or condition can comprise a cancer.
  • the method comprises administering to the subject a RAS polypeptide.
  • the method comprises administering to the subject a recombinant nucleic acid encoding the RAS polypeptide.
  • the RAS polypeptide comprises a RAS 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 TCR or the recombinant nucleic acid encoding the TCR.
  • the TCR recognizes and binds to a peptide:MHC complex.
  • the peptide:MHC complex comprises a RAS epitope sequence.
  • the peptide:MHC complex comprises a human MHC encoded by an HLA allele. In some embodiments, the peptide:MHC complex comprises (i) a RAS epitope sequence and (ii) a human MHC encoded by an HLA allele. In some embodiments, the method comprises administering to the subject the RAS polypeptide concurrently with the TCR or an immune cell comprising the TCR. In some embodiments, the method comprises administering to the subject the RAS polypeptide concurrently with 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 recombinant nucleic acid encoding the RAS polypeptide concurrently with 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 RAS 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 RAS polypeptide prior to the TCR or an immune cell comprising the TCR.
  • the method comprises administering to the subject the RAS polypeptide prior 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 RAS polypeptide prior 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 RAS 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 RAS polypeptide subsequent to the TCR or an immune cell comprising the TCR. In some embodiments, the method comprises administering to the subject the RAS 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 WSGR Docket No.50401-795.601 nucleic acid encoding the RAS polypeptide subsequent to the TCR or an immune cell comprising the TCR.
  • the method comprises administering to the subject the recombinant nucleic acid encoding the RAS 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 by an immune cell.
  • the method comprises administering a cell comprising the RAS polypeptide or nucleic acid encoding the RAS polypeptide concurrently with administering to the subject a TCR, a recombinant nucleic acid encoding the TCR or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the RAS peptide sequence, and (ii) a human MHC encoded by an HLA allele.
  • the method comprises administering a cell comprising the RAS polypeptide or nucleic acid encoding the RAS polypeptide subsequent to administering to the subject a TCR, a recombinant nucleic acid encoding the TCR, or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the RAS peptide sequence, and (ii) a human MHC encoded by an HLA allele.
  • the method comprises administering a cell comprising the RAS polypeptide or nucleic acid encoding the RAS polypeptide prior to administering to the subject a TCR, a recombinant nucleic acid encoding the TCR, or a cell comprising the TCR or the recombinant nucleic acid encoding the TCR, wherein the TCR recognizes and binds to a peptide:MHC complex, the peptide:MHC complex comprising (i) the RAS peptide sequence, and (ii) a human MHC encoded by an HLA allele.
  • the RAS polypeptide or the recombinant nucleic acid encoding the RAS 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 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 RAS polypeptide or the recombinant nucleic acid encoding the RAS 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 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 an immune cell comprising the TCR or the recombinant nucleic acid encoding the TCR.
  • 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, WSGR Docket No.50401-795.601 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 RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide.
  • 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 RAS polypeptide or the recombinant nucleic acid encoding the RAS polypeptide.
  • the RAS polypeptide is administered at least 1 hour after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS 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 embodiments, the RAS polypeptide is administered at least 4 hours after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 8 hours after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the RAS polypeptide is administered at least 12 hours after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 1 day after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 2 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 5 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 10 days after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the RAS polypeptide is administered at least 20 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 30 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 1 month after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 2 months after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the RAS polypeptide is administered at least 3 months after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the RAS polypeptide is administered at least 6 months after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the RAS polypeptide is administered at least 1 year after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the RAS polypeptide is administered at least 2 years after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the RAS 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 RAS polypeptide is administered at least 1 hour after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 2 hours after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS 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 the RAS polypeptide is administered at least 8 hours after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 12 hours after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 1 day after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the recombinant nucleic acid encoding the RAS polypeptide is administered at least 2 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 5 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS 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 RAS polypeptide is administered at least 20 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 30 days after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 1 month after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the recombinant nucleic acid encoding the RAS polypeptide is administered at least 2 months after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 3 months after the subject has been administered the TCR or an immune cell comprising the TCR. In some WSGR Docket No.50401-795.601 embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 6 months after the subject has been administered the TCR or an immune cell comprising the TCR.
  • the recombinant nucleic acid encoding the RAS polypeptide is administered at least 1 year after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide is administered at least 2 years after the subject has been administered the TCR or an immune cell comprising the TCR. In some embodiments, the recombinant nucleic acid encoding the RAS 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 RAS 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 embodiments, the RAS 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 embodiments, the RAS 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 RAS 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 embodiments, the RAS 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 embodiments, the RAS 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.
  • the RAS 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. In some embodiments, the RAS 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 embodiments, the RAS 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.
  • the RAS 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 embodiments, the RAS polypeptide is administered at least 30 days after the subject has been WSGR Docket No.50401-795.601 administered the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR.
  • the RAS 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 embodiments, the RAS 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. In some embodiments, the RAS 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.
  • the RAS 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. In some embodiments, the RAS 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 embodiments, the RAS 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 RAS 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 RAS 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 RAS 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 embodiments, the recombinant nucleic acid encoding the RAS 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 RAS 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 embodiments, the recombinant nucleic acid encoding the RAS 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 WSGR Docket No.50401-795.601 encoding the RAS 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 embodiments, the recombinant nucleic acid encoding the RAS 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 RAS 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 embodiments, the recombinant nucleic acid encoding the RAS 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.
  • the recombinant nucleic acid encoding the RAS 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 embodiments, the recombinant nucleic acid encoding the RAS 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 RAS 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 embodiments, the recombinant nucleic acid encoding the RAS 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 RAS 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 embodiments, the recombinant nucleic acid encoding the RAS 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 RAS 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 embodiments, the recombinant nucleic acid encoding the RAS 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 RAS WSGR Docket No.50401-795.601 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 RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 2 hours after the subject has been administered the RAS polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 4 hours after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 8 hours after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 12 hours after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 1 day after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 2 days after the subject has been administered the RAS polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 5 days after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 10 days after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 20 days after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 30 days after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 1 month after the subject has been administered the RAS polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 2 months after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 3 months after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 6 months after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 1 year after the subject has been administered the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 2 years after the subject has been administered the RAS polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 3 years or more after the subject has been administered the RAS polypeptide.
  • WSGR Docket No.50401-795.601 [0498]
  • 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 RAS 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 hours after the subject has been administered the RAS polypeptide.
  • 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 RAS polypeptide. In some embodiments, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 8 hours after the subject has been administered the RAS polypeptide. In some embodiments, 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 RAS 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 day after the subject has been administered the RAS polypeptide. In some embodiments, 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 RAS polypeptide. In some embodiments, the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 5 days after the subject has been administered the RAS 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide. In some embodiments, 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 RAS 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide.
  • the recombinant nucleic acid encoding the TCR or an WSGR Docket No.50401-795.601 immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 6 months after the subject has been administered the RAS polypeptide. In some embodiments, 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 RAS 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide. [0499] In some embodiments, 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 RAS 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide.
  • 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide.
  • 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide.
  • 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide. In some WSGR Docket No.50401-795.601 embodiments, the TCR or an immune cell comprising the TCR is administered at least 2 months after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide.
  • 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 RAS polypeptide. In some embodiments, 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 RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 1 year after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide.
  • the TCR or an immune cell comprising the TCR is administered at least 2 years after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, the TCR or an immune cell comprising the TCR is administered at least 3 years or more after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide. [0500] In some embodiments, 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 recombinant nucleic acid encoding the RAS 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 hours after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, 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 recombinant nucleic acid encoding the RAS polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 8 hours after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, 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 RAS 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 day after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, 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 RAS polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic acid encoding the TCR is administered at least 5 days after the subject has WSGR Docket No.50401-795.601 been administered the recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, 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 RAS polypeptide.
  • the recombinant nucleic acid encoding the TCR or an immune cell comprising the recombinant nucleic encoding the TCR is administered at least 20 days after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, 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 RAS 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 recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, 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 RAS polypeptide.
  • 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 RAS polypeptide. In some embodiments, 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 RAS 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 year after the subject has been administered the recombinant nucleic acid encoding the RAS polypeptide. In some embodiments, 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 RAS polypeptide.
  • 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 RAS polypeptide.
  • the TCR is expressed by a cell.
  • 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. In some embodiments, the method comprises administering two or more recombinant nucleic acids encoding the two or more different TCRs. In some embodiments, the two or more TCRs comprise a first TCR and a second TCR. In some embodiments, the two or more different TCRs are expressed on the surface of two different immune cells. In some embodiments, 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 having a G12V mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA- A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA- DRB1*07:01, HLA-A*03:02, and HLA-A*03:05
  • the second TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12D mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*08:02, HLA-C*03:04, HLA- C*05:01, HLA-A*-3:01, HLA
  • the TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12V mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA- A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA- DRB1*07:01, HLA-A*03:02, and HLA-A*03:05
  • the second TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12C mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*-3:01, HLA-A*11:01, HLA- A*68:01, and HLA-C*03:03.
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12D mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*08:02, HLA-C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA-A*11:01, and HLA-A*68:01.
  • the second TCR binds to a peptide:MHC complex comprising an epitope sequence having a G12C mutation and an MHC encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*-3:01, HLA-A*11:01, HLA- A*68:01, and HLA-C*03:03.
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 27, 58-62 and 65-72 and an MHC encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA- A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA- DRB1*07:01, HLA-A*03:02, and HLA-A*03:05 and the second TCR binds to a peptide:MHC WSGR Docket No.50401-795.601 complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 28, 75- 79 and an MHC encoded by an HLA allele selected from the group consisting of HLA-B
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 27, 58-62 and 65-72 and an MHC encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA-DRB1*07:01 and, HLA-A*03 and the second TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096 and an MHC encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*-3:01, HLA-A*11:01
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 28, 75-79 and an MHC encoded by an HLA allele selected from the group consisting of HLA- B*07:02, HLA-C*08:02, HLA-C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA-A*11:01, and HLA- A*68:01 and the second TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096 and an MHC encoded by an HLA allele selected from the group consisting HLA-DRB1*11:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • the RAS polypeptide does not comprise a full-length RAS protein sequence.
  • the RAS polypeptide comprises a RAS epitope sequence.
  • the RAS 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 NOs: 58 or 59.
  • the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NOs: 58 or 59.
  • the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NOs: 58 or 59. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NOs: 58 or 59. In some cases, the HLA allele is an HLA-A allele. In some cases, the HLA allele is selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*03:02, HLA-A*03:05, HLA-B*40:01, and HLA-A*68:01.
  • the RAS 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: 60.
  • the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 60.
  • the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO 60.
  • the RAS epitope sequence comprises a WSGR Docket No.50401-795.601 sequence of SEQ ID NO: 60.
  • the HLA allele is an HLA-C allele. In some embodiments, the HLA allele is an HLA-C*01:02 allele.
  • the RAS 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 NOs: 61 or 62. In some embodiments, the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NOs: 61 or 62.
  • the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NOs: 61 or 62. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NOs: 61 or 62. In some embodiments, the HLA allele is an HLA-C allele. In some embodiments, the HLA allele is an HLA-C*03:03, or HLA-C*03:04. [0507] In some embodiments, the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NOs: 58 or 59.
  • the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NOs: 58 or 59. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NOs: 58 or 59. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NOs: 58 or 59. In some embodiments, the HLA allele is an HLA-A allele. In some embodiments, the HLA allele is an HLA-A*11:01.
  • the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 65-71. In some embodiments, the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 65-71. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 65-71. In some embodiments, the RAS epitope sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 65-71. In some embodiments, the HLA allele is an HLA-DRB1 allele.
  • the HLA allele is an HLA-DRB1*07:01.
  • the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NOs: 58 or 72. In some embodiments, the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NOs: 58 or 72. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NOs: 58 or 72. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NOs: 58 or 72. In some embodiments, the HLA allele is an HLA-A allele.
  • the HLA allele is selected from the group consisting of A*03:01, HLA-A*03:02, and HLA-A*03:05.
  • WSGR Docket No.50401-795.601 [0510]
  • the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NO: 75. In some embodiments, the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 75. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 75. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NO: 75.
  • the HLA allele is an HLA-B allele. In some embodiments, the HLA allele is an HLA-B*07:02. In some embodiments, the HLA allele is an HLA- C allele. In some embodiments, the HLA allele is selected from the group consisting of HLA- C*08:02, HLA-C*03:04, and HLA-C*05:01. [0511] In some embodiments, the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NO: 76. In some embodiments, the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 76.
  • the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 76. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NO: 76. In some embodiments, the HLA allele is an HLA-A allele. In some embodiments, the HLA allele is selected from the group consisting of HLA-A*-3:01, HLA-A*11:01, and HLA-A*68:01. [0512] In some embodiments, the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NO: 77.
  • the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 77. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 77. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NO: 77. In some embodiments, the HLA allele is an HLA-A allele. In some embodiments, the HLA allele is HLA-A*11:01. [0513] In some embodiments, the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NOs: 78 or 79.
  • the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NOs: 78 or 79. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NOs: 78 or 79. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NOs: 78 or 79. In some embodiments, the HLA allele is an HLA-C allele. In some embodiments, the HLA allele is HLA-C*08:02. [0514] In some embodiments, the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NO: 29.
  • the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 29. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to WSGR Docket No.50401-795.601 a sequence of SEQ ID NO: 29. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NO: 29. In some embodiments, the HLA allele is an HLA-DRB1 allele. In some embodiments, the HLA allele is HLA-DRB1*11:01. [0515] In some embodiments, the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NO: 81.
  • the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 81. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 81. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NO: 81. In some embodiments, the HLA allele is an HLA-A allele. In some embodiments, the HLA allele is selected from a group consisting of HLA-A*3:01, HLA-A*11:01, and HLA-A*68:01.
  • the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NO: 1096. In some embodiments, the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 1096. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 1096. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NO: 1096. In some embodiments, the HLA allele is an HLA-A allele.
  • the HLA allele is selected from a group consisting of HLA- A*3:01, HLA-A*11:01, and HLA-A*68:01.
  • the RAS epitope sequence comprises a sequence with at least 70% sequence identity to a sequence of SEQ ID NO: 82. In some embodiments, the RAS epitope sequence comprises a sequence with at least 80% sequence identity to a sequence of SEQ ID NO: 82. In some embodiments, the RAS epitope sequence comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 82. In some embodiments, the RAS epitope sequence comprises a sequence of SEQ ID NO: 82.
  • the HLA allele is an HLA-C allele. In some embodiments, the HLA allele is HLA-C*03:03. [0518]
  • the RAS polypeptide comprises a multiepitopic polypeptide. In some embodiments, the multiepitopic polypeptide does not comprise a full-length RAS polypeptide. In some embodiments, the sequence of the multiepitopic polypeptide is the same RAS epitope sequence. In some embodiments, the multiepitopic polypeptide comprises two or more different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 2 different RAS epitope sequences.
  • the multiepitopic polypeptide comprises at least 3 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 4 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 5 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide WSGR Docket No.50401-795.601 comprises at least 6 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 8 different RAS epitope sequences. In some embodiments, the multiepitopic polypeptide comprises at least 9 different RAS epitope sequences.
  • the multiepitopic polypeptide comprises at least 10 or more different RAS epitope sequences. In some embodiments, the different RAS epitope sequences are separated. In some embodiments, the different RAS epitope sequences are separated by linker sequences. In some embodiments, the RAS polypeptide is a multiepitopic polypeptide. In some embodiments, the multiepitopic polypeptide is a RAS polypeptide. [0519] In some embodiments, antigen presenting cells (APCs) of subject administered the RAS polypeptide present a RAS epitope sequence as a peptide:MHC complex.
  • APCs antigen presenting cells
  • antigen presenting cells of subject administered the nucleic acid encoding the RAS polypeptide comprising the multiepitopic polypeptide present a RAS epitope sequence as a peptide:MHC complex.
  • the APCs present more of a RAS epitope as a peptide:MHC complex compared to the APCs of a subject administered a full length RAS polypeptide or recombinant nucleic acid encoding the full-length RAS 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 RAS polypeptide or a recombinant nucleic acid encoding the full-length RAS polypeptide.
  • the multiepitopic polypeptide comprises a first RAS amino acid sequence comprising a first RAS epitope sequence.
  • the multiepitopic polypeptide comprises a second RAS amino acid sequence comprising a second RAS epitope sequence.
  • the first RAS epitope sequence and the second RAS epitope sequence are different. In some embodiments, the first RAS epitope sequence and the second RAS epitope sequence is separated by a linker.
  • the first amino acid sequence is the first epitope sequence. In some embodiments the second amino acid sequence is the second epitope sequence. In some embodiments, the first amino acid sequence is the first epitope sequence, and the second amino acid sequence is the second epitope sequence. In some embodiments, the first amino acid sequence consists of the first epitope sequence. In some embodiments, the second amino acid sequence consists of 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 RAS amino acid sequence comprises a first RAS mutation.
  • the second RAS amino acid sequence comprises a second RAS mutation.
  • the first RAS mutation is different from the second RAS mutation.
  • the first RAS amino acid sequence comprises a first RAS mutation
  • the second RAS amino acid sequence comprises a second RAS mutation.
  • the first RAS amino acid sequence comprises a first RAS mutation
  • the second RAS amino acid sequence comprises a second RAS mutation different from the first RAS mutation.
  • the first RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the second RAS mutation is selected from the group consisting of G12V, G12D, and G12C.
  • the first RAS amino acid sequence is processed into one or more epitopes comprising the first RAS mutation within a cell.
  • the first RAS amino acid sequence is processed into one or more epitopes comprising the first RAS mutation within a cell.
  • the first RAS mutation is G12V, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 27, 58-62 and 65-72.
  • the first RAS mutation is G12D, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 28, 75-79. In some embodiments, the first RAS mutation is G12C, and the one or more epitopes are selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096. [0525] In some embodiments, the first RAS 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 RAS polypeptide.
  • the second RAS 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 RAS polypeptide.
  • the first RAS 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 RAS polypeptide.
  • the second RAS 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 RAS polypeptide.
  • the first RAS amino acid sequence and the second RAS amino acid sequence is separated by a linker.
  • the first amino acid sequence comprises the first RAS epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the first RAS epitope sequence from the full length RAS polypeptide.
  • the second amino acid sequence comprises the second RAS epitope sequence and one or more residues flanking the N-terminus or the C-terminus of the second RAS epitope sequence from the full length RAS polypeptide.
  • the multiepitopic polypeptide does not comprise more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive amino acids from the full-length RAS polypeptide.
  • the multiepitopic polypeptide comprises a first RAS epitope sequence.
  • the first RAS epitope sequence is operably linked to a second RAS epitope sequence.
  • the first RAS epitope sequence and the second RAS epitope sequence are the same.
  • the first RAS epitope sequence and second RAS epitope sequence are different.
  • the first RAS epitope sequence and second RAS epitope sequence are presentable by different HLA alleles. In some embodiments, the first RAS epitope sequence and second RAS epitope sequence are presented by different HLA alleles. In some embodiments, the first RAS epitope sequence and second RAS epitope sequence are predicted to bind to different HLA alleles. In some embodiments, the first RAS epitope sequence and second RAS epitope sequence are predicted to be presented by different HLA alleles. [0530] In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 500nM.
  • a first RAS epitope sequence binds to a first HLA allele with a KD less than 400nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 300nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele. In some embodiments, a first RAS epitope sequence is predicted to bind to a first HLA allele. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 200nM.
  • a first RAS epitope sequence binds to a first HLA allele with a KD less than 100nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 50nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD less than 1nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele. In some embodiments, a first RAS epitope sequence is predicted to bind to a second HLA allele.
  • a first RAS epitope sequence binds to a second HLA allele with a KD of more than 200nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 300nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 500nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 600nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 700nM.
  • a first RAS epitope sequence binds to a second HLA allele with a KD of more than 800nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 900nM. In some embodiments, a first RAS epitope sequence binds to a second HLA allele with a KD of more than 1000nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD of less than 1nM and binds to second HLA allele with WSGR Docket No.50401-795.601 a KD of more than 200nM.
  • a first RAS epitope sequence binds to a first HLA allele with a KD of less than 50nM and binds to second HLA allele with a KD of more than 300nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD of less than 100nM and binds to second HLA allele with a KD of more than 500nM. In some embodiments, a first RAS epitope sequence binds to a first HLA allele with a KD of less than 200 nM and binds to second HLA allele with a KD of more than 600nM.
  • the first RAS epitope sequence is operably linked to the second RAS epitope sequence. In some aspects, the first RAS epitope sequence is operably linked to the second RAS epitope sequence 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112. [0532] In some embodiments, the multiepitopic polypeptide comprises a first RAS epitope sequence, operably linked to a second RAS epitope sequence, operably linked to a third RAS epitope sequence.
  • the multiepitopic polypeptide comprises a first amino acid sequence comprising a first RAS epitope sequence, operably linked to a second amino acid sequence comprising a second RAS epitope sequence, operably linked to a third amino acid sequence comprising a third RAS epitope sequence.
  • the first RAS epitope sequence, the second RAS epitope sequence, or the third RAS epitope sequence comprises a RAS mutation selected from the group consisting of G12V, G12D, and G12C.
  • the first RAS epitope sequence, the second RAS epitope sequence, and the third RAS epitope sequence comprises a RAS mutation selected from the group consisting of G12V, G12D, and G12C. In some embodiments, the first RAS epitope sequence, the second RAS epitope sequence, and the third RAS epitope sequence comprise different RAS mutations. In some embodiments, the first RAS epitope sequence comprises a G12V mutation. In some embodiments, the first RAS epitope sequence comprises a G12D mutation. In some embodiments, the first RAS epitope sequence comprises a G12C mutation. In some embodiments, the second RAS epitope sequence comprises a G12V mutation.
  • the second RAS epitope sequence comprises a G12D mutation. In some embodiments, the second RAS epitope sequence comprises a G12C mutation. In some embodiments, the third RAS epitope sequence comprises a G12V mutation. In some embodiments, the third RAS epitope sequence comprises a G12D mutation. In some embodiments, the third RAS epitope sequence comprises a G12C mutation.
  • the first RAS epitope sequence is presentable by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA- A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA- DRB1*07:01, HLA-A*03:02, and HLA-A*03:05.
  • the first RAS epitope sequence is presented by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA- C*01:02, HLA-C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-A*03:02, and HLA-A*03:05.
  • the first RAS epitope sequence binds to an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA- A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA- A*03:02, and HLA-A*03:05.
  • the first RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA allele selected from the group consisting of HLA- A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA- C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-A*03:02, and HLA-A*03:05.
  • HLA- A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA- C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-A*03:02, and HLA-A*03:05 HLA- A*11:01, HLA-A*03
  • the first RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA- A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA- DRB1*07:01, HLA-A*03:02, and HLA-A*03:05.
  • the second RAS epitope sequence is presentable by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*03:04, HLA- C*05:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*08:02.
  • the second RAS epitope sequence is presented by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*08:02.
  • the second RAS epitope sequence binds to an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*08:02.
  • the second RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA- C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*08:02.
  • the second RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*03:04, HLA- C*05:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*08:02.
  • the third RAS epitope sequence is presentable by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • the third RAS epitope WSGR Docket No.50401-795.601 sequence is presented by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • the third RAS epitope sequence binds to an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • the third RAS epitope sequence is predicted to bind to an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • the third RAS epitope sequence is predicted to be presented by an MHC molecule encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • the subject does not express an HLA that binds to each RAS epitope of the multiepitopic polypeptide. In some cases, the subject only expresses HLA alleles that bind to a subset of RAS epitope sequences of the multiepitopic polypeptide.
  • the RAS 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 one or more sequences selected from the group consisting of SEQ ID NOs: 27- 29.
  • the RAS polypeptide comprises a sequence with at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises a sequence with at least 80% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises a sequence with at least 90% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises one or more sequences selected from the group consisting of SEQ ID NOs: 27- 29.
  • the RAS 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 selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises a sequence with at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises a sequence with at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 27-29. WSGR Docket No.50401-795.601 [0540] In some embodiments, the RAS 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 one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises one or more copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS 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 selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27- 29. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence with at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises one or more copies of a sequence selected from the group consisting of SEQ ID NOs: 27-29. [0542] In some embodiments, the RAS 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 one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises two copies of a sequence with at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 80% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises two copies of one or more sequences with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises two copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS 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 each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 70% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises two copies of a sequence with at least 80% sequence identity to each sequence of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises two copies of one or more sequences with at least 90% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises two copies of each sequence of SEQ ID NOs: 27-29. [0544] In some embodiments, the RAS 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 one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises three copies of a sequence with at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 80% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises three copies of one or more sequences with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises three copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS 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 each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 70% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises three copies of a sequence with at least 80% sequence identity to each sequence of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises three copies of one or more sequences with at least 90% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises three copies of each sequence of SEQ ID NOs: 27-29. [0546] In some embodiments, the RAS 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 one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises four copies of a sequence with at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least WSGR Docket No.50401-795.601 80% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises four copies of one or more sequences with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises four copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. [0547] In some embodiments, the RAS 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 each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 70% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises four copies of a sequence with at least 80% sequence identity to each sequence of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises four copies of one or more sequences with at least 90% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises four copies of each sequence of SEQ ID NOs: 27-29. [0548] In some embodiments, the RAS 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 one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises five copies of a sequence with at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 80% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises five copies of one or more sequences with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises five copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS 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 each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 70% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises five copies of a sequence with at least 80% sequence identity to each sequence of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises five copies of one or more sequences with at least 90% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises five copies of each sequence of SEQ ID NOs: 27-29.
  • WSGR Docket No.50401-795.601 [0550] In some embodiments, the RAS 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 one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS polypeptide comprises six or more copies of a sequence with at least 70% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 80% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises six or more copies of one or more sequences with at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises six or more copies of one or more sequences selected from the group consisting of SEQ ID NOs: 27-29.
  • the RAS 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 each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 70% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises six or more copies of a sequence with at least 80% sequence identity to each sequence of SEQ ID NOs: 27- 29.
  • the RAS polypeptide comprises six or more copies of one or more sequences with at least 90% sequence identity to each sequence of SEQ ID NOs: 27-29. In some embodiments, the RAS polypeptide comprises six or more copies of each sequence of SEQ ID NOs: 27-29.
  • the RAS 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: 27, 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: 29, 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: 28.
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 27, operably linked to the sequence of SEQ ID NO: 29, operably linked to the sequence of SEQ ID NO: 28.
  • the RAS 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: 27, operably linked to a WSGR Docket No.50401-795.601 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: 29 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
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 27, operably linked to the sequence of SEQ ID NO: 29 via a linker, and the sequence of SEQ ID NO: 29 operably linked to the sequence of SEQ ID NO: 28 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the linker is a cleavable linker. 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS 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: 29, 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: 28, 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: 27.
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 29, operably linked to the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 27.
  • the RAS 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: 29, 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: 28 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:
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 29, operably linked to the sequence of SEQ ID NO: 28 via a linker, and the sequence of SEQ ID NO: 28 operably linked to the sequence of SEQ ID NO: 27 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the linker is a cleavable linker. 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS 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: 28, 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: 27, 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: 29.
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 27, operably linked to the sequence of SEQ ID NO: 29.
  • the RAS 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: 28, 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: 27 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:
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 27 via a linker, and the sequence of SEQ ID NO: 27 operably linked to the sequence of SEQ ID NO: 29 via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a sequence WSGR Docket No.50401-795.601 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.
  • the linker comprises a sequence of SEQ ID NO: 31. In some embodiments, the linker is a cleavable linker. 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS 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: 27, 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: 28, 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: 29.
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 27, operably linked to the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 29.
  • the RAS 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: 27, 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: 28 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:
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 27, operably linked to the sequence of SEQ ID NO: 28 via a linker, and the sequence of SEQ ID NO: 28 operably linked to the sequence of SEQ ID NO: 29 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • 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 WSGR Docket No.50401-795.601 identity to a sequence of SEQ ID NO: 112.
  • the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS 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: 28, 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: 29, 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: 27.
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 29, operably linked to the sequence of SEQ ID NO: 27.
  • the RAS 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: 28, 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: 29 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:
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 28, operably linked to the sequence of SEQ ID NO: 29 via a linker, and the sequence of SEQ ID NO: 29 operably linked to the sequence of SEQ ID NO: 27 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the linker is a cleavable linker. 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS 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: 29, operably WSGR Docket No.50401-795.601 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: 27, 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: 28.
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 29, operably linked to the sequence of SEQ ID NO: 27, operably linked to the sequence of SEQ ID NO: 28.
  • the RAS 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: 29, 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: 27 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:
  • the RAS polypeptide comprises, from N terminus to C terminus, the sequence of SEQ ID NO: 29, operably linked to the sequence of SEQ ID NO: 27 via a linker, and the sequence of SEQ ID NO: 27 operably linked to the sequence of SEQ ID NO: 28 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: 31.
  • the linker comprises a sequence of SEQ ID NO: 31.
  • the linker is a cleavable linker. 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS polypeptide comprises at least two copies of a multiepitopic polypeptide, and wherein each copy 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: 29, 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: 28, and 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: 27.
  • the RAS polypeptide comprises at least two copies of a multiepitopic polypeptide, and wherein each copy comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 29, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises three copies of the multiepitopic polypeptide.
  • the RAS polypeptide comprises five copies of the multiepitopic polypeptide.
  • the RAS polypeptide comprises at three copies of a multiepitopic polypeptide, and wherein each copy 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: 29, 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: 28, and 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: 27.
  • the RAS polypeptide comprises at three copies of a multiepitopic polypeptide, and wherein each copy comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 29, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises at four copies of a multiepitopic polypeptide, and wherein each copy 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: 29, 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: 28, and 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: 27.
  • the RAS polypeptide comprises at four copies of a multiepitopic polypeptide, and wherein each copy comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 29, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide comprises at five copies of a multiepitopic polypeptide, and wherein each copy 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: 29, 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 WSGR Docket No.50401-795.601 least 95%, or 100% sequence identity to the sequence of SEQ ID NO: 28, and 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: 27.
  • the RAS polypeptide comprises at five copies of a multiepitopic polypeptide, and wherein each copy comprises, from N terminus to C terminus, a sequence of SEQ ID NO: 29, operably linked to a sequence of SEQ ID NO: 28, and operably linked to a sequence of SEQ ID NO: 27.
  • the RAS polypeptide further comprises a secretory domain (Sec) sequence.
  • the Sec sequence is at the N terminus of the multiepitopic polypeptide.
  • 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: 32.
  • the Sec sequence comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 32.
  • the Sec sequence comprises a sequence of SEQ ID NO: 32. [0569] In some cases, the Sec sequence is operably linked to the multiepitopic polypeptide. In some embodiments, 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: 30. In some embodiments, the linker comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 30. In some cases, the linker comprises a sequence of SEQ ID NO: 30. 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: 31.
  • the linker comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 31. In some cases, the linker comprises a sequence of SEQ ID NO: 31. 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: 112. In some embodiments, the linker comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 112. In some cases, the linker comprises a sequence of SEQ ID NO: 112.
  • the RAS polypeptide further comprises an MHC Class I trafficking domain (MITD) sequence.
  • the MITD sequence is at the C terminus of the multiepitopic polypeptide.
  • 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: 33.
  • the MITD sequence comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: WSGR Docket No.50401-795.601 33.
  • the Sec sequence comprises a sequence of SEQ ID NO: 33.
  • the multiepitopic polypeptide is operably linked to the MITD sequence 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: 30.
  • the linker comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 30.
  • the linker comprises a sequence of SEQ ID NO: 30In 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: 112. In some embodiments, the linker comprises a sequence of SEQ ID NO: 112. [0571] In some embodiments, the RAS 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 sequence identity to a sequence of SEQ ID NO: 25.
  • the RAS 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 sequence identity to a sequence of SEQ ID NO: 26. In some embodiments, the RAS 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 sequence identity to a sequence of SEQ ID NO: 113.
  • the recombinant nucleic acid encoding the RAS 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: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 14.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 14.
  • the recombinant nucleic acid encoding the RAS 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: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 15.
  • the WSGR Docket No.50401-795.601 recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS 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: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 16.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 16.
  • the recombinant nucleic acid encoding the RAS 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: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 17.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 17.
  • the recombinant nucleic acid encoding the RAS 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: 18.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence WSGR Docket No.50401-795.601 having at least 60% sequence identity to a sequence of SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the RAS 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: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS 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: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS 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: 21.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 21.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 21.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 21.
  • the recombinant nucleic acid encoding the RAS 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: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 70% sequence identity to a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 22. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS 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: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 9. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 14.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 18.
  • the recombinant nucleic WSGR Docket No.50401-795.601 acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 21.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 22. [0582] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 14.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS 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: 14-23, 84, 86, 88, 92-94, 96-98, 100- 102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS 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: 14. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 15.
  • the recombinant nucleic acid encoding the RAS 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: 16.
  • the recombinant nucleic acid encoding the RAS WSGR Docket No.50401-795.601 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: 17.
  • the recombinant nucleic acid encoding the RAS 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: 18. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 19.
  • the recombinant nucleic acid encoding the RAS 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: 20. In some embodiments, the recombinant nucleic acid encoding the RAS 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: 21.
  • the recombinant nucleic acid encoding the RAS 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: 22.
  • the recombinant nucleic acid encoding the RAS 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: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104- 106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 16.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ WSGR Docket No.50401-795.601 ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence with at least 90% sequence identity to a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14- 23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 19.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises one or more copies of a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid WSGR Docket No.50401-795.601 encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises two copies of a sequence of SEQ ID NO: 22. [0587] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of a sequence of SEQ ID NO: 22. WSGR Docket No.50401-795.601 [0589] In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 15. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 18.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 20. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of one or more sequences selected from the group consisting of SEQ ID NOs: 14-23, 84, 86, 88, 92-94, 96-98, 100-102, 104-106, 108-110. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 14. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 15.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 16. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 18. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 19. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 20.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 21. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of a sequence of SEQ ID NO: 22.
  • the recombinant nucleic acid encoding the RAS 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 WSGR Docket No.50401-795.601 SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, 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: 20-23, 88, 94, 98, 102, 106, and 110, and operably linked to a sequence 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%
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, and operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence of any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, and operably linked to a sequence of any one of SEQ ID NO: 17-19, 86, 93, 97, 101, 105, and 109.
  • the recombinant nucleic acid encoding the RAS 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: 14-16, 84, 92, 96, 100, 104, and 108, 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: 20-23, 88, 94, 98, 102, 106, and 110, 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 any one of SEQ ID NOs:
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, via a linker sequence and the sequence having at least 90% sequence identity to any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence a sequence having at least 90% WSGR Docket No.50401-795.601 sequence identity to any one of SEQ ID NO: 17-19, 86, 93, 97, 101, 105, and 109 via a linker sequence.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence of any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, via a linker sequence and the sequence of any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 operably linked to a sequence of any one of SEQ ID NO: 17-19, 86, 93, 97, 101, 105, and 109 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: 5.
  • 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: 6.
  • 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. In some embodiments, the linker sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 9. 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: 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: 11. 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: 12.
  • 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: 13.
  • the linker sequence comprises a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence of SEQ ID NO: 5.
  • the linker sequence comprises a sequence of SEQ ID NO: 6.
  • the linker sequence comprises a sequence of SEQ ID NO: 8.
  • the linker sequence comprises a sequence of SEQ ID NO: 9.
  • the linker sequence comprises a sequence of SEQ ID NO: 10.
  • the linker sequence comprises a sequence of SEQ ID NO: 11. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 12. In some embodiments, the linker sequence comprises a sequence of SEQ ID NO: 13. WSGR Docket No.50401-795.601 [0598] In some embodiments, the recombinant nucleic acid encoding the RAS 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: 20-23, 88, 94, 98, 102, 106, and 110, 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: 17-19,
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110 operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14- 16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence of any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence of any one of SEQ ID NO: 14-16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS 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: 20-23, 88, 94, 98, 102, 106, and 110, 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: 17-19, 86, 93, 97, 101, 105, and 109, 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 any one of SEQ ID NOs:
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence a sequence having at least 90% sequence WSGR Docket No.50401-795.601 identity to any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, via a linker sequence and the sequence having at least 90% sequence identity to any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, via a linker sequence.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence of any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, via a linker sequence and the sequence of any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence of any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, 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: 5.
  • 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: 6.
  • 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. 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: 9.
  • 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. 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: 11.
  • 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: 13.
  • the recombinant nucleic acid encoding the RAS 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: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence a sequence having at least 60%, at WSGR Docket No.50401-795.601 least 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: 14-16, 84, 92, 96, 100, 104, and 108, 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 least 85%, at least 90%,
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, and operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NO: 20-23, 88, 94, 98, 102, 106, and 110.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence of any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, and operably linked to a sequence of any one of SEQ ID NO: 20-23, 88, 94, 98, 102, 106, and 110.
  • the recombinant nucleic acid encoding the RAS 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: 17-19, 86, 93, 97, 101, 105, and 109, 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: 14-16, 84, 92, 96, 100, 104, and 108, 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 any one of
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, via a linker sequence and the sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NO: 20-23, 88, 94, 98, 102, 106, and 110, via a linker sequence.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, operably linked to a sequence of any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, via a linker sequence and the sequence of any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108, operably linked to a sequence of any one of SEQ ID NO: 20-23, 88, 94, 98, 102, 106, and 110, 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: 5.
  • 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: 6.
  • 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. 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: 9.
  • 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. 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: 11.
  • 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.
  • 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: 13.
  • the recombinant nucleic acid encoding the RAS 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: 20-23, 88, 94, 98, 102, 106, and 110, 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: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence a sequence having at least 60%, at least 65%, at least 70%, at least 75%, WSGR Docket No.50401-795.601 at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to any one of SEQ ID NOs: 20
  • 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: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 14-16, 84, 92, 96, 100, 104, and 108.
  • each string of sequences comprises, from 5’ end to 3’ end, a sequence of any one of SEQ ID NOs: 20-23, 88, 94, 98, 102, 106, and 110, operably linked to a sequence of any one of SEQ ID NOs: 17-19, 86, 93, 97, 101, 105, and 109, and operably linked to a sequence of any one of SEQ ID NO: 14-16, 84, 92, 96, 100, 104, and 108.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises three copies of the string of sequences.
  • the recombinant nucleic acid encoding the RAS polypeptide comprises four copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises five copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises six copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises seven copies of the string of sequences. In some embodiments, the recombinant nucleic acid encoding the RAS polypeptide comprises eight copies of the string of sequences.
  • the recombinant nucleic acid encoding the RAS 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: 3.
  • the string of sequences is operably linked to the Sec sequence.
  • 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: 4. 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: 7. In some embodiments, the linker comprises a sequence of SEQ ID NO: 4. In some embodiments, the linker comprises a sequence of SEQ ID NO: 7.
  • the recombinant nucleic acid encoding the RAS polypeptide further comprises a sequence encoding an MHC I Trafficking Domain (MITD) sequence.
  • 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: 24 or 90.
  • the string of sequences is operably linked to the MITD sequence.
  • 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: 4.
  • 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: 7.
  • the linker comprises a sequence of SEQ ID NO: 4.
  • the linker comprises a sequence of SEQ ID NO: 7.
  • 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: 2.
  • 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: 91. 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: 95.
  • 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: 99. 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: 103.
  • 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: 107. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60%, at least 65%, at least 70%, at WSGR Docket No.50401-795.601 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: 111.
  • the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 2. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 91. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 95. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 99.
  • the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 103. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 107. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 60% sequence identity to a sequence of SEQ ID NO: 111. [0619] In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2.
  • the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 91. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 95. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 99. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 103.
  • the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 107. In some embodiments, the recombinant nucleic acid comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 111. [0620] In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 1. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 2. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 91. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 95.
  • the recombinant nucleic acid comprises a sequence of SEQ ID NO: 99. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 103. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 107. In some embodiments, the recombinant nucleic acid comprises a sequence of SEQ ID NO: 111. WSGR Docket No.50401-795.601 [0621] In some embodiments, the subject expresses more of the RAS epitope when administered with the codon optimized recombinant nucleic acid than administered with wild-type recombinant nucleic acid.
  • the TCR comprises a beta chain construct. In some embodiments, the TCR comprises an alpha chain construct. In some embodiments, the TCR comprises a beta chain construct and an alpha chain construct. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3). In some embodiments 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: 57. In some embodiments the TCR beta chain construct comprises a CDR3 having an amino acid sequence set forth in SEQ ID NO: 57. In some embodiments, the TCR beta chain construct comprises a variable region.
  • CDR3 complementarity determining region 3
  • 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: 52 or SEQ ID NO: 53. 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: 55. In some embodiments, the TCR beta chain construct comprises a CDR1 having an amino acid set forth in SEQ ID NO: 55. 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: 56. In some embodiments, the TCR beta chain construct comprises a CDR2 having an amino acid sequence set forth in SEQ ID NO: 56. [0623] 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: 46. In some embodiments, the alpha chain construct comprises a CDR1 having an amino acid sequence set forth in SEQ ID NO: 46. 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: 47. In some embodiments, the alpha chain construct comprises a CDR2 having an amino acid sequence set forth in SEQ ID NO: 47.
  • the alpha 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: 100. In some embodiments, the alpha chain construct comprises a CDR3 having an amino acid WSGR Docket No.50401-795.601 sequence set forth in SEQ ID NO: 48. In some embodiments, the alpha chain construct comprises a variable region. In some embodiments the alpha 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: 43 or SEQ ID NO: 44.
  • the alpha chain construct comprises a variable region having an amino acid sequence set forth in SEQ ID NO: 43 or SEQ ID NO: 44.
  • 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: 49 or SEQ ID NO: 51.
  • the TCR comprises a beta chain having an amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO: 51.
  • 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: 40 or SEQ ID NO: 42.
  • the TCR comprises an alpha chain having an amino acid sequence set forth in SEQ ID NO: 40 or SEQ ID NO: 42. [0625] In some embodiments, the TCR comprises a beta chain construct. In some embodiments, the TCR comprises an alpha chain construct. In some embodiments, the TCR comprises a beta chain construct and an alpha chain construct. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 3 (CDR3). In some embodiments 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 of any one of TCR beta chain CDR3 presented in Tables 8A-8D.
  • CDR3 complementarity determining region 3
  • the TCR beta chain construct comprises a CDR3 having an amino acid sequence of any one TCR beta chain CDR3 presented in Tables 8A-8D. In some embodiments, the TCR beta chain construct comprises a variable region. In some embodiments 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 of any one TCR beta variable domain presented in Tables 8A-8D. 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 of any one TCR beta chain CDR1 presented in Tables 8A-8D.
  • the TCR beta chain construct comprises a CDR1 having an amino acid sequence of any one TCR beta chain CDR1 presented in Tables 8A-8D. In some embodiments, the TCR beta chain construct comprises a CDR2. In some embodiments, 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 any one TCR beta chain CDR2 presented in Tables 8A-8D. In some embodiments, the TCR beta chain WSGR Docket No.50401-795.601 construct comprises a CDR2 having an amino acid sequence of any one TCR beta chain CDR2 presented in Tables 8A-8D. [0626] In some embodiments, the TCR comprises an alpha chain construct.
  • 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. In some embodiments, 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 of any one TCR alpha chain CDR1 presented in Tables 8A-8D. In some embodiments, the alpha chain construct comprises a CDR1 having an amino acid sequence of any one TCR alpha chain CDR1 presented in Tables 8A- 8D.
  • 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 of any one TCR alpha chain CDR2 presented in Tables 8A-8D. In some embodiments, the alpha chain construct comprises a CDR2 having an amino acid sequence of any one TCR alpha chain CDR2 presented in Tables 8A-8D. In some embodiments, the alpha 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 of any one TCR alpha chain CDR3 presented in Tables 8A-8D.
  • the alpha chain construct comprises a CDR3 having an amino acid sequence of any one TCR alpha chain CDR3 presented in Tables 8A-8D. In some embodiments, the alpha chain construct comprises a variable region. In some embodiments the alpha 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 of any one TCR alpha variable domain presented in Tables 8A-8D. In some embodiments the alpha chain construct comprises a variable region having an amino acid sequence of any one TCR alpha variable domain presented in Tables 8A-8D.
  • 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 of any one TCR beta chain presented in Tables 8A-8D. In some embodiments, the TCR comprises a beta chain having an amino acid sequence of any one TCR beta chain presented in Tables 8A-8D. 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 of any one TCR alpha chain presented in Tables 8A-8D. In some embodiments, the TCR comprises an alpha chain having an amino acid sequence of any one TCR alpha chain presented in Tables 8A-8D. [0628] Table 8A.
  • 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 multiepitopic polypeptide comprises a second amino acid sequence comprising a second epitope sequence from the cancer protein.
  • the first epitope sequence and the second epitope sequence are the same.
  • the first epitope sequence and the second epitope sequence are different.
  • 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 subject has been previously treated with a TCR. In some embodiments, the subject has been previously treated with a recombinant 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 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 therapy comprises a cell comprising the TCR. In some embodiments, the therapy comprises a cell comprising a recombinant nucleic acid encoding the TCR. In some embodiments, the TCR recognizes and binds to a peptide:MHC complex. In some embodiments, the peptide:MHC complex comprises an epitope sequence form a cancer protein and a human MHC encoded by an HLA allele. In some embodiments, the subject has been previously treated with a multiepitopic polypeptide. In some embodiments, 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 WSGR Docket No.50401-795.601 subject has been previously treated with a cell comprising a 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 multiepitopic polypeptide comprises a second amino acid sequence comprising a second epitope sequence from the cancer protein.
  • the first epitope sequence and the second epitope sequence are the same.
  • the first epitope sequence and the second epitope sequence are different. In some embodiments, the first epitope sequence or the second epitope sequence are linked by a linker. In some embodiments, the multiepitopic polypeptide does not comprise the full-length cancer protein. In some embodiments the first epitope sequence or the second epitope sequence is the epitope sequence recognized by the TCR. [0634] Provided herein is a method of treating a subject with a disease or condition. In some embodiments, the disease or condition is a cancer. In some embodiments, the method comprises administering to the subject a first therapy. In some embodiments the first therapy comprises a multiepitopic polypeptide.
  • the first therapy comprises a recombinant nucleic acid encoding the multiepitopic polypeptide. In some embodiments, the first therapy comprises a cell comprising a multiepitopic polypeptide. In some embodiments, the first therapy comprises a cell comprising a recombinant nucleic acid encoding the multiepitopic polypeptide. In some embodiments, the multiepitopic polypeptide comprises a first amino acid sequence comprising a first epitope sequence from a cancer protein. In some embodiments, the multiepitopic polypeptide comprises a second amino acid sequence comprising a second epitope sequence from the cancer protein. In some embodiments, the first epitope sequence and the second epitope sequence are the same.
  • the first epitope sequence and the second epitope sequence are different. In some embodiments, the first epitope sequence or the second epitope sequence are linked by a linker. In some embodiments, the multiepitopic polypeptide does not comprise the full-length cancer protein. In some embodiments, the method comprises administering to the subject a second therapy. In some embodiments, the second therapy comprises a TCR. In some embodiments, the second therapy comprises a recombinant nucleic acid encoding the TCR. In some embodiments, the second therapy comprises a cell comprising a TCR. In some embodiments, the second therapy comprises a cell comprising 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 peptide:MHC complex comprises the second epitope sequence and a human MHC encoded by an HLA allele.
  • the first therapy is administered prior to the second therapy. In some embodiments, the first therapy is administered concurrently with the second therapy. In some embodiments, the first therapy is administered subsequent to the second therapy.
  • presentation of the first and/or second epitope sequence as a peptide:MHC complex by antigen presenting cells (APCs) of the subject is higher than the presentation of the first and/or second epitope sequence as the peptide:MHC complex by the APCs of a subject administered the full-length cancer protein or a recombinant nucleic acid encoding the full-length cancer protein.
  • the cancer protein is RAS.
  • 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 to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 27, 58-62 and 65-72 and an MHC encoded by an HLA allele selected from the group consisting of HLA-A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA-C*03:03, HLA-C*03:04, HLA-DRB1*07:01, HLA-A*03:02, and HLA-A*03:05 and the second TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 28, 75-79
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 27, 58- 62 and 65-72 and an MHC encoded by an HLA allele selected from the group consisting of HLA- A*11:01, HLA-A*03:01, HLA-A*30:01, HLA-A*68:01, HLA-B*40:01, HLA-C*01:02, HLA- C*03:03, HLA-C*03:04, HLA-DRB1*07:01 and, HLA-A*03 and the second TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096 and an MHC encoded by an HLA allele selected from the group consisting of HLA-DRB1*11:01, HLA-A*-3:01, HLA-A*11:
  • the first TCR binds to a peptide:MHC complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 28, 75-79 and an MHC encoded by an HLA allele selected from the group consisting of HLA-B*07:02, HLA-C*08:02, HLA-C*03:04, HLA-C*05:01, HLA-A*-3:01, HLA-A*11:01, and HLA-A*68:01 and the second TCR binds to a peptide:MHC WSGR Docket No.50401-795.601 complex comprising an epitope sequence selected from the group consisting of SEQ ID NOs: 29, 81, 82, and 1096 and an MHC encoded by an HLA allele selected from the group consisting HLA- DRB1*11:01, HLA-A*-3:01, HLA-A*11:01, HLA-A*68:01, and HLA-C*03:03.
  • 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.
  • the APC has been incubated with the multiepitopic polypeptide.
  • the APC has been incubated with the recombinant nucleic acid encoding the multiepitopic polypeptide.
  • the multiepitopic polypeptide comprises at least 2, 3, 4, 5, or more different epitope sequences derived from a cancer protein. [0637]
  • binding of the TCR to the peptide:MHC complex results in production of a cytokine by the immune cell.
  • the cytokine is IFN- ⁇ .
  • the cytokine is IL-2.
  • the cytokine is TNF- ⁇ . In some embodiments, production of the cytokine is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to an otherwise identical TCR recognizing an irrelevant peptide:MHC complex. In some embodiments, production of the cytokine is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more compared to an otherwise identical APC presenting a wild type RAS antigen.
  • T Cell Activation [0638]
  • the methods and compositions disclosed herein can comprise RAS-specific T cells that can be delivered to a subject having cancer.
  • RAS -specific T cells can be generated by NEO-STIM, a process of T cell activation and/or expansion.
  • Generating antigen specific (e.g., RAS -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 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 WSGR Docket No.50401-795.601 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 methods provided herein can be used to expand antigen specific CD8+ T cells having memory phenotype.
  • the therapeutic compositions can comprise antigen specific CD8+ T cells.
  • the therapeutic compositions can comprise antigen specific memory T cells.
  • T cells can be activated ex vivo with a composition comprising antigenic peptides or polynucleotides encoding the antigenic peptides.
  • T cells can be activated ex vivo with a composition comprising antigen loaded antigen- presenting cells.
  • the APCs and/or T cells are derived from a biological sample which is obtained from a subject.
  • 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.
  • the compositions provided herein comprise T cells that are stimulated by APCs, such as APCs pre-loaded with antigen peptides.
  • compositions can comprise a population of immune cells comprising T cells from a sample (e.g., a WSGR Docket No.50401-795.601 biological sample), wherein the T cells comprise APC-stimulated T cells.
  • a sample e.g., a WSGR Docket No.50401-795.601 biological sample
  • 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 a percentage of the at least one antigen specific T cell in the composition is at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%.
  • the biological sample comprises less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%.1%, 2%, 3%, 4%, 5%, or less than 10% antigen activated T cells of the total cell count in the biological sample that is derived from peripheral blood or leukapheresis. In some embodiments, the biological sample comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% antigen activated T cells of the total cell count in the biological sample that is derived from peripheral blood.
  • the biological sample comprises antigen naive 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 percentage of at least one antigen specific CD8+ T cell in the composition is less 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% in the biological sample derived from peripheral blood or leukapheresis.
  • a percentage of at least one antigen specific CD4+ T cell in the composition is at least 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%, of in the biological sample derived from peripheral blood or leukapheresis.
  • a percentage of the at least one antigen specific T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of the total immune cells.
  • a percentage of at least one antigen specific CD8+ T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of the total immune cells.
  • a percentage of at least one antigen specific CD4+ T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of the total immune cells. In some embodiments, a percentage of antigen specific T cells in the biological sample is at most about 0.5%. In some embodiments, a percentage of antigen specific WSGR Docket No.50401-795.601 CD8+ T cells in the biological sample is at most about 0.5%. In some embodiments, a percentage of antigen specific CD4+ T cells in the biological sample is at most about 0.5% in the biological sample.
  • the NEO-STIM process can comprise methods of preparing antigen loaded APCs.
  • 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- ⁇ , IL-1 ⁇ , IL-15, PGE1, IL-6, IFN- ⁇ , INF- ⁇ , R848, LPS, ss-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.
  • the APC is an artificial APC.
  • Immune cells can be characterized by cell surface molecules.
  • 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.
  • 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-2 ( ⁇ 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 WSGR Docket No.50401-795.601 differentiation molecule 11b (CD11b).
  • the second chain of ⁇ M ⁇ 2 is the common integrin ⁇ 2 subunit known as CD18, and integrin ⁇ M ⁇ 2 thus belongs to the ⁇ 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. It mediates inflammation by regulating leukocyte adhesion and migration and has been implicated in several immune processes such as phagocytosis, cell-mediated cytotoxicity, chemotaxis and cellular activation. It is involved in the complement system due to its capacity to bind inactivated complement component 3b (iC3b).
  • iC3b inactivated complement component 3b
  • CD11c also known as Integrin, alpha X (complement component 3 receptor 4 subunit) (ITGAX), is a gene that encodes for CD11c.
  • CD11c is an integrin alpha X chain protein. 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).
  • CD11c is a type I transmembrane protein found at high levels on 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.
  • CD14 exists in two forms, one anchored to the membrane by a glycosylphosphatidylinositol tail (mCD14), the other a soluble form (sCD14). Soluble CD14 either appears after shedding of mCD14 (48 kDa) or is directly secreted from intracellular vesicles (56 kDa). CD14 acts as a co-receptor (along with the Toll-like receptor TLR 4 and MD-2) for the detection of bacterial lipopolysaccharide (LPS).
  • mCD14 glycosylphosphatidylinositol tail
  • sCD14 soluble form
  • Soluble CD14 either appears after shedding of mCD14 (48 kDa) or is directly secreted from intracellular vesicles (56 kDa).
  • CD14 acts as a co-receptor (along with the Toll-like receptor TLR 4 and MD-2) for the detection of bacterial lipopolysacc
  • CD14 can bind LPS only in the presence of lipopolysaccharide-binding protein (LBP). Although LPS is considered its main ligand, CD14 also recognizes other pathogen-associated molecular patterns such as lipoteichoic acid.
  • LBP lipopolysaccharide-binding 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+CD25hi 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.
  • WSGR Docket No.50401-795.601 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- ⁇ , IL-1 ⁇ , PGE1, IL-6, IL-7, IL-15, IFN- ⁇ , IFN- ⁇ , R848, LPS, ss-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. In some embodiments, 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 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.
  • 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. [0668] Monocyte APC precursors can also be obtained from a tissue source by using an APC precursor-adhering substrate.
  • 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.
  • Monocyte APC precursors can also be obtained from
  • 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. Typically, these cytokines 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
  • 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%.
  • 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.
  • the differentiation of immature APCs from the precursors 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.
  • 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. [0676] 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- ⁇ , IL-1 ⁇ , or CD40 ligand), bacterial products (e.g., LPS or BCG), and the like.
  • cytokines e.g., TNF- ⁇ , IL-1 ⁇ , or 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.
  • 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 differentiation or WSGR Docket No.50401-795.601 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- ⁇ ) to inhibit immature APC maturation.
  • 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. [0679] 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 ( ⁇ 196 °C) or its vapor ( ⁇ 165 °C).
  • Frozen cells are preferably thawed quickly (e.g., in a water bath maintained at 37-41 °C) and chilled immediately upon thawing. It can be desirable to treat the cells in order to prevent cellular clumping upon thawing. To prevent clumping, various procedures can be used, including the addition before and/or after freezing of DNAse, low molecular weight dextran and citrate, hydroxyethyl starch, and the like.
  • a composition for T cell activation comprises a population of immune cells that has been depleted of one or more types of immune cells.
  • a composition can comprise a population of immune cells that has been depleted of one or more types of immune cells that express one or more proteins, such as one or more cell surface receptors.
  • a composition comprises a population of immune cells from a biological sample comprising at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, wherein an amount of CD14 and/or CD25 expressing immune cells in the population is proportionally different from an amount of immune cells expressing CD14 and/or CD25 in the biological sample.
  • a composition can comprise a population of immune cells from a biological sample comprising at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, wherein an amount of CD14 expressing immune cells in the population is proportionally different from an amount of immune cells expressing CD14 in the biological sample.
  • a composition can comprise a population of immune cells from a biological sample comprising at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, wherein an amount of CD25 expressing immune cells in the population is proportionally different from an amount of immune cells expressing CD25 in the biological sample.
  • a composition can comprise a population of immune cells from a biological sample comprising at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, wherein an amount of CD14 and CD25 expressing immune cells in the population is proportionally different from an amount of immune cells expressing CD14 and CD25 in the biological sample.
  • TCR T-cell receptor
  • a composition can comprise a population of immune cells from a biological sample, wherein an amount of immune cells expressing CD14 and CD25 in the population is proportionally less than an amount of immune cells expressing CD14 and CD25 in the biological sample.
  • a method of preparing antigen-specific T cells ex vivo the method comprises (a) depleting CD14+ cells and/or CD25+ cells from a population of immune cells comprising antigen-presenting cells (APCs) and T cells, thereby forming a CD14 and/or CD25 depleted population of immune cells comprising a first population of APCs and T cells, wherein the population of immune cells is from a biological sample from a human subject; (b) incubating the first population of APCs and T cells from (a) for a first time period in the presence of: (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (ii) (A) a polypeptide comprising
  • the expanded population of cells comprises from at least 5x105 to at least 5x1011 total cells. In some embodiments, the expanded population of cells comprises at least 1x106 total cells. In some embodiments, the expanded population of cells comprises at least 5x106 total cells. In some embodiments, the expanded population of cells comprises at least 1x107 total cells. In some embodiments, the expanded population of cells comprises at least 5x107 total cells. In some embodiments, the expanded population of cells comprises at least 1x108 total cells. In some embodiments, the expanded population of cells comprises at least 5x108 total cells. In some embodiments, the expanded population of cells comprises at least 1x109 total cells. In some embodiments, the expanded population of cells comprises at least 5x109 total cells.
  • the expanded population of cells comprises at least 1x1010 total cells. In some embodiments, the expanded population of cells comprises at least 5x1010 total cells. In some embodiments, the expanded population of cells comprises at least 1x1011 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 1x108 to 1x1011 total cells. In some embodiments, the expanded population of cells comprises from 0,75x108 to 1.25x1010 total cells. In some embodiments the expanded population of cells comprises from 5x108 to 1x109 total cells. In some embodiments, the expanded population of cells comprises 5x108 to 1x109 total cells. In some embodiments, the expanded population of cells comprises 5x108 to 2x109 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. In some embodiments, 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. [0686] In some embodiments, the cell population is enriched for CD11c+ cells. In some embodiments, the antigen loaded APC comprises dendritic cells (DCs).
  • DCs dendritic cells
  • 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 depleting CD14+ 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.
  • the same protein encoded by the genome of a cancer cell is RAS.
  • 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 polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different epitope sequences from two or more different proteins encoded by the genome of a cancer cell.
  • the method comprises introducing the polynucleotide encoding the polypeptide or the mRNA encoding the polypeptide into the APCs of the first population of APCs and T cells.
  • introducing comprises electroporating.
  • WSGR Docket No.50401-795.601 introducing comprises nucleofecting.
  • introducing is carried out without separating the T cells from the APCs of the first population of APCs and T cells.
  • the method is performed in less than 35 days. In some embodiments, 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. In some embodiments, 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 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 0.1% 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 0.5% 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 1% 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 5% 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 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.
  • 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 WSGR Docket No.50401-795.601 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+ 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. In some embodiments, 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.
  • 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. In some embodiments, at least 70% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from na ⁇ ve CD4+ T cells.
  • the expanding comprises contacting the population of cells comprising stimulated T cells with a second population of mature APCs. In some embodiments, the second population of mature APCs have been incubated with FLT3L.
  • the second WSGR Docket No.50401-795.601 population of mature APCs have been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs.
  • the second population of mature APCs have been incubated with FLT3L for 2 days.
  • the second population of mature APCs have been incubated with FLT3L for 3 days.
  • the second population of mature APCs have been incubated with FLT3L for 4 days.
  • the second population of mature APCs have been incubated with FLT3L for 5 days.
  • the second population of mature APCs present the peptide consisting of the epitope sequence from the protein encoded by the genome of the cancer cells.
  • the second population of mature APCs have been incubated with FLT3L and present the peptide consisting of the epitope sequence from the protein encoded by the genome of the cancer cell.
  • expanding comprises expanding the population of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells.
  • depleting CD14+ 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.
  • depleting 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 CD25 binding agent.
  • 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. In some embodiments, when HLA-binding peptides are predicted or known, a longer immunogenic peptide could consist of (1) individual binding peptides with extensions of 2-5 amino acids toward the N- and C-terminus of each WSGR Docket No.50401-795.601 corresponding gene product; or (2) a concatenation of some or all of the binding peptides with extended sequences for each.
  • a longer antigen peptide when sequencing reveals a long (>10 residues) epitope sequence, e.g., an epitope derived from a protein encoded by a gene in a cancer cell (e.g., due to a frameshift, read-through or intron inclusion that leads to a novel peptide sequence), a longer antigen peptide could consist of the entire stretch of novel antigen-specific amino acids as either a single longer peptide or several overlapping longer peptides. In some embodiments, use of a longer peptide is presumed to allow for endogenous processing by patient cells and can lead to more effective antigen presentation and induction of T cell responses.
  • each of the plurality of antigenic peptide comprises the same antigenic epitope. In some embodiments the plurality of antigenic peptide comprises more than one antigenic epitope.
  • the one or more polynucleotides encoding the plurality of antigen peptides is DNA.
  • the one or more polynucleotides encoding the plurality of antigen peptides is inserted in one or more mammalian expression vectors.
  • the one or more polynucleotides encoding the plurality of antigen peptides is messenger RNA.
  • the present disclosure provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising a modified nucleoside.
  • the present disclosure provides gene therapy vectors comprising the RNA, oligoribonucleotide, and polyribonucleotide.
  • the present disclosure provides gene therapy methods and gene transcription silencing methods comprising same.
  • the polynucleotide encodes a single antigenic peptide.
  • the one polynucleotide encodes more than one antigenic peptide.
  • the polynucleotide is messenger RNA.
  • each messenger RNA comprises coding sequence for two or more antigenic peptides in tandem.
  • each messenger RNA comprises a coding sequence for two, three, four, five, six, seven, eight, nine or ten or more antigenic peptides in tandem.
  • an mRNA comprises a 5'-UTR, a protein coding region, and a 3'-UTR. mRNA only possesses limited half-life in cells and in vitro.
  • the mRNA is self-amplifying mRNA.
  • mRNA can be generated by in vitro transcription from a DNA template.
  • the in vitro transcription methodology is known to the skilled person.
  • the stability and translation efficiency of RNA can be modified.
  • RNA can be stabilized, and its translation increased by one or more modifications having a stabilizing effect and/or increasing translation efficiency of RNA. Such modifications are described, for example, in PCT/EP2006/009448 incorporated herein by reference.
  • an mRNA can include multiple antigenic epitopes.
  • a mRNA of a large portion of, or even the entire coding region of a gene comprising sequences encoding antigenic peptides are delivered into an immune cell for endogenous processing and presentation of antigens.
  • 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.
  • the mRNA is 400-700 nucleotides long. In some embodiments, the mRNA is 450-600 nucleotides long. In some embodiments, the mRNA is at least 200 nucleotides long. In some embodiments the mRNA is greater than 250 nucleotides, greater than 300 nucleotides, greater than 350 nucleotides, greater than 400 nucleotides, greater than 450 nucleotides, greater than 500 nucleotides, greater than 550 nucleotides, greater than 600 nucleotides, greater than 650 nucleotides, greater than 700 nucleotides, greater than 750 nucleotides, greater than 800 nucleotides, greater than 850 nucleotides long, greater than 900 nucleotides long greater than 950 nucleotides long, greater than 1000 nucleotides long, greater than 2000 nucleotides long, greater than 3000 nucleotides long, greater than 4000 nucleotides long
  • mRNA encoding one or more antigenic peptide is modified, wherein the modification relates to the 5’-UTR.
  • the modification relates to providing an RNA with a 5'-cap or 5’- cap analog in the 5’-UTR.
  • the term “5'-cap” refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5' to 3' triphosphate linkage. In some embodiments, this guanosine is methylated at the 7-position.
  • RNA 5'-cap refers to a naturally occurring RNA 5'- cap, to the 7-methylguanosine cap (m G).
  • the term “5'-cap” WSGR Docket No.50401-795.601 includes a 5'-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and/or enhance translation of RNA if attached thereto, in vivo and/or in a cell.
  • mRNA is capped co-transcriptionally.
  • the mRNA encoding one or more antigenic peptides comprise a 3’- UTR comprising a poly A tail.
  • the poly A tail is 100-200 bp long. In some embodiments, the poly A tail is longer than 20 nucleotides. In some embodiments, the poly A tail is longer than 50 nucleotides. In some embodiments, the poly A tail is longer than 60 nucleotides. In some embodiments, the poly A tail is longer than 70 nucleotides. In some embodiments, the poly A tail is longer than 80 nucleotides. In some embodiments, the poly A tail is longer than 90 nucleotides. In some embodiments, the poly A tail is longer than 100 nucleotides. In some embodiments, the poly A tail is longer than 110 nucleotides. In some embodiments, the poly A tail is longer than 120 nucleotides.
  • the poly A tail is longer than 130 nucleotides. In some embodiments, the poly A tail is longer than 140 nucleotides. In some embodiments, the poly A tail is longer than 150 nucleotides. In some embodiments, the poly A tail is longer than 160 nucleotides. In some embodiments, the poly A tail is longer than 170 nucleotides. In some embodiments, the poly A tail is longer than 180 nucleotides. In some embodiments, the poly A tail is longer than 190 nucleotides. In some embodiments, the poly A tail is longer than 200 nucleotides. In some embodiments, the poly A tail is longer than 210 nucleotides.
  • the poly A tail is longer than 220 nucleotides. In some embodiments, the poly A tail is longer than 230 nucleotides. In some embodiments, the poly A tail is longer than 100 nucleotides. In some embodiments, the poly A tail is longer than 240 nucleotides. In some embodiments, the poly A tail is longer than 100 nucleotides. In some embodiments, the poly A tail is about 250 nucleotides. [0712] In some embodiments, the poly A tail comprises 100-250 adenosine units. In some embodiments, the poly A tail comprises 120-130 adenine units. In some embodiments, the poly A tail comprises 120 adenine units.
  • the poly A tail comprises 121 adenine units. In some embodiments, the poly A tail comprises 122 adenine units. In some embodiments, the poly A tail comprises 123 adenine units. In some embodiments, the poly A tail comprises 124 adenine units. In some embodiments, the poly A tail comprises 125 adenine units. In some embodiments, the poly A tail as 129 bases. [0713] In some embodiments, the coding sequence for two consecutive antigenic peptides are separated by a spacer or linker. [0714] In some embodiments, the spacer or linker comprises up to 5000 nucleotide residues. An exemplary spacer sequence is GGCGGCAGCGGCGGCGGCGGCAGCGGCGGC.
  • Another exemplary spacer sequence is GGCGGCAGCCTGGGCGGCGGCGGCAGCGGC.
  • Another exemplary spacer sequence is GGCGTCGGCACC.
  • Another exemplary spacer sequence is WSGR Docket No.50401-795.601 CAGCTGGGCCTG.
  • Another exemplary spacer is a sequence that encodes a lysine, such as AAA or AAG.
  • Another exemplary spacer sequence is CAACTGGGATTG.
  • the mRNA comprises one or more additional structures to enhance antigen epitope processing and presentation by APCs.
  • the linker or spacer region can contain cleavage sites.
  • cleavage sites ensure cleavage of the protein product comprising strings of epitope sequences into separate epitope sequences for presentation.
  • the preferred cleavage sites are placed adjacent to certain epitopes in order to avoid inadvertent cleavage of the epitopes within the sequences.
  • the design of epitopes and cleavage regions on the mRNA encoding strings of epitopes are non-random.
  • an mRNA encoding an antigen peptide of the present disclosure is administered to a subject in need thereof.
  • the mRNA to be administered comprises at least one modified nucleoside-phosphate.
  • T cells are activated with antigenic peptides by artificial antigen- presenting cells.
  • artificial scaffolds are used to activate a T cell with antigenic peptides, the artificial scaffolds are loaded with antigenic peptides couples with an MHC antigen to which the antigenic peptide can bind with high affinity.
  • the additional structures comprise encoding specific domains from the proteins selected from a group MITD, SP1, and 10th Fibronectin Domain: 10FnIII.
  • the cells derived from peripheral blood or from leukapheresis are contacted with the plurality of antigen peptides, or one or more polynucleotides encoding the plurality of antigen peptides once or more than once to prepare the antigen loaded APCs.
  • the method comprises incubating the APC or one or more of the APC preparations with a first medium comprising at least one cytokine or growth factor for a first time period.
  • the method comprises incubating one or more of the APC preparations with at least one peptide for a second time period.
  • the enriched cells further comprise CD1c+ cells.
  • the cell population is enriched for CD11c+ and CD141+ cells.
  • the cell population comprising the antigen loaded APCs comprises greater than 1%, 2%, 3%, 4%, 5%, 6,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more CD11c+ cells.
  • the cell population comprising the antigen loaded APCs comprises less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4% or lower CD11b+ expressing cells.
  • the cell population comprising the antigen loaded APCs comprises greater than 1%, 2%, 3%, 4%, 5%, 6,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% antigenic peptide expressing cells that are CD11c+.
  • the cell population comprising the antigen loaded APCs comprises greater than 1%, 2%, 3%, 4%, 5%, 6,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% antigenic peptide expressing cells that are CD11c+ CD1c+, or CD141+ cells.
  • the antigen loaded APCs comprise mature APCs.
  • the method comprises obtaining a biological sample from a subject comprising at least one APC and at least one PBMC or at least on T cell.
  • the method comprises depleting cells expressing CD14 and/or CD25 and/or CD19 from a biological sample, thereby obtaining a CD14 and/or CD25 and/or CD19 cell depleted sample.
  • the method comprises incubating a CD14 and/or CD25 and/or CD19 cell depleted sample with FLT3L for a first time period.
  • the method comprises incubating at least one peptide with a CD14 and/or CD25 and/or CD19 cell depleted sample for a second time period, thereby obtaining a first matured APC peptide loaded sample.
  • the NEO-STIM process can comprise methods of preparing antigen activated (e.g., RAS activated) T cells using antigen loaded APCs.
  • the antigen loaded APC (APC) prepared by the methods described above is incubated with T cells to obtain antigen activated T cells.
  • the method can comprise generating at least one antigen specific T cell where the antigen is an antigen derived from a cancer cell.
  • the generating at least one antigen specific T cell comprises generating a plurality of antigen specific T cells.
  • the T cells are obtained from a biological sample from a subject.
  • the T cells are obtained from a biological sample from the same subject from whom the APCs are derived. In some embodiments, the T cells are obtained from a biological sample from a different subject than the subject from whom the APCs are derived. [0739] In some embodiments, the APCs and/or T cells are derived from a biological sample which is peripheral blood mononuclear cells (PBMC). In some embodiments, the APCs and/or T cells are derived from a biological sample which is a leukapheresis sample. [0740] In some embodiments, the APC comprises a dendritic cell (DC).
  • DC dendritic cell
  • the APC is derived from a CD14+ monocyte, or is a CD14 enriched APC, or is a CD141 enriched APC.
  • the CD14+ monocyte is enriched from a biological sample from a subject comprising peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the APC is PBMC.
  • the PBMC is freshly isolated PBMC.
  • the PBMC is frozen PBMC.
  • the PBMC is autologous PBMC isolated from the subject or the patient.
  • the PBMC is loaded with antigens, where the antigens can be peptides or polypeptides or polynucleotides, such as mRNA, that encode the peptides and polypeptides.
  • PBMCs monoocytes, DCs phagocytic cells
  • Peptides or polypeptides loaded on the PBMCs can be supplemented with adjuvants to increase immunogenicity.
  • the PBMC is loaded with nucleic acid antigens. Nucleic acid antigens can be in the form of mRNA, comprising sequences encoding one or more antigens.
  • mRNA antigen loading does not require adjuvant supplementation, because, for example, RNA can act as a self-adjuvant.
  • PBMCs are directly isolated or thawed from a frozen sample, and subjected to incubating with one or more antigens.
  • the PBMC sample is not further cultured for differentiation or subjected to further maturation of one or more cell components within the PBMC, (for example, maturation of antigen-presenting cells, or differentiation of monocytes to dendritic cells), before exposing the PBMCs to one or more antigens or nucleic acid encoding the one or more antigens.
  • one or more cell types are depleted or removed from the freshly isolated PBMC cell population or a freshly thawed PBMC population before exposing or incubating the cells to one or more antigens or nucleic acid encoding the one or more antigens.
  • CD14+ cells are depleted from the PBMC.
  • CD25+ cells are depleted from the PBMC.
  • CD11b+ cells are depleted from the PBMC.
  • the CD14+ and CD25+ cells are depleted from the PBMCs, before incubating with one or more antigens or one or more nucleic acids encoding the one or more antigens.
  • the CD11b+, and/or the CD14+ and/or CD25+ cells are depleted from the PBMC.
  • a method provided herein comprises preparing antigen-specific T cells by depleting CD14+ cells and/or CD25+ cells from a PBMC sample from a human subject containing about the same percentage of immature dendritic cells (DCs) as the percentage of immature DCs in the peripheral blood of the human subject.
  • a method provided herein comprises preparing antigen-specific T cells by depleting CD14+ cells and/or CD25+ cells from a PBMC sample from a human subject containing about the same percentage of mature DCs as the percentage of mature DCs in the peripheral blood of the human subject.
  • a method provided herein comprises preparing antigen-specific T cells by depleting CD14+ cells and/or CD25+ cells from a PBMC sample from a human subject containing about the same ratio of immature DCs to mature DCs as the ratio of immature DCs to mature DCs in the peripheral blood of the human subject.
  • a method provided herein comprises preparing antigen-specific T cells by depleting CD14+ cells and/or CD25+ cells from a PBMC sample from a human subject that has not been subject to a step of maturing immature DCs into mature DCs.
  • the antigen is from a cancer cell.
  • the CD14+ monocyte is stimulated with one or more cytokines or growth factors.
  • one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, TNF- ⁇ , IL-1 ⁇ , PGE1, IL-6, IL-7, IL-15, IFN- ⁇ , IFN- ⁇ , R848, LPS, ss-rna40, poly I:C, or a combination thereof.
  • the CD14+ monocyte is from a second biological sample comprising PBMCs.
  • the second biological sample is from the same subject.
  • the biological sample comprises peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the at least one antigen-specific T cell is stimulated in a medium comprising IL-7, IL-15, an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof.
  • IDO inhibitor is epacadostat, navoximod, 1-methyltryptophan, or a combination thereof.
  • the subject is administered FLT3L prior to obtaining the biological sample for preparing the APCs and/or T cells.
  • the T cells are obtained from a biological sample from a subject as described in the previous sections of this disclosure.
  • the biological sample is freshly obtained from a subject or is a frozen sample.
  • the incubating is in presence of at least one cytokine or growth factor, which comprises GM-CSF, IL-4, FLT3L, TNF- ⁇ , IL-1 ⁇ , PGE1, IL-6, IL-7, IL-15, IFN- ⁇ , IFN- ⁇ , IL- 15, R848, LPS, ss-rna40, poly I:C, or any combination thereof.
  • cytokine or growth factor which comprises GM-CSF, IL-4, FLT3L, TNF- ⁇ , IL-1 ⁇ , PGE1, IL-6, IL-7, IL-15, IFN- ⁇ , IFN- ⁇ , IL- 15, R848, LPS, ss-rna40, poly I:C, or any combination thereof.
  • a method comprises stimulating T cells with IL-7, IL-15, or a combination thereof. In some embodiments, a method comprises stimulating T cells with IL-7, IL-15, or a combination thereof, in the presence of an IDO inhibitor, a PD-1 antibody or IL-12. In some embodiments, the stimulated T cell is expanded in presence of the one or more antigen epitope WSGR Docket No.50401-795.601 sequence or APCs loaded with the one or more antigen or epitope sequence, or APCs loaded with (e.g.
  • nucleic acid sequences such as mRNA sequences
  • one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, TNF- ⁇ , IL-1 ⁇ , PGE1, IL-6, IL-7, IL-15, IFN- ⁇ , IFN- ⁇ , R848, LPS, ss-rna40, poly I:C, or a combination thereof, FLT3L, under suitable T cell growth conditions ex vivo.
  • the method further comprises administering the antigen specific T cells to a subject.
  • the method comprises incubating the APC prepared as described in the previous sections with T cells in presence of a medium comprising the at least one cytokines or growth factor to generate antigen activated T cells.
  • the incubating comprises incubating a first APC preparation of the APC preparations to the T cells for more than 7 days.
  • the incubated T cells are stimulated T cells that expand in vitro on presence of the APC preparation, cytokines and growth factors for more than 7 days.
  • the incubating comprises incubating a first APC preparation of the APC preparations to the T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days.
  • the first time period of the one or more time periods is about 1, 23, 4, 5, 6, 7, 8, or 9 days.
  • a total time period of the separate time periods is less than 28 days.
  • a total time period of the separate time periods is from 20-27 days.
  • a total time period of the separate time periods is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 days.
  • a method comprises incubating a first APC preparation of the APC preparations with the T cells for more than 7 days.
  • a method comprises incubating a first APC preparation of the APC preparations with the T cells for more than 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In some embodiments, a method comprises incubating a first APC preparation of the APC preparations with the T cells for from 7-20, 8-20, 9-20, 10-20, 11- 20, or 12-20 days. In some embodiments, a method comprises incubating a first APC preparation of the APC preparations with the T cells for about 10-15 days. [0764] In some embodiments, a method comprises incubating a second APC preparation of the APC preparations to the T cells for 5-9 days.
  • a method comprises incubating a second APC preparation of the APC preparations to the T cells for 5, 6, 7, 8, or 9 days. In some embodiments, the method further comprises removing the one or more cytokines or growth factors of the second medium after the third time period and before a start of the fourth time period.
  • WSGR Docket No.50401-795.601 [0765]
  • a method comprises incubating a third APC preparation of the APC preparations to the T cells for 5-9 days. In some embodiments, the method comprises incubating a third APC preparation of the APC preparations to the T cells for 5, 6, 7, 8, or 9 days.
  • the method comprises incubating a first APC preparation of the APC preparations with the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days, incubating a second APC preparation of the APC preparations to the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days, and incubating a third APC preparation of the APC preparations to the T cells for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days.
  • the method is performed ex vivo.
  • the T cells are cultured in a medium containing a cytokine.
  • an example of cytokines includes IL-7.
  • an example of cytokines includes IL-15.
  • an example of cytokines includes IL-7 and IL-15.
  • the T cells are cultured in a medium comprising IL-7, and/or IL-15.
  • the cytokine in a T cell culture or a medium has a final concentration of at least 0.05 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.8 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 12 ng/mL, 15 ng/mL, 18 ng/mL, or 20 ng/mL.
  • the IL-7 in a T cell culture or a medium has a final concentration of at least 0.05 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.8 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 12 ng/mL, 15 ng/mL, 18 ng/mL, or 20 ng/mL.
  • the IL-15 in a T cell culture or a medium has a final concentration of at least 0.05 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.3 ng/mL, 0.4 ng/mL, 0.5 ng/mL, 0.8 ng/mL, 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 12 ng/mL, 15 ng/mL, 18 ng/mL, or 20 ng/mL.
  • the T cells are cultured in a medium further containing FLT3L.
  • the FLT3L in a T cell culture or a medium has a final concentration of in a T cell culture or a medium has a final concentration of at least 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 12 ng/mL, 15 ng/mL, 18 ng/mL, 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL, 100 ng/mL, or 200 ng/mL.
  • the T cells are incubated, induced, or stimulated in a medium containing FLT3L for a first period time. In some embodiments, the T cells are incubated, induced, or stimulated in a medium containing additionally added FLT3L for a second period time. In some embodiments, the T cells are incubated, induced, or stimulated in a medium containing additional added FLT3L for a third period time. In some embodiments, the T cells are incubated, induced, or stimulated in a medium containing additional added FLT3L for a fourth, a fifth, or a sixth period time, with freshly added FLT3L in each time period.
  • the T cells are cultured in presence of an antigen, e.g., an antigen presented by an APC, wherein the media comprises high potassium [K]+ content.
  • the T cells are cultured in presence of high [K]+ content in the media for at least a period of time during the incubation with APCs or T cells.
  • the [K]+ content in the media is altered for at least a period of time during the incubation with APCs or T cells.
  • the content in the media is kept constant over the period of T cell ex vivo culture.
  • the [K]+ content in the T cell culture medium is ⁇ 5 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 6 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 7 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 8 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 9 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 10 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 11 mM.
  • the [K]+ content in the T cell culture medium is ⁇ 12 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 13 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 14 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 15 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 16 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 17 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 18 mM.
  • the [K]+ content in the T cell culture medium is ⁇ 19 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 20 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 22 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 25 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 30 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 35 mM. In some embodiments, the [K]+ content in the T cell culture medium is ⁇ 40 mM.
  • the [K]+ content in the T cell culture medium is about 40 mM. [0769] In some embodiments, the [K]+ content in the T cell culture medium is about 40 mM for at least a period of time during the incubation of T cells with antigen.
  • the antigen can be presented by the antigen loaded APCs.
  • the T cells in the presence of [K]+ are tested for T effector functions, CD8+ cytotoxicity, cytokine production, and for memory phenotype. In some embodiments, T cells are grown in the presence of high [K]+ express effector T cell phenotype. In some embodiments, T cells grown in presence of high [K]+ express memory cell marker.
  • T cells grown in presence of high [K]+ do not express T cell exhaustion markers.
  • the stimulated T cell is a population of immune cells comprising the activated T cells stimulated with APCs comprising an antigenic peptide-MHC complex.
  • a method can comprise incubating a population of immune cells from a biological sample with APCs comprising a peptide-MHC complex, thereby obtaining a stimulated immune cell sample; determining expression of one or more cell markers of at least one immune cell of the stimulated immune cell sample; and determining binding of the at least one immune cell of the stimulated immune cell sample to a peptide-MHC complex; wherein determining expression of certain cell surface markers or other determinant markers, such as intracellular factors, or released agents, such as cytokines etc., and determining binding to the antigen-MHC complex are performed simultaneously.
  • the one or more cell markers comprise TNF- ⁇ , IFN- ⁇ , LAMP- 1, 4-1BB, IL-2, IL-17A, Granzyme B, PD-1, CD25, CD69, TIM3, LAG3, CTLA-4, CD62L, CD45RA, CD45RO, FoxP3, or any combination thereof.
  • the one or more cell markers comprise a cytokine.
  • the one or more cell markers comprise a degranulation marker.
  • the one or more cell markers comprise a cell-surface marker.
  • the one or more cell markers comprise a protein.
  • determining binding of the at least one immune cell of the stimulated immune cell sample to the peptide-MHC complex comprises determining binding of the at least one immune cell of the stimulated immune cell sample to an MHC tetramer comprising the peptide and the MHC of the peptide-MHC complex.
  • the MHC is a class I MHC or a class II MHC.
  • the peptide-MHC complex comprises one or more labels. [0771]
  • activation of T cell is verified by detecting the release of a cytokine by the activated T cell.
  • the cytokine is one or more of: TNF- ⁇ , IFN- ⁇ , IL-2, or IL- 18.
  • the activation of T cell is verified by its specific antigen binding and cytokine release. In some embodiments, the activation of T cells is verified by its ability to kill infected cells in vitro. A sample of activated T cells can be used to verify the activation status of the T cells. In some embodiments, a sample from the T cells is withdrawn from the T cell culture to determine the cellular composition and activation state by flow cytometry.
  • a percentage of the at least one antigen specific T cell in the composition is at least about 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% of total T cells or total immune cells.
  • the percentage of the at least one antigen specific T cell in the composition is about 5%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 7%.
  • the percentage of the at least one antigen specific T cell in the composition is about 10%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 12%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 15%. In some embodiments, the percentage of the at least one antigen WSGR Docket No.50401-795.601 specific T cell in the composition is about 20%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 25%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 30%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 40%.
  • the percentage of the at least one antigen specific T cell in the composition is about 50%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 60%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 70%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 80%. In some embodiments, the percentage of the at least one antigen specific T cell in the composition is about 90%.
  • a percentage of at least one antigen specific CD8+ T cell in the composition is at least about 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% of total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 5%.
  • the percentage of the at least one antigen specific CD8+ T cells in the composition is about 7%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 10%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 12%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 15%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 20%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 25%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 30%.
  • the percentage of the at least one antigen specific CD8+ T cells in the composition is about 40%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 50%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 60%. In some embodiments, the percentage of the at least one antigen specific CD8+ T cells in the composition is about 70% of total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.
  • a percentage of the at least one antigen specific T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.
  • a percentage of at least one antigen specific CD8+ T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.
  • a percentage of at least one antigen specific CD4+ T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.
  • the antigen is an overexpressed antigen, a viral antigen, a bacterial antigen, a protozoan antigen, a helminth antigen, or a combination thereof.
  • the number of at least one antigen specific CD8+ T cell in the composition is at least about 1x10 ⁇ 6, 2x10 ⁇ 6, 5x10 ⁇ 6, 1x10 ⁇ 7, 2x10 ⁇ 7, 5x10 ⁇ 7, 1x10 ⁇ 8, 2x10 ⁇ 8, or 5x10 ⁇ 8, antigen specific CD8+ T cells.
  • a number of at least one antigen specific CD4+ T cell in the composition is at least about 1x10 ⁇ 6, 2x10 ⁇ 6, 5x10 ⁇ 6, 1x10 ⁇ 7, 2x10 ⁇ 7, 5x10 ⁇ 7, 1x10 ⁇ 8, 2x10 ⁇ 8, or 5x10 ⁇ 8, antigen specific CD4+ T cells.
  • Method of T cell Manufacturing Provided herein are methods for antigen specific T cell manufacturing.
  • methods of preparing T cell compositions such as therapeutic T cell compositions.
  • a method can comprise expanding or inducing antigen specific T cells.
  • Preparing (e.g., inducing or expanding) T cells can also refer to manufacturing T cells, and broadly encompasses procedures to isolate, stimulate, culture, induce, and/or expand any type of T cells (e.g., CD4+ T cells and CD8+ T cells).
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprising incubating an APC with a population of immune cells from a biological sample depleted of cells expressing CD14 and/or CD25.
  • the method comprises preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating an APC with a population of immune cells from a biological sample depleted of cells expressing CD11b and/or CD19.
  • the WSGR Docket No.50401-795.601 method comprises incubating an APC with a population of immune cells from a biological sample depleted of cells expressing any CD11b and/or CD19 and/or CD14 and/or CD25 or any combination thereof.
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating an FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APC with a population of immune cells from a biological sample.
  • TCR T-cell receptor
  • FLT3L FMS-like tyrosine kinase 3 receptor ligand
  • a method of preparing a pharmaceutical composition comprising at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising: incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first time period; and thereafter incubating at least one T cell of the biological sample with an APC.
  • TCR T-cell receptor
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods of less than 28 days from incubating the population of immune cells with a first APC preparation of the one or more APC preparations, wherein at least one antigen specific memory T cell is expanded, or at least one antigen specific na ⁇ ve T cell is induced.
  • TCR T-cell receptor
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with 3 or less APC preparations for 3 or less separate time periods, wherein at least one antigen specific memory T cell is expanded or at least one antigen specific na ⁇ ve T cell is induced.
  • TCR T-cell receptor
  • a method of preparing antigen specific T cells comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods, thereby stimulating T cells to become antigen specific T cells, wherein a percentage of antigen specific T cells is at least 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% of total CD4+ T cells, total CD8+ T cells,
  • a method of preparing antigen specific T cells comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells from a biological sample with 3 or less APC preparations for 3 or less WSGR Docket No.50401-795.601 separate time periods, thereby stimulating T cells to become antigen specific T cells.
  • a method of preparing antigen specific T cells comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells from a biological sample with 2 or less APC preparations for 2 or less separate time periods, thereby stimulating T cells to become antigen specific T cells.
  • a method that comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods, thereby stimulating T cells to become antigen specific T cells, wherein the APC preparation is a PBMC cell population from which cells expressing one or more cell surface markers are depleted prior to antigen loading of the APC population.
  • the APC preparation is a PBMC cell population from which cells expressing one or more cell surface markers are depleted prior to antigen loading of the APC population.
  • CD14+ cells are depleted prior to antigen loading of an APC population.
  • CD25+ cells are depleted prior to antigen loading of an APC population.
  • CD11b+ cells are depleted prior to antigen loading of an APC population.
  • CD19+ cells are depleted prior to antigen loading of an APC population.
  • CD3+ cells are depleted prior to antigen loading of an APC population.
  • CD25+ cells and CD14+ cells are depleted prior to antigen loading of an APC population.
  • CD11b+ and CD25+ cells are depleted prior to antigen loading of an APC population.
  • CD11b+ and CD14+ cells are depleted prior to antigen loading of an APC population.
  • CD11b+, CD14+ and CD25+ cells are depleted prior to antigen loading of an APC population.
  • CD11b+, and CD19+ cells are depleted prior to antigen loading of an APC population.
  • CD11b+, CD19+ and CD25+ cells are depleted prior to antigen loading of an APC population.
  • CD11b+, CD14+, CD19+ and CD25+ cells are depleted prior to antigen loading of an APC population.
  • the method comprises adding to any of the depleted APC population described above, an APC enriched cell PBMC-derived population that are depleted of CD3+ cell.
  • the APC enriched cell PBMC-derived population is depleted of CD3+ and cells depleted of any one or more of CD11b+, CD14+, CD19+, or CD25+.
  • a biological sample comprises peripheral blood mononuclear cells (PBMCs).
  • the method comprises adding to a PBMC sample, a composition comprising one or more antigenic peptides or nucleic acids encoding the same, thereby loading the APCs within the PBMCs with antigens for antigen presentation to T cells in the PBMC.
  • a method comprises: (a) obtaining a biological sample from a subject comprising at least one antigen-presenting cell (APC); (b) enriching cells expressing CD11c from the biological sample, thereby obtaining a CD11c+ cell enriched sample; (c) incubating the CD11c+ cell enriched sample with at least one cytokine or growth factor for a first time period; (d) incubating at WSGR Docket No.50401-795.601 least one peptide with the CD11c+ enriched sample of (c) for a second time period, thereby obtaining an APC peptide loaded sample; (e) incubating the APC peptide loaded sample with one or more cytokines or growth factors for a third time period, thereby obtaining a matured APC sample; (f) incubating APCs of the matured APC sample with a CD11b and/or CD14 and/or CD25 depleted sample comprising PBMCs for a fourth time
  • a method comprises: (a) obtaining a biological sample from a subject comprising at least one antigen-presenting cell (APC); (b) enriching cells expressing CD14 from the biological sample, thereby obtaining a CD14+ cell enriched sample; (c) incubating the CD14+ cell enriched sample with at least one cytokine or growth factor for a first time period; (d) incubating at least one peptide with the CD14+ enriched sample of (c) for a second time period, thereby obtaining an APC peptide loaded sample; (e) incubating the APC peptide loaded sample with one or more cytokines or growth factors for a third time period, thereby obtaining a matured APC sample; (f) incubating APCs of the matured APC sample with a CD14 and/or CD25 depleted sample comprising PBMCs for a fourth time period; (g) incubating the PBMCs with APCs of a matured APC
  • a method comprises: (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD11b and/or CD19 from the biological sample, thereby obtaining a CD11b and/or CD19 cell depleted sample; (c) incubating the CD11b and/or CD19 cell depleted sample with FLT3L for a first time period; (d) incubating at least one peptide with the CD11b and/or CD19 cell depleted sample of (c) for a second time period, thereby obtaining an APC peptide loaded sample; (e) incubating the APC peptide loaded sample with the at least one PBMC for a third time period, thereby obtaining a first stimulated PBMC sample; (f) incubating a PBMC of the first stimulated PBMC sample with an APC of a matured APC sample for a fourth time period, thereby obtaining a
  • a method comprises: (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD11b and/or CD19 and/or CD14 and/or CD25 from the biological sample, thereby obtaining a CD11b and/or CD19 cell depleted sample; (c) incubating the CD11b and/or CD19 and/or CD14 and/or CD25 cell WSGR Docket No.50401-795.601 depleted sample with FLT3L for a first time period; (d) incubating at least one peptide with the CD11b and/or CD19 and/or CD14 and/or CD25 cell depleted sample of (c) for a second time period, thereby obtaining an APC peptide loaded sample; (e) incubating the APC peptide loaded sample with the at least one PBMC for a third time period, thereby obtaining a first stimulated PBMC sample; (a) obtaining a biological sample
  • a method comprises: (a) obtaining a biological sample from a subject comprising at least one APC and at least one PBMC; (b) depleting cells expressing CD14 and/or CD25 from the biological sample, thereby obtaining a CD14 and/or CD25 cell depleted sample; (c) incubating the CD14 and/or CD25 cell depleted sample with FLT3L for a first time period; (d) incubating at least one peptide with the CD14 and/or CD25 cell depleted sample of (c) for a second time period, thereby obtaining an APC peptide loaded sample; (e) incubating the APC peptide loaded sample with the at least one PBMC for a third time period, thereby obtaining a first stimulated PBMC sample; (f) incubating a PBMC of the first stimulated PBMC sample with an APC of a matured APC sample for a fourth time period, thereby obtaining a second stimulated
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating an APC with a population of immune cells from a biological sample depleted of cells expressing CD14 and/or CD25.
  • TCR T-cell receptor
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods of less than 28 days from incubating the population of immune cells with a first APC preparation of the one or more APC preparations, wherein at least one antigen specific memory T cell is expanded, or at least one antigen specific na ⁇ ve T cell is induced.
  • TCR T-cell receptor
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising incubating a population of immune cells from a biological sample with 3 or WSGR Docket No.50401-795.601 less APC preparations for 3 or less separate time periods, wherein at least one antigen specific memory T cell is expanded or at least one antigen specific na ⁇ ve T cell is induced.
  • TCR T-cell receptor
  • a method of preparing antigen specific T cells comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises contacting a population of immune cells (e.g., PBMCs) to APCs.
  • a method of preparing antigen specific T cells comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells (e.g., PBMCs) with APCs for a time period.
  • the population of immune cells is from a biological sample.
  • the population of immune cells is from a sample (e.g., a biological sample) depleted of CD14 expressing cells.
  • the population of immune cells is from a sample (e.g., a biological sample) depleted of CD25 expressing cells. In some embodiments, the population of immune cells is from a sample (e.g., a biological sample) depleted of CD14 expressing cells and CD25 expressing cells.
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating an FMS- like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APC with a population of immune cells from a biological sample.
  • TCR T-cell receptor
  • a method of preparing a pharmaceutical composition comprising at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence, the method comprising: incubating FMS-like tyrosine kinase 3 receptor ligand (FLT3L) with a population of immune cells from a biological sample for a first time period; and thereafter incubating at least one T cell of the biological sample with an APC.
  • TCR T-cell receptor
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) with FMS-like tyrosine kinase 3 receptor ligand (FLT3L).
  • a sample e.g., a biological sample
  • FMS-like tyrosine kinase 3 receptor ligand FMS-like tyrosine kinase 3 receptor ligand
  • a method of preparing at least one antigen specific T cell comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) with FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs.
  • TCR T-cell receptor
  • FLT3L FMS-like tyrosine kinase 3 receptor ligand
  • TCR T-cell receptor
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises contacting a population of immune cells from a sample (e.g., a biological sample) to one or more APC preparations.
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) to one or more APC preparations for one or more separate time periods.
  • a method of preparing at least one antigen specific T cell comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells from a sample (e.g., a biological sample) to one or more APC preparations for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 separate time periods.
  • the one or more separate time periods is less than 28 days calculated from incubating the population of immune cells with a first APC preparation of the one or more APC preparations.
  • a method of preparing antigen specific T cells comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells to APCs for a time period, wherein the population of immune cells is from a biological sample comprising PBMCs.
  • a method of preparing antigen specific T cells comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells to APCs for a time period, wherein the population of immune cells is from a biological sample depleted of CD14 and/or CD25 expressing cells.
  • a method of preparing antigen specific T cells comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells from a biological sample with FMS-like tyrosine kinase 3 receptor ligand (FLT3L)- stimulated APCs for a time period.
  • TCR T-cell receptor
  • TCR T-cell receptor
  • a method of preparing antigen specific T cells comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods, thereby inducing or expanding antigen specific T cells, wherein the one or more separate time periods is less than 28 days calculated from incubating the population of immune cells with a WSGR Docket No.50401-795.601 first APC preparation of the one or more APC preparations.
  • TCR T-cell receptor
  • incubating a population of immune cells from a biological sample with one or more APC preparations for one or more separate time periods is performed in a medium containing IL-7, IL-15, or a combination thereof.
  • the medium further comprises an indoleamine 2,3-dioxygenase-1 (IDO) inhibitor, an anti-PD-1 antibody, IL-12, or a combination thereof.
  • IDO indoleamine 2,3-dioxygenase-1
  • the IDO inhibitor can be epacadostat, navoximod, 1-Methyltryptophan, or a combination thereof.
  • the IDO inhibitor can increase the number of antigen-specific CD8+ cells.
  • the IDO inhibitor can maintain the functional profile of memory CD8+ T cell responses.
  • the PD-1 antibody can increase the absolute number of antigen-specific memory CD8+ T cell responses.
  • the PD-1 antibody can increase proliferation rate of the cells treated with such antibody.
  • the additional of IL- 12 can result in an increase of antigen-specific cells and/or an increase in the frequency of CD8+ T cells.
  • a method of preparing antigen specific T cells comprising a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells comprising from a biological sample with one or more APC preparations for one or more separate time periods, thereby expanding or inducing antigen specific T cells, wherein a percentage of antigen specific T cells, antigen specific CD4+ T cells, or antigen specific CD8+ T cells is at least 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%,
  • a method of preparing antigen specific T cells comprises a T-cell receptor (TCR) specific to at least one antigen peptide sequence comprises incubating a population of immune cells from a biological sample with 3 or less APC preparations for 3 or less separate time periods, thereby stimulating T cells to become antigen specific T cells.
  • the population of immune cells is from a biological sample depleted of CD14 and/or CD25 expressing cells.
  • the APCs are FMS-like tyrosine kinase 3 receptor ligand (FLT3L)-stimulated APCs.
  • the APCs comprise one or more APC preparations.
  • the APC preparations comprise 3 or less APC preparations. In some embodiments, the APC preparations are incubated with the immune cells sequentially within one or more separate time periods.
  • the biological sample is from a subject. In some embodiments, the subject is a human. For example, the subject can be a patient or a donor. In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is cancer.
  • the antigen specific T cells comprise CD4+ and/or CD8+ T cells. In some WSGR Docket No.50401-795.601 embodiments, the antigen specific T cells comprise CD4 enriched T cells and/or CD8 enriched T cells.
  • a CD4+ T cell and/or CD8+ T cell can be isolated from, enriched from, or purified from a biological sample from a subject comprising PBMCs.
  • the antigen specific T cells are na ⁇ ve CD4+ and/or na ⁇ ve CD8+ T cells.
  • the antigen specific T cells are memory CD4+ and/or memory CD8+ T cells.
  • each of the at least one antigen peptide sequence binds to a protein encoded by an HLA allele expressed by the subject. In some embodiments, each of the at least one antigen peptide sequence is not present in non-cancer cells of the subject.
  • each of the at least one antigen peptide sequences is encoded by genome of a cancer cell. In some embodiments, one or more of the at least one antigen peptide sequence has a length of from 8-50 naturally occurring amino acids. In some embodiments, the at least one antigen peptide sequence comprises a plurality of antigen peptide sequences. In some embodiments, the plurality of antigen peptide sequences comprises from 2-50, 3-50, 4-50, 5-5-, 6-50, 7-50, 8-50, 9-50, or 10-50 antigen peptide sequences. [0809] In some embodiments, the APCs comprise APCs loaded with one or more antigen peptides comprising one or more of the at least one antigen peptide sequence.
  • the APCs are autologous APCs or allogenic APCs. In some embodiments, the APCs comprise dendritic cells (DCs).
  • a method comprises depleting CD14 and/or CD25 expressing cells from the biological sample. In some embodiments, depleting CD14+ cells comprises contacting a CD14 binding agent to the APCs. In some embodiments, the APCs are derived from CD14+ monocytes. In some embodiments, the APCs are enriched from the biological sample. For example, an APC can be isolated from, enriched from, or purified from a biological sample from a subject comprising PBMCs.
  • the APCs are stimulated with one or more cytokines or growth factors.
  • the one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, or a combination thereof.
  • the one or more cytokines or growth factors comprise IL-4, IFN- ⁇ , LPS, GM-CSF, TNF- ⁇ , IL-1 ⁇ , PGE1, IL-6, IL-7 or a combination thereof.
  • the APCs are from a second biological sample. In some embodiments, the second biological sample is from the same subject.
  • a percentage of antigen specific T cells in the method is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells.
  • a percentage of antigen specific T cells in the method is from about 0.1% to about 5%, from about 5 % to 10%, from about 10% to 15%, from about 15% to 20%, from about 20% to 25%, from about 25% to 30%, from about 30% to 35%, WSGR Docket No.50401-795.601 from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, from about 50% to about 55%, from about 55% to about 60%, from about 60% to 65%, or from about 65% to about 70% of total T cells or total immune cells.
  • a percentage of antigen specific CD8+ T cells in the method is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells.
  • a percentage of antigen specific na ⁇ ve CD8+ T cells in the method is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells.
  • a percentage of antigen specific memory CD8+ T cells in the method is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells.
  • a percentage of antigen specific CD4+ T cells in the method is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells.
  • a percentage of antigen specific CD4+ T cells in the method is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of total T cells or total immune cells.
  • a percentage of antigen specific T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.
  • a percentage of antigen specific CD8+ T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.
  • a percentage of antigen specific na ⁇ ve CD8+ T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.
  • a percentage of antigen specific memory CD8+ T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.
  • a percentage of antigen specific CD4+ T cells in the biological sample is at most about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.
  • a biological sample is freshly obtained from a subject or is a frozen sample.
  • a method comprises incubating one or more of the APC preparations with a first medium comprising at least one cytokine or growth factor for a first time period.
  • the first time period is at lease 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, or 18 days.
  • the first time period is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days.
  • the first time period is at least 1, 23, 4, 5, 6, 7, 8, or 9 days.
  • the first time period is no more than 3, 4, 5, 6, 7, 8, 9, or 10 days.
  • the at least one cytokine or growth factor comprises GM-CSF, IL-4, FLT3L, TNF- ⁇ , IL-1 ⁇ , PGE1, IL-6, IL-7, IFN- ⁇ , LPS, IFN- ⁇ , R848, LPS, ss-rna40, poly I:C, or any combination thereof.
  • a method comprises incubating one or more of the APC preparations with at least one peptide for a second time period. In some embodiments, the second time period is no more than 1 hour.
  • a method comprises incubating one or more of the APC preparations with a second medium comprising one or more cytokines or growth factors for a third time period, thereby obtaining matured APCs.
  • the one or more cytokines or growth factors comprises GM-CSF (granulocyte macrophage colony-stimulating factor), IL-4, FLT3L, IFN- ⁇ , LPS, TNF- ⁇ , IL-1 ⁇ , PGE1, IL-6, IL-7, IFN- ⁇ , R848 (resiquimod), LPS, ss-rna40, poly I:C, CpG, or a combination thereof.
  • the third time period is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days. In some embodiments, the third time period is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days. In some embodiments, the third time period is no more than 2, 3, 4, or 5 days. In some embodiments, the third time period is at least 1, 2, 3, or 4 days. [0818] In some embodiments, the method further comprises removing the one or more cytokines or growth factors of the second medium after the third time period and before a start of the fourth time period.
  • the NEO-STIM process may comprise methods of generating antigen loaded PBMCs for induction of T cells (e.g., RAS-specific T cells).
  • the methods provided herein comprise isolating PBMCs from a human blood sample, and directly loading the PBMCs with antigens. PBMCs directly contacted with antigens can readily take up antigens by phagocytosis and present antigens to T cells that can be in the culture or added to the culture.
  • the methods provided herein comprise isolating PBMCs from a human blood sample, and nucleofecting or electroporating a polynucleotide, such as an mRNA, that encodes one or more antigens into the PBMCs.
  • antigens delivered to PBMCs instead of antigen-presenting cells maturing to DCs, provides a great advantage in terms of time and manufacturing efficiency.
  • the PBMCs can be further depleted of one or more cell types.
  • the PBMCs can be depleted of CD3+ cells for an initial period of antigen loading and the CD3+ cells returned to the culture for the PBMCs to stimulate the CD3+ T cells.
  • the PBMCs can be depleted of CD25+ cells. In some embodiments, the PBMCs can be depleted of CD14+ cells. In some embodiments, the PBMCs can be depleted of CD19+ cells. In some embodiments, the PBMCs can be depleted of both CD14 and CD25 expressing WSGR Docket No.50401-795.601 cells. In some embodiments, CD11b+ cells are depleted from the PBMC sample before antigen loading. In some embodiments, CD11b+ and CD25+ cells are depleted from the PBMC sample before antigen loading.
  • the PBMCs isolated from a human blood sample can be handled as minimally as possible prior to loading with antigens. Increased handling of PBMCs, for example freezing and thawing cells, multiple cell depletion steps, etc., can impair cell health and viability.
  • the PBMCs are allogeneic to the subject of therapy. In some embodiments the PBMCs are allogeneic to the subject of adoptive cell therapy with antigen specific T cells.
  • the PBMCs are HLA-matched for the subject of therapy.
  • the PBMCs are allogeneic, and matched for the subject’s HLA subtypes, whereas the CD3+ T cells are autologous.
  • the PBMCs are loaded with the respective antigens (e.g., derived from analysis of a peptide presentation analysis platform such as RECON), cocultured with subject’s PBMC comprising T cells in order to stimulate antigen specific T cells.
  • mRNA is used as the immunogen for uptake and antigen presenting.
  • the mRNA comprises shortmer constructs, encoding 9-10 amino acid peptides comprising an epitope. In some embodiments, the mRNA comprises longmer constructs, encoding bout 25 amino acid peptides. In some embodiments, the mRNA comprises a concatenation of multiple epitopes. In some embodiments, the concatemers can comprise one or more epitopes from the same antigenic protein. In some embodiments, the concatemers can comprise one or epitopes from several different antigenic proteins. Several embodiments are described in the Examples section.
  • Antigen loading of PBMCs by antigen loading can comprise various mechanisms of delivery ad incorporation of nucleic acid into the PBMCs.
  • the delivery or mechanism of incorporation includes transfection, electroporation, nucleofection, chemical delivery, for example, lipid encapsulated, or liposome mediated delivery.
  • Use of antigen loaded PBMCs to stimulate T cells can save the maturation time needed in a method that generates DCs from a PBMC sample prior to T cell stimulation.
  • use of antigen loaded PBMCs for example, mRNA loaded PBMCs as APCs reduces the total manufacturing time by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
  • use of antigen loaded PBMCs as APCs reduces the total manufacturing time by 3 days. In some embodiments, use of antigen loaded PBMCs as APCs reduces the total manufacturing time by 4 days. In some embodiments, use of antigen loaded PBMCs as APCs reduces the total manufacturing time by 5 days. In some embodiments, use of antigen loaded PBMCs as APCs reduces the total manufacturing time WSGR Docket No.50401-795.601 by 6 days. In some embodiments, use of antigen loaded PBMCs as APCs reduces the total manufacturing time by 7 days.
  • mRNA loaded PBMCs can stimulate T cells and generate higher antigen specific T cells. In some embodiments, mRNA loaded PBMCs can stimulate T cells and generate higher yield of antigen specific T cells. In some embodiments, mRNA loaded PBMCs can stimulate T cells and generate antigen specific T cells that have higher representation of the input antigens, i.e., reactive to diverse antigens. In some embodiments, mRNA loaded PBMCs can stimulate T cells that have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigen reactivity in the pool of expanded cells.
  • the mRNA loaded PBMCs can stimulate T cells that have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigen reactivity than conventional antigen loaded APCs (such as peptide loaded DCs).
  • T-Cell Receptors [0827] Also disclosed herein are T cell receptors (TCRs) and recombinant nucleic acids encoding the same.
  • TCRs T cell receptors
  • recombinant nucleic acid encoding a TCR that recognizes epitopes (e.g., epitopes derived from RAS) derived from a protein encoded by a gene of a cancer cell.
  • the T-cell receptor can comprise a TCR beta chain construct and a TCR alpha chain construct.
  • TCR The ability of T cells to recognize antigens associated with infectious organisms or cancers is conferred by its TCR, which is made up of both an alpha ( ⁇ ) chain and a beta ( ⁇ ) chain or a gamma ( ⁇ ) and a delta ( ⁇ ) chain.
  • the proteins which make up these chains are encoded by DNA, which employs a unique mechanism for generating the diversity of the TCR.
  • This multi-subunit immune recognition receptor can associate with the CD3 complex and bind peptides presented by the MHC class I and II proteins on the surface of antigen-presenting cells (APCs).
  • APCs antigen-presenting cells

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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/US2025/026433 2024-04-25 2025-04-25 Compositions de vaccins ras et méthodes d'utilisation Pending WO2025227066A2 (fr)

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