WO2017173321A1 - Neoantigens and methods of their use - Google Patents

Neoantigens and methods of their use Download PDF

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Publication number
WO2017173321A1
WO2017173321A1 PCT/US2017/025462 US2017025462W WO2017173321A1 WO 2017173321 A1 WO2017173321 A1 WO 2017173321A1 US 2017025462 W US2017025462 W US 2017025462W WO 2017173321 A1 WO2017173321 A1 WO 2017173321A1
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WO
WIPO (PCT)
Prior art keywords
cell
peptide
cells
neoantigenic peptide
isolated
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.)
Ceased
Application number
PCT/US2017/025462
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English (en)
French (fr)
Inventor
Michael Steven ROONEY
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
Neon Therapeutics 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
Priority to AU2017240745A priority Critical patent/AU2017240745B2/en
Priority to RS20250878A priority patent/RS67172B1/sr
Priority to KR1020227041564A priority patent/KR20220163523A/ko
Priority to CR20180519A priority patent/CR20180519A/es
Priority to US16/094,851 priority patent/US20190307868A1/en
Priority to PL17776811.6T priority patent/PL3436048T3/pl
Priority to EP17776811.6A priority patent/EP3436048B1/en
Priority to SG11201808196UA priority patent/SG11201808196UA/en
Priority to CA3018748A priority patent/CA3018748A1/en
Priority to FIEP17776811.6T priority patent/FI3436048T3/fi
Priority to RU2018138163A priority patent/RU2773273C2/ru
Priority to BR112018070183-1A priority patent/BR112018070183A2/pt
Priority to DK17776811.6T priority patent/DK3436048T3/da
Priority to ES17776811T priority patent/ES3036912T3/es
Priority to CN202211177112.5A priority patent/CN115558030A/zh
Priority to CN201780034080.4A priority patent/CN109310739A/zh
Priority to SI201731623T priority patent/SI3436048T1/sl
Priority to JP2018551996A priority patent/JP2019513373A/ja
Application filed by Neon Therapeutics Inc filed Critical Neon Therapeutics Inc
Priority to KR1020187031742A priority patent/KR20180129899A/ko
Publication of WO2017173321A1 publication Critical patent/WO2017173321A1/en
Priority to IL261880A priority patent/IL261880B/en
Priority to PH12018502047A priority patent/PH12018502047A1/en
Anticipated expiration legal-status Critical
Priority to CONC2018/0011668A priority patent/CO2018011668A2/es
Priority to AU2021269272A priority patent/AU2021269272B2/en
Priority to IL292094A priority patent/IL292094B2/en
Priority to JP2022088325A priority patent/JP7491965B2/ja
Priority to IL299926A priority patent/IL299926B1/en
Priority to US18/311,155 priority patent/US20230405102A1/en
Priority to US18/311,158 priority patent/US20230414735A1/en
Priority to AU2023241320A priority patent/AU2023241320B2/en
Priority to JP2024080050A priority patent/JP2024105596A/ja
Priority to IL323656A priority patent/IL323656A/en
Priority to AU2025242131A priority patent/AU2025242131A1/en
Ceased legal-status Critical Current

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Definitions

  • the field of the present invention relates to immunotherapeutic peptides, nucleic acids encoding the peptides, peptide binding agents, and their use, for example, in the immunotherapy of cancer.
  • the invention provides neoantigenic peptides, useful alone or in combination with other tumor-associated peptides, anti-cancer, or immunomodulatory agents to treat cancer.
  • Tumor vaccines are typically composed of tumor antigens and immunostimulatory molecules (e.g., adjuvants, cytokines or TLR ligands) that work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells.
  • immunostimulatory molecules e.g., adjuvants, cytokines or TLR ligands
  • Such vaccines contain either shared tissue restricted tumor antigens or a mixture of shared and patient-specific antigens in the form of whole tumor cell preparations.
  • the shared tissue restricted tumor antigens are ideally immunogenic proteins with selective expression in tumors across many individuals and are commonly delivered to patients as synthetic peptides or recombinant proteins.
  • whole tumor cell preparations are delivered to patients as autologous irradiated cells, cell lysates, cell fusions, heat-shock protein preparations or total mRNA. Since whole tumor cells are isolated from the autologous patient, the cells may include patient-specific tumor antigens as well as shared tumor antigens. Finally, there is a third class of tumor antigens, neoantigens, that has rarely been used in vaccines, which consists of proteins with tumor-specific mutations (which can be patient-specific or shared) that result in altered amino acid sequences.
  • Such mutated proteins are: (a) unique to the tumor cell as the mutation and it's corresponding protein are present only in the tumor; (b) avoid central tolerance and are therefore more likely to be immunogenic; (c) provide an excellent target for immune recognition including by both humoral and cellular immunity.
  • the use of personalized neoantigens requires sequencing of each patient's genome and then production of a patient-specific neoantigen composition. Accordingly, there is still a need for developing additional cancer therapeutics.
  • an isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCz, wherein each A and C represents an amino acid corresponding to the native polypeptide, y is at least 1 and B represents an amino acid substitution or insert of the native polypeptide, x + y + z is at least 8, the at least 8 contiguous amino acids comprises By, and the native polypeptide is encoded by a gene selected from the group consisting of: (a) ABL1, wherein AxByCz is (i)
  • the native polypeptide is encoded by the EGFR, ERBB3 or FGFR3 gene and at least one By is expressed extracellularly.
  • an isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCz, wherein each A is an amino acid corresponding to the native polypeptide; By is absent; each C is an amino acid encoded by a frameshift of a sequence encoding the native polypeptide; x + y + z is at least 8; the at least 8 contiguous amino acids comprises at least one Cz; and the native polypeptide is encoded by a gene selected from the group consisting of: (a) APC, wherein Cz is (i)
  • AxByCz or Cz is SIQVMRAQMNQIQSVEGQPLARRPRATGRTKRCQPRDVTKKTCNSNDGKKREWEKRKQILGGGGK YKEYFLKRILIRKAMTVLAGDKKGLGRFMRCVQSETKAVSLQLPLGR; (o) ESRP1, wherein AxByCz is (i) LDFLGEFATDIRTOGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKKHEGQIC, or (ii) LDFLGEFATDIRTHGVHMVLNHQGRPSGDAFIQMKSADRAFMAAQKCHKKT; (p) F AMI IB, wherein AxByCz is SIQVMRAQMNQIQSVEGQPLARRPRATGRTKRCQPRDVTKKTCNSNDGKKREWEKRKQILGGGGK YKEYFLKRILIRKAMTVLAGDKKGLGRFMRCVQSETKAVSLQLPLGR; (o)
  • AxByCz is (i) MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKN, or (ii) MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKLMLLRLNLRKMCGPF; (y) SEC63, wherein AxByCz is (i)
  • an isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCz, wherein each A is an amino acid corresponding to a first native polypeptide; each C is an amino acid corresponding to a second native polypeptide, or a cryptic exon or exon of a splice variant of the first native polypeptide, each B is an amino acid that is not an amino acid corresponding to the first native polypeptide, the second native polypeptide, or the cryptic exon of the first native polypeptide, and x + y + z is at least 8, wherein y is absent and the at least 8 contiguous amino acids comprises at least one Ax and at least one Cz, or y is at least 1 and the at least 8 con
  • the first native polypeptide is encoded by a C I lorf95 gene
  • the second native polypeptide is encoded by an RELA gene
  • y is 1
  • AxByCz is
  • the first native polypeptide is encoded by a CBFB gene and the second native polypeptide is encoded by an MYH1 1 gene, y is 0, and AxByCz is
  • the first native polypeptide is encoded by a CD74 gene and the second native polypeptide is encoded by an ROS 1 gene, y is 0, and AxByCz is
  • the first native polypeptide is encoded by an EGFR gene and the second native polypeptide is encoded by (i) an SEPT14 gene, y is 0, and AxByCz is
  • the first native polypeptide is encoded by a EML4 gene
  • the second native polypeptide is encoded by an ALK gene
  • y is 1
  • AxByCz is
  • SWENSDDSRNKLSKIPSTPKLIPKVTKTADKHKDVIINQAKMSTREKNSQVYRRKHQELQAMQMEL QSPEYKLSKLRTSTIMTDYNPNYCFAGKTSSISDL (g) the first native polypeptide is encoded by a FGFR3 gene, the second native polypeptide is encoded by an TACC3 gene, y is 0, and AxByCz is
  • the first native polypeptide is encoded by a NAB gene
  • the second native polypeptide is encoded by an STAT6 gene
  • y is at least 1
  • AxByCz is RDNTLLLRRVELFSLSRQVARESTYLSSLKGSRLHPEELGGPPLKKLKQEATSKSQIMSLWGLVSKMP PEKVQRLYVDFPQHLRHLLGDWLESQPWEFLVGSDAFCC
  • the second native polypeptide is encoded by an ERG, y is 0, and (i) the first native polypeptide is encoded by a NDRGl gene, and AxByCz is
  • the first native polypeptide is encoded by a TMPRSS2 gene, and AxByCz is MALNSEALSVVSEDQSLFECAYGTPHLAKTEMTASSSSDYGQTSKMSPRVPQQDW;
  • the first native polypeptide is encoded by a PML gene, the second native polypeptide is encoded by an RARA gene, y is 1, and AxByCz is (i)
  • the first native polypeptide is encoded by a RU X1 gene
  • the second native polypeptide is encoded by an CBFA2T1 (RU X1T1) gene
  • y is 1
  • AxByCz is
  • the first native polypeptide is encoded by a AR-v7 gene
  • the cryptic exon or the exon of a splice variant is encoded by the AR-v7 gene
  • y is 0, and
  • AxByCz is SCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGEKFRVGNCKHLKMTRP
  • an isolated neoantigenic peptide comprising a tumor-specific neoepitope, wherein the isolated neoantigenic peptide is not a native polypeptide, wherein the neoepitope comprises at least 8 contiguous amino acids of an amino acid sequence represented by: AxByCz wherein each A is an amino acid corresponding to a first native polypeptide; each B is an amino acid that is not an amino acid corresponding to the first native polypeptide or the second native polypeptide, each C is an amino acid encoded by a frameshift of a sequence encoding a second native polypeptide; x + y + z is at least 8, wherein y is absent and the at least 8 contiguous amino acids comprises at least one Cz, or y is at least 1 and the at least 8 contiguous amino acids comprises at least one By and/or at least one Cz; and (a) the first native polypeptide is encoded by an amino acid sequence represented by: AxByCz wherein
  • the second native polypeptide is encoded by a LRRC69 gene, y is 1, and AxByCz is MAGAPPPASLPPCSLISDCCASNQRDSVGVGPSEPGN IKICNESASRK
  • the first native polypeptide is encoded by an EEF1DP3 gene
  • the second native polypeptide is encoded by a FRY gene
  • y is 1
  • AxByCz is
  • HGWRPFLPVRARSRWNRRLDVTVANGRSWKYGWSLLRVPQVNGIQVLNVSLKSSSNVISY (c) the first native polypeptide is encoded by a MAD1L1 gene, the second native polypeptide is encoded by a MAFK gene, y is 0, and AxByCz is
  • the first native polypeptide is encoded by a PPP1R1B gene
  • the second native polypeptide is encoded by a STARD3 gene
  • y is 1
  • AxByCz is
  • the isolated neoantigenic peptide comprises a sequence according to Table 1.
  • x + y + z is at most 500, at most 250, at most 150, at most 125, or at most 100
  • x + y + z is at least 8, at least 50, at least 100, at least 200, or at least 300.
  • z is at most 500, at most 250, at most 150, at most 125, or at most 100.
  • z is at least 8, at least 50, at least 100, at least 200, or at least 300.
  • the isolated neoantigenic peptide is from about 8 to about 500 amino acids in length.
  • the isolated neoantigenic peptide is from about 8 to about 100 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 8 to about 50 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 15 to about 35 amino acids in length.
  • the isolated neoantigenic peptide is from about 8 and about 15 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 8 and about 1 1 amino acids in length. In embodiments, the isolated neoantigenic peptide is 9 or 10 amino acids in length. In embodiments, the isolated neoantigenic peptide binds major histocompatibility complex (MHC) class I. In embodiments, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 500 nM or less. In embodiments, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 250 nM or less.
  • MHC major histocompatibility complex
  • the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 50 nM or less. In embodiments, the isolated neoantigenic peptide is from about 8 and about 30 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 8 to about 25 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 15 to about 24 amino acids in length. In embodiments, the isolated neoantigenic peptide is from about 9 to about 15 amino acids in length. In embodiments, the isolated neoantigenic peptide binds MHC class II. In embodiments, the isolated
  • neoantigenic peptide binds MHC class II with a binding affinity of 1000 nM or less. In embodiments, the isolated neoantigenic peptide binds MHC class I with a binding affinity of about 500 nM or less. In embodiments, the isolated neoantigenic peptide further comprises flanking amino acids. In embodiments, the flanking amino acids are not native flanking amino acids In embodiments, the isolated neoantigenic peptide has a total length of at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least
  • the isolated neoantigenic peptide has a total length of at most 8, at most 9, at most 10, at most 1 1, at most 12, at most 13, at most 14, at most 15, at most 16, at most
  • the isolated neoantigenic peptide is a first neoantigenic peptide linked to at least a second neoantigenic peptide.
  • the isolated neoantigenic peptide is linked to the at least second neoantigenic peptide by a poly -glycine or poly-serine linker.
  • the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 1000 nM.
  • the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 500 nM.
  • isolated neoantigenic peptide and the second neoantigenic peptide bind to human leukocyte antigen (HLA) -A, -B, -C, -DP, -DQ, or -DR.
  • HLA human leukocyte antigen
  • the isolated neoantigenic peptide binds a class I HLA and the second neoantigenic peptide binds a class II HLA.
  • the isolated neoantigenic peptide binds a class II HLA and the second neoantigenic peptide binds a class I HLA.
  • the isolated neoantigenic peptide further comprises a modification which increases in vivo half-life, cellular targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, antigen presentation, or a combination thereof.
  • the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticulate encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, the addition of a surface active material, the addition of amino acid mimetics, or the addition of unnatural amino acids.
  • the isolated neoantigenic peptide further comprises a modification which increases cellular targeting to antigen presenting cells.
  • the antigen presenting cells are dendritic cells.
  • the dendritic cells are targeted using DEC205, XCRl, CD 197, CD80, CD86, CD123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD141, CD1 lc, CD83, TSLP receptor, Clec9a, or CD la marker.
  • the dendritic cells are targeted using the CD141, DEC205, Clec9a, or XCRl marker.
  • the dendritic cells are autologous cells.
  • one or more of the dendritic cells are bound to a T cell.
  • the T cell is an autologous T cell.
  • the isolated neoantigenic peptide is not a isolated neoantigenic peptide listed in Table 2.
  • the isolated neoantigenic peptide is linked to at least one additional neoantigenic peptide listed in Table 1 or 2.
  • an in vivo delivery system comprising an isolated neoantigenic peptide described herein.
  • the delivery system includes cell-penetrating peptides, nanoparticulate encapsulation, virus like particles, liposomes, or any combination thereof.
  • the cell-penetrating peptide is TAT peptide, herpes simplex virus VP22, transportan, Antp, or any combination thereof.
  • a cell comprising an isolated neoantigenic peptide described herein.
  • the cell is an antigen presenting cell. In embodiments, the cell is a dendritic cell. In
  • the cell is an autologous cell. In embodiments, the cell is bound to a T cell. In embodiments, the T cell is an autologous T cell.
  • composition comprising an isolated neoantigenic peptide described herein.
  • the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least
  • neoantigenic peptides comprising a tumor-specific neoepitope according to
  • the composition comprises from about 2 to about 20 neoantigenic peptides, or from about 2 to about 30 neoantigenic peptides.
  • the neoantigen is specific for an individual subject's tumor.
  • the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 additional neoantigenic peptides.
  • the composition comprises from about 4 to about 20 additional neoantigenic peptides, from about 4 to about 30 additional neoantigenic peptides.
  • the additional neoantigenic peptides is specific for an individual subject's tumor.
  • the subject specific neoantigenic peptide is selected by identifying sequence differences between the genome, exome, and/or transcriptome of the subject's tumor sample and the genome, exome, and/or transcriptome of a non-tumor sample.
  • the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells.
  • the sequence differences are determined by Next Generation Sequencing.
  • the polynucleotide encoding an isolated neoantigenic peptide described herein.
  • the polynucleotide is DNA.
  • the polynucleotide is RNA.
  • the RNA is a self-amplifying RNA.
  • the RNA is modified to increase stability, increase cellular targeting, increase translation efficiency, adjuvanticity, cytosol accessibility, and/or decrease cytotoxicity.
  • the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, codon optimization, increased GC -content, incorporation of modified nucleosides, incorporation of 5'-cap or cap analog, and/or incorporation of an unmasked poly-A sequence.
  • a cell comprising the polynucleotide described herein.
  • a vector comprising the polynucleotide described herein.
  • the polynucleotide is operably linked to a promoter.
  • the vector is a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion.
  • the vector is derived from an adeno-associated virus, herpesvirus, lentivirus, or a pseudotype thereof.
  • an in vivo delivery system comprising the isolated polynucleotide described herein.
  • the delivery system includes spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids, or nanoparticles.
  • a cell comprising a vector or delivery system described herein.
  • the cell is an antigen presenting cell.
  • the cell is a dendritic cell.
  • the cell is an immature dendritic cell.
  • composition comprising at least one polynucleotide described herein.
  • the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least
  • the composition comprises from about 2 and about 20 of the isolated polynucleotides, or from about 2 to about 30 of the isolated polynucleotides.
  • the neoantigenic peptides are encoded by a vector comprising one or more of the the isolated polynucleotides.
  • the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 additional neoantigenic polynucleotides encoding for additional neoantigenic peptides.
  • one or more of the additional neoantigenic peptides are encoded by a vector comprising one or more of the additional neoantigenic polynucleotides.
  • the composition comprises from about 4 to about 20 additional neoantigenic polynucleotides, or from about 4 to about 30 additional neoantigenic polynucleotides.
  • the isolated polynucleotides and the additional neoantigenic polynucleotides are linked.
  • the polynucleotides are linked using nucleic acids that encode a poly-glycine or poly-serine linker.
  • the additional neoantigenic peptide is specific for an individual subject 's tumor.
  • the subject specific neoantigenic peptide is selected by identifying sequence differences between the genome, exome, and/or transcriptome of the subject's tumor sample and the genome, exome, and/or transcriptome of a non-tumor sample.
  • the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells.
  • the sequence differences are determined by Next Generation Sequencing.
  • TCR T cell receptor
  • the MHC of the MHC -peptide is MHC class I or class II.
  • TCR is a bispecific TCR further comprising a domain comprising an antibody or antibody fragment capable of binding an antigen.
  • the antigen is a T cell-specific antigen.
  • the antigen is CD3.
  • the antibody or antibody fragment is an anti-CD3 scFv.
  • a chimeric antigen receptor comprising: (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding at least one neoantigenic peptide described hereinor an MHC-peptide complex comprising at least one neoantigenic peptide described herein.
  • CD3-zeta is the T cell activation molecule.
  • the chimeric antigen receptor further comprises at least one costimulatory signaling domain.
  • the signaling domain is CD28, 4-1BB, ICOS, OX40, ITAM, or Fc epsilon Rl-gamma.
  • the antigen recognition moiety is capable of binding the isolated neoantigenic peptide in the context of MHC class I or class II.
  • the neoantigenic peptide is located in the extracellular domain of a tumor associated polypeptide.
  • the MHC of the MHC-peptide is MHC class I or class II.
  • T cell comprising the T cell receptor or chimeric antigen receptor described herein, optionally wherein the T cell is a helper or cytotoxic T cell.
  • the T cell is a T cell of a subject.
  • a T cell comprising a T cell receptor (TCR) capable of binding at least one neoantigenic peptide described herein or an MHC-peptide complex comprising at least one neoantigenic peptide described herein, wherein the T cell is a T cell isolated from a population of T cells from a subject that has been incubated with antigen presenting cells and one or more of the at least one neoantigenic peptide described herein for a sufficient time to activate the T cells.
  • the T cell is a CD8+ T cell, a helper T cell or cytotoxic T cell.
  • the population of T cells from a subject is a population of CD8+ T cells from the subject.
  • the one or more of the at least one neoantigenic peptide described herein is a subject-specific neoantigenic peptide.
  • the subject-specific neoantigenic peptide has a different tumor neo-epitope that is an epitope specific to a tumor of the subject.
  • the subject-specific neoantigenic peptide is an expression product of a tumor-specific non-silent mutation that is not present in a non-tumor sample of the subject. In embodiments, the subject-specific neoantigenic peptide binds to a HLA protein of the subject. In embodiments, the subject-specific neoantigenic peptide binds to a HLA protein of the subject with an IC50 less than 500 nM.
  • the activated CD8+ T cells are separated from the antigen presenting cells. In embodiments, the antigen presenting cells are dendritic cells or CD40L-expanded B cells. In embodiments, the antigen presenting cells are non-transformed cells.
  • the antigen presenting cells are non-infected cells. In embodiments, the antigen presenting cells are autologous. In embodiments, the antigen presenting cells have been treated to strip endogenous MHC -associated peptides from their surface. In embodiments, the treatment to strip the endogenous MHC -associated peptides comprises culturing the cells at about 26°C. In embodiments, the treatment to strip the endogenous MHC -associated peptides comprises treating the cells with a mild acid solution. In embodiments, the antigen presenting cells have been pulsed with at least one neoantigenic peptide described herein.
  • pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 ⁇ g/ml of each of the at least one neoantigenic peptide described herein.
  • ratio of isolated T cells to antigen presenting cells is between about 30: 1 and 300: 1.
  • the incubating the isolated population of T cells is in the presence of IL-2 and IL-7.
  • the MHC of the MHC-peptide is MHC class I or class II.
  • a method for activating tumor specific T cells comprising: isolating a population of T cells from a subject; and incubating the isolated population of T cells with antigen presenting cells and at least one neoantigenic peptide described herein for a sufficient time to activate the T cells.
  • the T cell is a CD8+ T cell, a helper T cell or cytotoxic T cell.
  • the population of T cells from a subject is a population of CD8+ T cells from the subject.
  • the one or more of the at least one neoantigenic peptide described herein is a subject-specific neoantigenic peptide.
  • the subject- specific neoantigenic peptide has a different tumor neo-epitope that is an epitope specific to a tumor of the subject.
  • the subject-specific neoantigenic peptide is an expression product of a tumor- specific non-silent mutation that is not present in a non-tumor sample of the subject.
  • the subject-specific neoantigenic peptide binds to a HLA protein of the subject.
  • the subject- specific neoantigenic peptide binds to a HLA protein of the subject with an IC50 less than 500 nM.
  • the method further comprises separating the activated T cells from the antigen presenting cells.
  • the method further comprises testing the activated T cells for evidence of reactivity against at least one of neoantigenic peptide of described herein.
  • the antigen presenting cells are dendritic cells or CD40L-expanded B cells.
  • the antigen presenting cells are non-transformed cells.
  • the antigen presenting cells are non-infected cells.
  • the antigen presenting cells are autologous.
  • the antigen presenting cells have been treated to strip endogenous MHC -associated peptides from their surface.
  • the treatment to strip the endogenous MHC -associated peptides comprises culturing the cells at about 26°C.
  • the treatment to strip the endogenous MHC -associated peptides comprises treating the cells with a mild acid solution.
  • the antigen presenting cells have been pulsed with at least one neoantigenic peptide described herein.
  • pulsing comprises incubating the antigen presenting cells in the presence of at least about 2 ⁇ g/ml of each of at least one neoantigenic peptide described herein.
  • ratio of isolated T cells to antigen presenting cells is between about 30: 1 and 300: 1.
  • the incubating the isolated population of T cells is in the presence of IL-2 and IL-7.
  • the MHC of the MHC- peptide is MHC class I or class II.
  • composition comprising activated tumor specific T cells produced by a method described herein.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of activated tumor specific T cell described herein, or produced by a method described herein.
  • the administering comprises administering from about 10 6 to 10 12 , from about 10 8 to 10 11 , or from about 10 9 to 10 10 of the activated tumor specific T cells.
  • a nucleic acid comprising a promoter operably linked to a polynucleotide encoding the T cell receptor described herein.
  • the TCR is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II.
  • MHC major histocompatibility complex
  • nucleic acid comprising a promoter operably linked to a polynucleotide encoding the chimeric antigen receptor described herein.
  • the antigen recognition moiety is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class
  • the neoantigenic peptide is located in the extracellular domain of a tumor associated polypeptide.
  • the nucleic acid comprises the CD3-zeta, CD28, CTLA-4, ICOS,
  • BTLA KIR
  • LAG3 CD 137
  • OX40 CD27
  • CD40L Tim-3
  • A2aR Tim-3
  • A2aR Tim-3
  • PD- 1 transmembrane region BTLA, KIR, LAG3, CD 137, OX40, CD27, CD40L, Tim-3, A2aR, or PD- 1 transmembrane region.
  • an antibody or antibody fragment capable of binding at least one neoantigenic peptide described herein or an MHC -peptide complex comprising at least one neoantigenic peptide described herein, optionally wherein the antibody fragment is a bi-specific T cell engager (BiTE).
  • the antibody or antibody fragment binds to an extracellular portion of the at least one neoantigenic peptide.
  • the native polypeptide is encoded by a gene selected from the group consisting of: ⁇ 2 ⁇ , wherein Cz is RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or
  • AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; BTK, wherein AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; BTK, wherein AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDYVREHKD NIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAA; BTK, wherein AxByCz is IPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLIMQLMPFGCLLDY
  • the antibody or antibody fragment is a bispecific antibody or antibody fragment.
  • one antigen binding domain of the bispecific antibody or antibody fragment is an anti-CD3 binding domain.
  • the modified cell is a T cell, tumor infiltrating lymphocyte, NK-T cell, TCR-expressing cell, CD4+ T cell, CD8+ T cell, or NK cell.
  • composition comprising the T cell receptor or chimeric antigen receptor described herein.
  • composition comprising autologous subject T cells containing the T cell receptor or chimeric antigen receptor described herein.
  • the composition further comprises an immune checkpoint inhibitor.
  • the composition further comprises at least two immune checkpoint inhibitors.
  • each of the immune checkpoint inhibitors inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDLl, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • each of the immune checkpoint inhibitors interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDLl, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • the T cells are PD-1 and/or CTLA4 knockout T cells, optionally ,wherein the PD-1 and/or CTLA4 knockout T cells are created using a CRISPR system.
  • the composition further comprises an immune modulator or adjuvant.
  • the immune modulator is a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB.
  • the immune modulator is at least one cancer cell or cancer cell extract.
  • the cancer cell is autologous to the subject in need of the composition.
  • the cancer cell has undergone lysis or been exposed to UV radiation.
  • the composition further comprises an adjuvant.
  • the adjuvant is selected from the group consisting of: Poly(I:C), Poly-ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312 VG, Montanide ISA 206 VG, Montanide ISA 50 V2, Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK, PepTel®.
  • the composition induces a humoral response when administered to a subject.
  • the composition induces a T helper cell type 1 when administered to a subject.
  • a method of inhibiting growth of a tumor cell expressing a tumor-specific neoepitope comprising contacting the tumor cell with the peptide, polynucleotide, delivery system, vector, composition, antibody, or cells described herein.
  • a method of prophylaxis of a subject comprising contacting a cell of the subject with the peptide, polynucleotide, delivery system, vector, composition, antibody, or cells described herein.
  • the native polypeptide is encoded by a gene selected from the group consisting of: ⁇ 2 ⁇ , wherein Cz is RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or
  • a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject the peptide, polynucleotide, vector, composition, antibody, or cells described herein.
  • the subject is a human.
  • the subject has cancer.
  • the cancer is selected from the group consisting of urogenital, gynecological, lung, gastrointestinal, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel Cell Carcinoma or bone and soft tissue sarcomas, haematologic neoplasias, multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, non-small cell lung cancer (NSCLC), breast cancer, metastatic colorectal cancers, hormone sensitive or hormone refractory prostate cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, oesophageal cancers, hepatocellular cancers, cholangiocellular cancers, head and neck squamous cell cancer soft
  • the peptide, polynucleotide, vector, composition, antibody, or cells described herein is for use in treating a corresponding cancer according to Table 1 or Table 2. In embodiments, the peptide, polynucleotide, vector, composition, antibody, or cells described herein is for use in treating a subject with an HLA type that is a corresponding
  • the subject has undergone surgical removal of the tumor.
  • the peptide, polynucleotide, vector, composition, or cells is administered via intravenous, intraperitoneal, intratumoral, intradermal, or subcutaneous administration.
  • the peptide, polynucleotide, vector, composition, or cells is administered into an anatomic site that drains into a lymph node basin.
  • administration is into multiple lymph node basins.
  • administration is by a subcutaneous or intradermal route.
  • peptide is administered.
  • administration is intratumorally.
  • polynucleotide, optionally RNA is administered.
  • the polynucleotide is administered intravenously.
  • the cell is a
  • the peptide or polynucleotide comprises an antigen presenting cell targeting moiety.
  • the cell is an autologous cell.
  • the method further comprises administering at least one immune checkpoint inhibitor to the subject.
  • the checkpoint inhibitor is a biologic therapeutic or a small molecule.
  • the checkpoint inhibitor is selected from the group consisting of a monoclonal antibody, a humanized antibody, a fully human antibody and a fusion protein or a combination thereof
  • the checkpoint inhibitors is a PD-1 antibody or a PD-L1 antibody.
  • the checkpoint inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, avelumab, durvalumab, atezolizumab, pembrolizumab, and any combination thereof.
  • the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3,
  • the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3,
  • two or more checkpoint inhibitors are administered.
  • at least one of the two or more checkpoint inhibitors is a PD-1 antibody or a PD-L1 antibody.
  • At least one of the two or more checkpoint inhibitors is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, avelumab, durvalumab, atezolizumab, and pembrolizumab.
  • the checkpoint inhibitor and the composition are administered simultaneously or sequentially in any order.
  • the peptide, polynucleotide, vector, composition, or cells is administered prior to the checkpoint inhibitor.
  • the peptide, polynucleotide, vector, composition, or cells is administered after the checkpoint inhibitor.
  • administration of the checkpoint inhibitor is continued throughout neoantigen peptide, polynucleotide, vector, composition, or cell therapy.
  • the neoantigen peptide, polynucleotide, vector, composition, or cell therapy is administered to subjects that only partially respond or do not respond to checkpoint inhibitor therapy.
  • the composition is administered intravenously or subcutaneously.
  • the checkpoint inhibitor is administered intravenously or subcutaneously.
  • the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the composition.
  • the composition is administered into the same draining lymph node as the checkpoint inhibitor.
  • the method further comprises administering an additional therapeutic agent to the subject either prior to, simultaneously with, or after treatment with the peptide, polynucleotide, vector, composition, or cells.
  • the additional agent is a chemotherapeutic agent, an immunomodulatory drug, an immune metabolism modifying drug, a targeted therapy, radiation an anti-angiogenesis agent, or an agent that reduces immune-suppression.
  • the chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor, an anti-metabolite, or an anti-mitotic agent.
  • the additional agent is an anti- glucocorticoid induced tumor necrosis factor family receptor (GITR) agonistic antibody or antibody fragment, ibrutinib, docetaxeol, cisplatin, a CD40 agonistic antibody or antibody fragment, an IDO inhibitor, or cyclophosphamide.
  • the method elicits a CD4+ T cell immune response or a CD8+ T cell immune response.
  • the method elicits a CD4+ T cell immune response and a CD8+ T cell immune response.
  • the immune response is cytotoxic and/or humoral immune response.
  • the method stimulates a T cell-mediated immune response in a subject.
  • the T cell-mediated immune response is directed against a target cell.
  • the target cell is a tumor cell.
  • the modified cells are transfected or transduced in vivo.
  • the modified cells are transfected or transduced ex vivo.
  • the modified cells are autologous subject T cells.
  • the autologous subject T cells are obtained from a subject that has received a neoantigen peptide or nucleic acid vaccine.
  • the neoantigen peptide or nucleic acid vaccine comprises at least one personalized neoantigen.
  • the neoantigen peptide or nucleic acid vaccine comprises at least one additional neoantigenic peptide listed in Table 1 or 2.
  • the subject received a chemotherapeutic agent, an immunomodulatory drug, an immune metabolism modifying drug, targeted therapy or radiation prior to and/or during receipt of the neoantigen peptide or nucleic acid vaccine.
  • the subject receives treatment with at least one checkpoint inhibitor.
  • the autologous T cells are obtained from a subject that has already received at least one round of T cell therapy containing a neoantigen.
  • the method further comprises adoptive T cell therapy.
  • the adoptive T cell therapy comprises autologous T cells.
  • the autologous T cells are targeted against tumor antigens.
  • the adoptive T cell therapy further comprises allogenic T cells.
  • the allogenic T cells are targeted against tumor antigens.
  • the adoptive T cell therapy is administered before the checkpoint inhibitor, after the checkpoint inhibitor, or simultaneously eith the checkpoint inhibitor.
  • a method for evaluating the efficacy of any of the cells described herein comprising: (i) measuring the number or concentration of target cells in a first sample obtained from the subject before administering the modified cell, (ii) measuring the number concentration of target cells in a second sample obtained from the subject after administration of the modified cell, and (iii) determining an increase or decrease of the number or concentration of target cells in the second sample compared to the number or concentration of target cells in the first sample.
  • treatment efficacy is determined by monitoring a clinical outcome; an increase, enhancement or prolongation of anti-tumor activity by T cells; an increase in the number of anti-tumor T cells or activated T cells as compared with the number prior to treatment; B cell activity; CD4+ T cell activity; or a combination thereof.
  • treatment efficacy is determined by monitoring a biomarker.
  • the biomarker is selected from the group consisting of CEA, Her-2/neu, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA 125, CA19.9, CA 15.3, leptin, prolactin, osteopontin, IGF-II, CD98, fascin, sPIgR, 14-3-3 eta, troponin I, circulating tumor cell R A or DNA, and b-type natriuretic peptide.
  • clinical outcome is selected from the group consisting of tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor expansion, recurrence or spread; or a combination thereof.
  • the treatment effect is predicted by presence of T cells or by presence of a gene signature indicating T cell inflammation or a combination thereof.
  • a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject: the peptide, polynucleotide, vector, composition, antibody, or cells described herein; and at least one checkpoint inhibitor.
  • the method further comprises administration of an immunomodulator or adjuvant.
  • the immunomodulator or adjuvant is selected from the group consisting of Poly(I:C), Poly-
  • ICLC ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune,
  • ISA 50 V2 Montanide ISA 51 VG, OK-432, OM-174, OM-197-MP-EC, ISA-TLR2 agonist, ONTAK,
  • PepTel® vector system PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles,
  • VEGF trap R848, beta-glucan, Pam3Cys, Pam3CSK4, acrylic or methacrylic polymers, copolymers of maleic anhydride, and QS21 stimulon.
  • a co-stimulatory ligand a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-lBB.
  • the immunomodulator or adjuvant is Poly-ICLC.
  • the checkpoint inhibitor is an anti-PDl antibody or antibody fragment.
  • the anti-PDl antibody or antibody fragment is nivolumab or pembolizumab.
  • the checkpoint inhibitor is an anti-PD-Ll antibody or antibody fragment.
  • the anti-PD-Ll antibody or antibody fragment is avelumab, durvalumab or atezolizumab.
  • the checkpoint inhibitor is an anti-CTLA4 antibody or antibody fragment.
  • the anti-CTLA4 antibody is ipilimumab or tremelimumab.
  • the method comprises administering both an anti-PDl antibody and an anti-CTLA4 antibody.
  • administration of the checkpoint inhibitor is initiated before initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In embodiments, administration of the checkpoint inhibitor is initiated after initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In embodiments, administration of the checkpoint inhibitor is initiated simultaneously with the initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell. In embodiments, the peptide,
  • polynucleotide, vector, composition, antibody, or cell is administered intravenously or subcutaneously.
  • the checkpoint inhibitor is administered intravenously or subcutaneously.
  • the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the peptide, polynucleotide, vector, composition, antibody, or cell.
  • the peptide, polynucleotide, vector, composition, antibody, or cell is administered into the same draining lymph node as the checkpoint inhibitor.
  • kits comprising the peptide, polynucleotide, vector, composition, antibody, cells, or composition described herein.
  • the cancer is selected from the group consisting of:
  • the cancer is selected from the group consisting of: prostate cancer, bladder, lung squamous, NSCLC, breast, head and neck, lung adenocarcinoma,
  • the cancer is selected from the group consisting of:
  • the cancer is selected from the group consisting of: melanoma, lung squamous, DLBCL, uterine, head and neck, uterine, liver, and CRC.
  • the cancer is selected from the group consisting of: lymphoid cancer; Burkitt lymphoma, neuroblastoma, prostate adenocarcinoma, colorectal adenocarcinoma; Uterine/Endometrium Adenocarcinoma; MSI+; endometrium serous carcinoma; endometrium carcinosarcoma-malignant mesodermal mixed tumour; glioma; astrocytoma; GBM, acute myeloid leukaemia associated with MDS; chronic lymphocytic leukaemia-small lymphocytic lymphoma; myelodysplastic syndrome; acute myeloid leukaemia; luminal NS carcinoma of breast; chronic myeloid leukaemia; ductal carcinoma of pancreas; chronic myelomonocytic leukaemia; myelofibrosis; myelodysplastic syndrome; prostate adenocarcinoma; essential thrombocythaemia; and me
  • the cancer is selected from the group consisting of: colorectal, uterine, endometrial, and stomach. In embodiments, the cancer is selected from the group consisting of: cervical, head and neck, anal, stomach, Burkitt's lymphoma, and nasopharyngeal carcinoma. In embodiments, the cancer is selected from the group consisting of: bladder, colorectal, and stomach. In embodiments, the cancer is selected from the group consisting of: lung, CRC, melanoma, breast, NSCLC, and CLL. In embodiments, the subject is a partial or non-responder to checkpoint inhibitor therapy.
  • the cancer is selected from the group consisting of: bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), breast cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukaemia (CLL), colorectal cancer (CRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), Prostate Cancer, skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), thyroid
  • the cancer is selected from the group consisting of: colorectal cancer, uterine cancer, endometrium cancer, stomach cancer, and Lynch syndrome.
  • the cancer is an MSI+ cancer.
  • the present invention is directed to an isolated neoantigenic peptide comprising a tumor-specific neoepitope defined in Table 1, wherein the isolated neoantigenic peptide is not a native polypeptide.
  • the present invention is also directed to an isolated neoantigenic peptide which comprises a tumor-specific neoepitope and is defined in Table 1.
  • the isolated neoantigenic peptide is between about 8 to about 50 amino acids in length. In another embodiment, the isolated neoantigenic peptide is between about 15 to about 35 amino acids in length. In another embodiment, the isolated neoantigenic peptide is about 15 amino acids or less in length. In another embodiment, the isolated neoantigenic peptide is between about 8 and about 1 1 amino acids in length. In another embodiment, the isolated neoantigenic peptide is 9 or 10 amino acids in length. In another embodiment, the isolated neoantigenic peptide binds major histocompatibility complex (MHC) class I. In another embodiment, the isolated neoantigenic peptide binds MHC class I with a binding affinity of less than about 500 nM.
  • MHC major histocompatibility complex
  • the isolated neoantigenic peptide is about 30 amino acids or less in length. In another embodiment, the isolated neoantigenic peptide is between about 6 and about 25 amino acids in length. In another embodiment, the isolated neoantigenic peptide is between about 15 and about 24 amino acids in length. In another embodiment, the isolated neoantigenic peptide is between about 9 and about 15 amino acids in length. In another embodiment, the isolated neoantigenic peptide binds MHC class II. In another embodiment, the isolated neoantigenic peptide binds MHC class II with a binding affinity of less than about 1000 nM.
  • the isolated neoantigenic peptide further comprises flanking amino acids.
  • the flanking amino acids are not native flanking amino acids.
  • the isolated neoantigenic peptide is linked to at least a second neoantigenic peptide.
  • the peptides are linked using a poly -glycine or poly-serine linker.
  • the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 1000 nM.
  • the second neoantigenic peptide binds MHC class I or class II with a binding affinity of less than about 500 nM.
  • both of the neoepitopes bind to human leukocyte antigen (HLA) -A, -B, -C, -DP, -DQ, or -DR.
  • HLA human leukocyte antigen
  • the isolated neoantigenic peptide binds a class I HLA and the second neoantigenic peptide binds a class II HLA.
  • the isolated neoantigenic peptide binds a class II HLA and the second neoantigenic peptide binds a class I HLA.
  • the isolated neoantigenic peptide further comprises modifications which increase in vivo half-life, cellular targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation.
  • the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticulate encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, the addition of a surface active material, the addition of amino acid mimetics, or the addition of unnatural amino acids.
  • the cells that are targeted are antigen presenting cells.
  • the antigen presenting cells are dendritic cells.
  • the dendritic cells are targeted using DEC205, XCR1, CD 197, CD80, CD86, CD 123, CD209, CD273, CD283, CD289, CD 184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, CD 141, CD 1 lc, CD83, TSLP receptor, Clec9a or CD la marker.
  • the dendritic cells are targeted using the CD 141, DEC205, or XCR1 marker.
  • the invention provides an in vivo delivery system comprising an isolated neoantigenic peptide described herein.
  • the delivery system includes cell-penetrating peptides, nanoparticulate encapsulation, virus like particles, or liposomes.
  • the cell- penetrating peptide is TAT peptide, herpes simplex virus VP22, transportan, or Antp.
  • the invention is directed to a cell comprising an isolated neoantigenic peptide described herein.
  • the cell is an antigen presenting cell.
  • the cell is a dendritic cell.
  • the invention is directed to a composition comprising an isolated neoantigenic peptide described herein.
  • the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
  • the composition comprises between 2 and 20 neoantigenic peptides.
  • the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least
  • the composition comprises between about 4 and about 20 additional neoantigenic peptides.
  • the additional neoantigenic peptide is specific for an individual patient's tumor.
  • the patient specific neoantigenic peptide is selected by identifying sequence differences between the genome, exome, and/or transcriptome of the patient's tumor sample and the genome, exome, and/or transcriptome of a non-tumor sample.
  • the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells.
  • the sequence differences are determined by Next Generation Sequencing.
  • the invention is directed to an isolated polynucleotide encoding an isolated neoantigenic peptide described herein.
  • the isolated polynucleotide is RNA, optionally a self-amplifying RNA.
  • the RNA is modified to increase stability, increase cellular targeting, increase translation efficiency, adjuvanticity, cytosol accessibility, and/or decrease cytotoxicity.
  • the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, codon optimization, increased GC-content, incorporation of modified nucleosides, incorporation of 5'-cap or cap analog, and/or incorporation of an unmasked poly-A sequence.
  • the invention is directed to a cell comprising a polynucleotide described herein.
  • the invention is directed to a vector comprising a polynucleotide described herein.
  • the polynucleotide is operably linked to a promoter.
  • the vector comprises a self-amplifying RNA replicon, plasmid, phage, transposon, cosmid, virus, or virion.
  • the vector is an adeno-associated virus, herpesvirus, lentivirus, or pseudotypes thereof.
  • the invention is directed to an in vivo delivery system comprising an isolated polynucleotide described herein.
  • the delivery system includes spherical nucleic acids, viruses, virus-like particles, plasmids, bacterial plasmids, or nanoparticles.
  • the invention is directed to a cell comprising a vector or delivery system described herein.
  • the cell is an antigen presenting cell.
  • the cell is a dendritic cell.
  • the cell is an immature dendritic cell.
  • the invention is directed to a composition comprising at least one
  • the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
  • the composition comprises between about 2 and about 20 polynucleotides that encode neoantigenic peptides. In another embodiment, the composition further comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least
  • the composition comprises between about 4 and about 20 additional neoantigenic polynucleotides.
  • the isolated polynucleotides and the additional neoantigenic polynucleotides are linked.
  • the polynucleotides are linked using nucleic acids that encode a poly-glycine or poly-serine linker.
  • at least one of the additional neoantigenic peptide is specific for an individual patient's tumor.
  • the patient specific neoantigenic peptide is selected by identifying sequence differences between the genome, exome, and/or transcriptome of the patient's tumor sample and the genome, exome, and/or transcriptome of a non-tumor sample.
  • the samples are fresh or formalin-fixed paraffin embedded tumor tissues, freshly isolated cells, or circulating tumor cells.
  • the sequence differences are determined by Next Generation Sequencing.
  • the invention is directed to a T cell receptor (TCR) capable of binding at least one neoantigenic peptide described herein.
  • TCR T cell receptor
  • the TCR is capable of binding the isolated neoantigenic peptide in the context of MHC class I or class II.
  • the invention is directed to a chimeric antigen receptor comprising: (i) a T cell activation molecule; (ii) a transmembrane region; and (iii) an antigen recognition moiety capable of binding an isolated neoantigenic peptide described herein.
  • the chimeric antigen receptor contains CD3-zeta as the T cell activation molecule.
  • the chimeric antigen receptor further comprises at least one costimulatory signaling domain.
  • the signaling domain is CD28, 4- IBB, ICOS, OX40, ITAM, or Fc epsilon Rl-gamma.
  • the antigen recognition moiety is capable of binding the isolated neoantigenic peptide in the context of MHC class I or class II.
  • the chimeric antigen receptor comprises the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD 137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region.
  • the tumor-specific epitope is located in the extracellular domain of a tumor associated polypeptide.
  • the invention is directed to a T cell comprising the T cell receptor or chimeric antigen receptor described herein.
  • the T cell is a helper or cytotoxic T cell.
  • the invention is directed to a nucleic acid comprising a promoter operably linked to a polynucleotide encoding a T cell receptor described herein.
  • the TCR is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II.
  • the nucleic acid comprises a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor described herein.
  • the antigen recognition moiety is capable of binding the at least one neoantigenic peptide in the context of major histocompatibility complex (MHC) class I or class II.
  • the tumor-specific epitope is located in the extracellular domain of a tumor associated polypeptide.
  • the nucleic acid comprises the CD3-zeta, CD28, CTLA-4, ICOS, BTLA, KIR, LAG3, CD 137, OX40, CD27, CD40L, Tim-3, A2aR, or PD-1 transmembrane region.
  • the invention is directed to an antibody capable of binding at least one neoantigenic peptide described herein.
  • the invention is directed to a modified cell transfected or transduced with a nucleic acid described herein.
  • the modified cell is a T cell, tumor infiltrating lymphocyte, NK-T cell, TCR-expressing cell, CD4 + T cell, CD8 + T cell, or NK cell.
  • the invention is directed to a composition comprising a T cell receptor or chimeric antigen receptor described herein.
  • the composition comprises autologous patient T cells containing a T cell receptor or chimeric antigen receptor.
  • the composition further comprises an immune checkpoint inhibitor.
  • the composition further comprises at least two immune checkpoint inhibitors.
  • each of the immune checkpoint inhibitors inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • each of the immune checkpoint inhibitors interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • the composition further comprises an immune modulator or adjuvant.
  • the immune modulator is a co-stimulatory ligand, a TNF ligand, an Ig superfamily ligand, CD28, CD80, CD86, ICOS, CD40L, OX40, CD27, GITR, CD30, DR3, CD69, or 4-1BB.
  • the immune modulator is at least one cancer cell or cancer cell extract.
  • the cancer cell is autologous to the subject in need of the composition.
  • the cancer cell has undergone lysis or been exposed to UV radiation.
  • the adjuvant induces a humoral when administered to a subject.
  • the adjuvant induces a T helper cell type 1 when administered to a subject.
  • the invention is directed to a method of inhibiting growth of a tumor cell expressing a tumor-specific neoepitope described herein, comprising contacting the tumor cell with the peptide, polynucleotide, delivery system, vector, composition, antibody, or cells of the invention.
  • the invention is directed to a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject the peptide, polynucleotide, vector, composition, antibody, or cells described herein.
  • the cancer is selected from adrenal, bladder, breast, cervical, colorectal, glioblasoma, head and neck, kidney chromophobe, kidney clear cell, kidney papillary, liver, lung adeno, lung squamous, ovarian, pancreatic, melanoma, stomach, uterine corpus endometrial,and uterine carcinosarcoma.
  • the cancer is selected from the group of: prostate cancer, bladder, lung squamous, NSCLC, breast, head and neck, lung adenocarcinoma, GBM, Glioma, CML, AML, supretentorial ependyomas, acute promyelocytic leukemia, solitary fibrous tumors, and crizotinib resistant cancer.
  • the cancer is selected from the group consisting of CRC, head and neck, stomach, lung squamous, lung adeno., Prostate, Bladder, stomach, renal cell carcinoma, and uterine.
  • the cancer is selected from the group consisting of melanoma, lung squamous, DLBCL, uterine, head and neck, uterine, liver, and CRC.
  • the cancer is selected from the group consisting of lymphoid cancer; Burkitt lymphoma, neuroblastoma, prostate adenocarcinoma, colorectal adenocarcinoma; Uterine/Endometrium Adenocarcinoma; MSI + ; endometrium serous carcinoma; endometrium carcinosarcoma-malignant mesodermal mixed tumour; glioma; astrocytoma; GBM, acute myeloid leukaemia associated with MDS; chronic lymphocytic leukaemia-small lymphocytic lymphoma; myelodysplastic syndrome; acute myeloid leukaemia; luminal NS carcinoma of breast; chronic myeloid leukaemia; ductal carcinoma of pancreas;
  • the cancer is selected from the group consisting of colorectal, uterine, endometrial, and stomach.
  • the cancer is selected from the group consisting of cervical, head and neck, anal, stomach, Burkitt' s lymphoma, and nasopharyngeal carcinoma.
  • the cancer is selected from the group consisting of bladder, colorectal, and stomach.
  • the cancer is selected from the group consisting of lung, CRC, melanoma, breast, NSCLC, and CLL.
  • the cancer is selected from the group consisting of adrenocortical carcinoma
  • ACC acute promyelocytic leukemia
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndromes
  • anal cancer astrocytoma
  • bladder cancer astrocytoma
  • bladder urothelial carcinoma astrocytoma
  • BLCA breast cancer
  • BRCA breast invasive carcinoma
  • BRCA breast invasive carcinoma
  • Burkitt's Lymphoma castration-resistant prostate cancer
  • cervical cancer cervical squamous cell carcinoma and endocervical adenocarcinoma
  • CLL chronic lymphocytic leukaemia
  • CML chronic myeloid leukaemia
  • CML chronic myelomonocytic leukaemia
  • colorectal adenocarcinoma colorectal cancer
  • CRC Crizotinib resistant non-small cell lung cancer
  • DLBCL diffuse large B-cell lymphoma
  • ductal carcinoma of pancreas endometrium carcinosarcoma-malignant mesodermal mixed tumour, endometrium serous carcinoma, essential thrombocythaemia, glioblastoma multiforme (GBM), Glioma, head and neck cancer, head and neck squamous cell carcinoma (HNSC), invasive lobular carcinoma (ILC) LumA breast cancer, kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), acute myeloid leukemia (LAML), liver hepatocellular carcinoma (LIHC), liver cancer, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), Luminal NS carcinoma of breast, lung cancer, lymphoid cancer, medullomyoblastoma, melanoma
  • myelodysplastic syndrome myelofibrosis, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer (NSCLC), ovarian serous cystadenocarcinoma (OV), pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), prostate cancer, renal cell carcinoma, skin cutaneous melanoma (SKCM), solitary fibrous tumors, stomach adenocarcinoma (STAD), stomach cancer, supretentorial ependyomas, thyroid adenocarcinoma (THCA), uterine corpus endometrioid carcinoma (UCEC), or uterine carcinosarcoma (UCS), uterine cancer, and uterine/endometrium adenocarcinoma.
  • NSCLC non-small cell lung cancer
  • PAAD pancreatic adenocarcinoma
  • PRAD prostate adenocarcinoma
  • STAD solitary
  • the cancer is selected from the group consisting of bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), breast cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukaemia (CLL), colorectal cancer (CRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), Prostate Cancer, skin cutaneous melanoma
  • BLCA bladder urothelial carcinoma
  • BRCA breast invasive carcinoma
  • CESC cervical squamous cell carcinoma and endocervical adenocarcinoma
  • CLL chronic lymphocytic leukaemia
  • CML colorectal cancer
  • SKCM stomach adenocarcinoma
  • STAD stomach adenocarcinoma
  • THCA thyroid adenocarcinoma
  • UCEC uterine corpus endometrioid carcinoma
  • the subject is a human.
  • the subject has cancer.
  • the cancer is selected from the group consisting of urogenital, gynecological, lung, gastrointestinal, head and neck cancer, malignant glioblastoma, malignant mesothelioma, non-metastatic or metastatic breast cancer, malignant melanoma, Merkel Cell Carcinoma or bone and soft tissue sarcomas, haematologic neoplasias, multiple myeloma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and acute lymphoblastic leukemia, non-small cell lung cancer (NSCLC), breast cancer, metastatic colorectal cancers, hormone sensitive or hormone refractory prostate cancer, colorectal cancer, ovarian cancer, hepatocellular cancer, renal cell cancer, pancreatic cancer, gastric cancer, oesophageal cancers, he
  • NSCLC non-small cell lung cancer
  • the subject has undergone surgical removal of the tumor.
  • the peptide, polynucleotide, vector, composition, or cells is administered via intravenous, intraperitoneal, intratumoral, intradermal, or subcutaneous administration.
  • the peptide, polynucleotide, vector, composition, or cells is administered into an anatomic site that drains into a lymph node basin.
  • the administration is into multiple lymph node basins.
  • the administration is by a subcutaneous or intradermal route.
  • a peptide is administered. In another embodiment, the administration is intratumorally. In another embodiment of the method, a polynucleotide, optionally RNA, is administered. In another embodiment, the polynucleotide is administered intravenously. In some embodiments of the method, a cell is administered. In another embodiment, the cell is a T cell or dendritic cell. In another embodiment, the peptide or polynucleotide comprises an antigen presenting cell targeting moiety.
  • At least one immune checkpoint inhibitor is also administered to the subject.
  • the checkpoint inhibitor is a biologic therapeutic or a small molecule.
  • the checkpoint inhibitor is selected from the group consisting of a monoclonal antibody, a humanized antibody, a fully human antibody and a fusion protein or a combination thereof.
  • the checkpoint inhibitor inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof.
  • two or more checkpoint inhibitors are administered.
  • the checkpoint inhibitors are: (i) ipilimumab or tremelimumab, and (ii
  • the checkpoint inhibitor and the composition are administered simultaneously or sequentially in any order.
  • a neoantigenic peptide, polynucleotide, vector, composition, or cells is administered prior to the checkpoint inhibitor.
  • a peptide, polynucleotide, vector, composition, or cells is administered after the checkpoint inhibitor.
  • the checkpoint inhibitor is continued throughout neoantigen peptide, polynucleotide, vector, composition, or cell therapy.
  • the neoantigen peptide, polynucleotide, vector, composition, or cell therapy is administered to subjects that only partially respond or do not respond to checkpoint inhibitor therapy.
  • the composition is administered intravenously or subcutaneously.
  • the checkpoint inhibitor is administered intravenously or subcutaneously.
  • the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the composition.
  • the composition is administered into the same draining lymph node as the checkpoint inhibitor.
  • an additional agent is administered.
  • the agent is a chemotherapeutic agent, an immunomodulatory drug, an immune metabolism modifying drug, a targeted therapy, radiation an anti-angiogenesis agent, or an agent that reduces immune-suppression.
  • the chemotherapeutic agent is an alkylating agent, a topoisomerase inhibitor, an antimetabolite, or an anti-mitotic agent.
  • the additional agent is an anti-glucocorticoid induced tumor necrosis factor family receptor (GITR) agonistic antibody or antibody fragment, ibrutinib, docetaxeol, cisplatin, or cyclophosphamide.
  • the administration elicits a CD4 + T cell immune response.
  • the administration elicits a CD4 + T cell immune response and a CD8 + T cell immune response.
  • the invention is directed to a method for stimulating an immune response in a subject, comprising administering an effective amount of modified cells or composition described herein.
  • the immune response is cytotoxic and/or humoral immune response.
  • the method stimulates a T cell -mediated immune response in a subject.
  • the T cell -mediated immune response is directed against a target cell.
  • the target cell is a tumor cell.
  • the modified cells are transfected or transduced in vivo.
  • the modified cells are transfected or transduced ex vivo.
  • the modified cells are autologous patient T cells.
  • the autologous patient T cells are obtained from a patient that has received a neoantigen peptide or nucleic acid vaccine.
  • the neoantigen peptide or nucleic acid vaccine comprises at least one personalized neoantigen.
  • the neoantigen peptide or nucleic acid vaccine comprises at least one additional neoantigenic peptide described herein.
  • the patient received a chemotherapeutic agent, an immunomodulatory drug, an immune metabolism modifying drug, targeted therapy or radiation prior to and/or during receipt of the neoantigen peptide or nucleic acid vaccine.
  • the patient receives treatment with at least one checkpoint inhibitor.
  • the autologous T cells are obtained from a patient that has already received at least one round of T cell therapy containing a neoantigen.
  • the method further comprises adoptive T cell therapy.
  • the adoptive T cell therapy comprises autologous T cells.
  • the autologous T cells are targeted against tumor antigens.
  • the adoptive T cell therapy further comprises allogenic T cells.
  • the allogenic T cells are targeted against tumor antigens.
  • the adoptive T cell therapy is administered before the checkpoint inhibitor.
  • the invention is directed to a method for evaluating the efficacy of treatment comprising: (i) measuring the number or concentration of target cells in a first sample obtained from the subject before administering the modified cell, (ii) measuring the number concentration of target cells in a second sample obtained from the subject after administration of the modified cell, and (iii) determining an increase or decrease of the number or concentration of target cells in the second sample compared to the number or concentration of target cells in the first sample.
  • the treatment efficacy is determined by monitoring a clinical outcome; an increase, enhancement or prolongation of anti-tumor activity by T cells; an increase in the number of anti-tumor T cells or activated T cells as compared with the number prior to treatment; B cell activity; CD4 T cell activity; or a combination thereof.
  • the treatment efficacy is determined by monitoring a biomarker.
  • the biomarker is selected from the group consisting of CEA, Her-2/neu, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA 125, CA19.9, CA 15.3, leptin, prolactin, osteopontin, IGF-II, CD98, fascin, sPIgR, 14-3-3 eta, troponin I, and b-type natriuretic peptide.
  • the clinical outcome is selected from the group consisting of tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor expansion, recurrence or spread; or a combination thereof.
  • the treatment effect is predicted by presence of T cells or by presence of a gene signature indicating T cell inflammation or a combination thereof.
  • the invention is directed to a method of treating cancer or initiating, enhancing, or prolonging an anti-tumor response in a subject in need thereof comprising administering to the subject: (a) the peptide, polynucleotide, vector, composition, antibody, or cells described herein; and (b) at least one checkpoint inhibitor.
  • the method further comprises administration of an
  • the immunomodulator or adjuvant is selected from the group consisting of Poly(I:C), Poly-ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS15,
  • the immunomodulator or adjuvant is Poly-ICLC.
  • the checkpoint inhibitor is an anti-PDl antibody or antibody fragment.
  • the inhibitor of the PD-1 pathway is nivolumab.
  • the checkpoint inhibitor is an anti- CTLA4 antibody or antibody fragment.
  • the anti-CTLA4 antibody is ipilimumab or tremelimumab.
  • the method comprises administering both an anti-PDl antibody and an anti-CTLA4 antibody.
  • the administration of the checkpoint inhibitor is initiated before initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell.
  • the administration of the checkpoint inhibitor is initiated after initiation of
  • the administration of the checkpoint inhibitor is initiated simultaneously with the initiation of administration of the peptide, polynucleotide, vector, composition, antibody, or cell.
  • the peptide, polynucleotide, vector, composition, antibody, or cell is administered intravenously or subcutaneously.
  • the checkpoint inhibitor is administered intravenously or subcutaneously.
  • the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the peptide, polynucleotide, vector, composition, antibody, or cell.
  • the peptide, polynucleotide, vector, composition, antibody, or cell is administered into the same draining lymph node as the checkpoint inhibitor.
  • the additional therapeutic agent is for example, a chemotherapeutic or biotherapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular cancer may be administered.
  • chemotherapeutic and biotherapeutic agents include, but are not limited to, an angiogenesis inhibitor, such ashydroxy angiostatin K 1-3, DL-a-Difluoromethy!- oroithine, endostatiii, fumagillin, genistein, minocycline, staurosporine, and thalidomide; a DNA
  • intercaitor/cross-linker such as Bleomycin, Carboplatin, Carrmistme, Chlorambucil, Cyclophosphamide, cis- Diammineplat nurn(D) dichloride (Cispiatin), Melphalan, Mitoxantrone, and Oxaliplatin
  • a DNA synthesis inhibitor such as ( ⁇ )-Amethopterin (Methotrexate), 3-Amino-l,2,4-beiizotriazine 1,4-dioxide, Aminopterin, Cytosine ⁇ -D-arabinofuraiioside, 5-Fmoro-5' ⁇ deoxyuridine, 5-Fhsorouracil, Ganciclovir, Hydroxyurea, and Mitomycin C
  • a DNA-RNA transcription regulator such as Aetinomycin D, Dau orubicin, Doxorubicin, Homoharringtonine, and Idarubicin
  • an ⁇ /. ⁇ inhibitor, such as S(-i-)
  • the therapeutic agent may be altretamine, amifostine, asparaginase, capecitabine, cladribine, cisapride, cyiarahirse, dacarbazine (DT1C), dactinomycin, dronabinol, epoetin alpha, "filgrastim, fludarabine, gemcitabine, granisetron, ifosfamide, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, metoclopramide, mitotane, omeprazole, ondansetron, pilocarpine, prochloroperazine, or topotecan hydrochloride.
  • the therapeutic agent may be a monoclonal antibody such as rituximab (Rituxan®), alemtuzumab (Campath®), Bevacizumab (Avastin®), Cetuximab (Erbitux®), panitumumab (Vectibix®), and trastuzumab (Herceptin®), Vemurafenib (Zelboraf®) imatinib mesylate (Gleevec®), erlotinib (Tarceva®), gefitinib (Iressa®), Vismodegib (ErivedgeTM), 90Y- ibritumomab tiuxetan, 1311-tosit.umomab, ado-trastuzumab emtansine, lapatinib (Tykerb®), pertuzumab (PerjetaTM), ado-trastuzumab emtansine ( a
  • the therapeutic agent is a neoantigen.
  • the therapeutic agent may be a cytokine such as interferons (INFs), interlcukins (ILs), or hematopoietic growth factors.
  • the therapeutic agent may be INF-a, IL-2, Aldesleukin, IL-2,
  • the therapeutic agent may be a targeted therapy such as toremifene (Fareston®), fulvestrant (Faslodex®), anastrozole (Arimidex®), exemestane (Aromasin®), letrozole (Femara®), ziv- aflibercept (Zaltrap®), Aiitretinoin (Panretin®), temsirolimus (Torisel®), Tretinoin (Vesanoid®), denileukin diftitox (Ontak®), vorinostat (Zoiinza®), romidepsin (Istodax®), bexarotene (Targretin®), pralatrexate (Foiotyn®), !enaliomide (Revlimi
  • toremifene Fareston®
  • fulvestrant Feslodex®
  • anastrozole Arimidex®
  • exemestane
  • the therapeutic agent may be an epigenetic targeted drug such as HDAC inhibitors, kinase inhibitors, DNA methyltransferase inhibitors, histone demethylase inhibitors, or histone methylation inhibitors.
  • the epigenetic drugs may be Azacitidine (Vidaza), Decitabine (Dacogen), Vorinostat (Zoiinza), Romidepsin (Istodax), or Ruxolitinib (Jakafi).
  • TAXOL paclitaxel
  • the invention is directed to a kit comprising any neoantigen therapeutic described herein.
  • the present invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members.
  • the present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
  • Figure 1 depicts and exemplary graph of 562 peptides with predicted affinity for select HLA Class I molecule between 1 nM and 100,000 nM (plotted on x-axis) that were synthesized. Actual affinity (IC 50 (nM)) was measured as described (y-axis). Thick vertical and horizontal lines denote 500 nM cutoff between weak and very weak predicted and observed binders, respectively. Diagonal dotted line depicts line-of-best fit (Graphpad Prism) with R 2 of 0.45.
  • Figure 2 depicts and exemplary graph of 275 peptides from Figure 1 that were tested for stability (Tin, hrs) on their respective HLA Class I molecules. Peptides were binned according to observed affinity from Figure 1. Median and interquartile range is shown for each bin.
  • Figure 3A depicts and exemplary graph of HLA-A02:01 + T cells co-cultured with monocyte-derived dendritic cells loaded with TMPRSS2: :ERG fusion neoepitope (ALNSEALSV; HLA-A02:01) for 10 days.
  • CD8 + T cells were analyzed for antigen-specificity for TMPRSS2: :ERG fusion neoepitope using multimers (initial: BV421 and PE).
  • Figure 3B depicts and exemplary graph of HLA-A02:01 + T cells co-cultured with monocyte-derived dendritic cells loaded with TMPRSS2: :ERG fusion neoepitope (ALNSEALSV; HLA-A02:01) for 10 days.
  • CD8 + T cells were analyzed for antigen-specificity for TMPRSS2: :ERG fusion neoepitope using multimers (validation: APC and BUV396).
  • Figure 4A depicts and exemplary graph of HLA-A02:01 + T cells co-cultured with monocyte-derived dendritic cells loaded with GATA3 frameshift neoepitope (SMLTGPPARV; HLA-A02:01) for 10 days.
  • CD8 + T cells were analyzed for antigen-specificity for GATA3 frameshift neoepitope using multimers (initial: APC and BUV396).
  • Figure 4B depicts and exemplary graph of HLA-A02:01 + T cells co-cultured with monocyte-derived dendritic cells loaded with GATA3 frameshift neoepitope (SMLTGPPARV; HLA-A02:01) for 10 days.
  • CD8 + T cells were analyzed for antigen-specificity for GATA3 frameshift neoepitope using multimers (validation: PE and BV421).
  • Figure 5A depicts and exemplary graph of HLA-A02:01 + T cells co-cultured with monocyte-derived dendritic cells loaded with ⁇ 2 ⁇ frameshift neoepitope (LLCVWVSSI; HLA-A02:01) for 10 days.
  • CD8 + T cells were analyzed for antigen-specificity for ⁇ 2 ⁇ frameshift neoepitope using multimers (initial: PE and APC).
  • Figure 5B depicts and exemplary graph of HLA-A02:01 + T cells co-cultured with monocyte-derived dendritic cells loaded with ⁇ 2 ⁇ frameshift neoepitope (LLCVWVSSI; HLA-A02:01) for 10 days.
  • CD8 + T cells were analyzed for antigen-specificity for ⁇ 2 ⁇ frameshift neoepitope using multimers (validation: PE and BV421).
  • Figure 6A depicts and exemplary graph of HLA-A02:01 + T cells co-cultured with monocyte-derived dendritic cells loaded with KRAS G12C neoepitope (KLVVVGACGV; HLA-A02:01) for 10 days.
  • CD8 + T cells were analyzed for antigen-specificity for KRAS G12C frameshift neoepitope using multimers (initial: BUV396 and BV421).
  • Figure 6B depicts and exemplary graph HLA-A02:01 + T cells co-cultured with monocyte-derived dendritic cells loaded with KRAS G12C neoepitope (KLVVVGACGV; HLA-A02:01) for 10 days.
  • CD8 + T cells were analyzed for antigen-specificity for KRAS G12C frameshift neoepitope using multimers
  • Figure 7 depicts and exemplary graph of T cells co-cultured with monocyte-derived dendritic cells loaded ⁇ 2 ⁇ frameshift neopeptides for 20 days (restimulation with fresh monocyte-derived dendritic cells on day 20).
  • CD4 + T cells were analyzed for antigen-specificity by intracellular cytokine staining after restimulation with monocyte-derived dendritic cells loaded with ⁇ 2 ⁇ frameshift peptide for 24 hours (right), compared to controls without peptide (left).
  • Figure 8 depicts and exemplary graph of T cells co-cultured with monocyte-derived dendritic cells loaded BTK C481 S neopeptide for 20 days (restimulation with fresh monocyte-derived dendritic cells on day 20).
  • CD4 + T cells were analyzed for antigen-specificity by intracellular cytokine staining after restimulation with monocyte-derived dendritic cells loaded with BTK C481 S neopeptide for 24 hours (right), compared to controls wild-type BTK peptide (left).
  • Figure 9 depicts and exemplary graph of T cells co-cultured with monocyte-derived dendritic cells loaded GATA3 frameshift neopeptides for 20 days (restimulation with fresh monocyte-derived dendritic cells on day 20).
  • CD4 + T cells were analyzed for antigen-specificity by intracellular cytokine staining after restimulation with monocyte-derived dendritic cells loaded with GATA3 frameshift peptide for 24 hours (right), compared to controls without peptide (left).
  • Described herein are novel immunotherapeutic agents and uses thereof based on the discovery of neoantigens arising from mutational events unique to an individual's tumor. Accordingly, the invention described herein provides peptides, polynucleotides encoding the peptides, and peptide binding agents, that can be used, for example, to stimulate an immune response to a tumor associated antigen, to create an immunogenic composition or cancer vaccine for use in treating disease.
  • the term “about” or “approximately” can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about” meaning within an acceptable error range for the particular value should be assumed.
  • Neoantigen means a class of tumor antigens which arise from tumor-specific changes in proteins. Neoantigens encompass, but are not limited to, tumor antigens which arise from, for example, substitution in the protein sequence, frame shift mutation, fusion polypeptide, in-frame deletion, insertion, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of polypepitdes.
  • Tumor specific neoepitope refers to an epitope that is not present in a reference such as a normal non-cancerous or germline cell but is found in cancer cells. This includes, in particular, situations wherein in a normal non-cancerous or germline cell a corresponding epitope is found, however, due to one or more mutations in a cancer cell the sequence of the epitope is changed so as to result in the neo-epitope.
  • a “reference” can be used to correlate and compare the results obtained in the methods of the invention from a tumor specimen.
  • the "reference” may be obtained on the basis of one or more normal specimens, in particular specimens which are not affected by a cancer disease, either obtained from a patient or one or more different individuals, for example, healthy individuals, in particular individuals of the same species.
  • a “reference” can be determined empirically by testing a sufficiently large number of normal specimens.
  • mutation refers to a change of or difference in the nucleic acid sequence (nucleotide substitution, addition or deletion) compared to a reference.
  • a “somatic mutation” can occur in any of the cells of the body except the germ cells (sperm and egg) and therefore 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.
  • non-synonymous mutation refers to a mutation, for example, 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 the translation of a non-native protein sequence. It is possible for different mutations in a gene to achieve the same altered reading frame.
  • affinity refers to a measure of the strength of binding between two members of a binding pair, for example, an HLA-binding peptide and a class I or II HLA.
  • K D is the dissociation constant and has units of molarity.
  • the affinity constant is the inverse of the dissociation constant.
  • An affinity constant is sometimes used as a generic term to describe this chemical entity. It is a direct measure of the energy of binding. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units. Affinity may also be expressed as the inhibitory concentration 50 (IC 50 ), that concentration at which 50% of the peptide is displaced. Likewise, ln(IC 5 o) refers to the natural log of the IC 50 .
  • K off refers to the off-rate constant, for example, for dissociation of an HLA-binding peptide and a class I or II HLA.
  • binding data results can be expressed in terms of "IC 50 .”
  • IC 50 is the concentration of the 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 (i.e., limiting HLA protein and labeled reference peptide concentrations), these values 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.
  • binding can be expressed relative to binding by a reference standard peptide.
  • a reference standard peptide For example, can be based on its IC 50 , relative to the IC 50 of a reference standard peptide.
  • Binding can also be determined using other assay systems including those using: live cells (e.g.,
  • ELISA systems e.g., Reay et al., EMBO J. 1 1 :2829 ( 1992)
  • surface plasmon resonance e.g., Khilko et al., J.
  • Cross-reactive binding indicates that a peptide is bound by more than one HLA molecule; a synonym is degenerate binding.
  • a derived epitope when used to discuss an epitope is a synonym for "prepared."
  • a derived epitope can be isolated from a natural source, or it 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 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.
  • a "diluent” includes sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is also a diluent for pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as diluents, for example, in injectable solutions.
  • an “epitope” is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by, for example, an immunoglobulin, T cell receptor, HLA molecule, or chimeric antigen receptor.
  • an epitope can be defined as a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins, chimeric antigen receptors, and/or Major Histocompatibility Complex (MHC) receptors.
  • 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 such as cyclohexylalanine. Throughout this disclosure, epitopes may be referred to in some cases as peptides or peptide epitopes.
  • proteins or peptides that comprise an epitope or an analog described herein as well as additional amino acid(s) are still within the bounds of the invention.
  • the peptide comprises a fragment of an antigen.
  • a peptide of the invention there is a limitation on the length of a peptide of the invention.
  • the embodiment that is length-limited occurs when the protein or peptide comprising an epitope described herein comprises a region (i.e., a contiguous series of amino acid residues) having 100% identity with a native sequence.
  • a region i.e., a contiguous series of amino acid residues
  • the region with 100% 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% 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, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.
  • HLA Human Leukocyte Antigen
  • HLA Human Leukocyte Antigen
  • MHC Major Histocompatibility Complex
  • HLA supertype or HLA family describes sets of HLA molecules grouped on the basis of shared peptide -binding specificities. HLA class I molecules that share somewhat similar binding affinity for peptides bearing certain amino acid motifs are grouped into such HLA supertypes.
  • HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules are synonyms.
  • nucleic in the context of two or more peptide sequences or antigen fragments, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • an "immunogenic" peptide or an "immunogenic” epitope or “peptide epitope” is a peptide that comprises an allele-specific motif such that the peptide will bind an HLA molecule and induce a cell-mediated or humoral response, for example, cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and/or B lymphocyte response.
  • CTL cytotoxic T lymphocyte
  • HTL helper T lymphocyte
  • B lymphocyte response for example, cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and/or B lymphocyte response.
  • 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 domain) and a T cell receptor (TCR) constant domain.
  • an immunoglobulin antigen binding domain e.g., an immunoglobulin variable domain
  • TCR T cell receptor
  • a "constant domain" of a TCR polypeptide includes a membrane-proximal TCR constant domain, and may also include a TCR transmembrane domain and/or a TCR cytoplasmic tail.
  • the CAR is a dimer that includes a first polypeptide comprising a immunoglobulin heavy chain variable domain linked to a TCR.beta.
  • the CAR is a dimer that includes a first polypeptide comprising a immunoglobulin heavy chain variable domain linked to a TCRa constant domain and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a ⁇ or ⁇ variable domain) linked to a TCR constant domain.
  • the CAR is a dimer that includes a first polypeptide comprising a immunoglobulin heavy chain variable domain linked to a TCRa constant domain and a second polypeptide comprising an immunoglobulin light chain variable domain (e.g., a ⁇ or ⁇ variable domain) linked to a TCR constant domain.
  • isolated or biologically pure refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides described herein do not contain some or all of the materials normally associated with the peptides in their in situ environment.
  • An “isolated” epitope refers to an epitope that does not include the whole sequence of the antigen from which the epitope was derived. Typically the “isolated” epitope does not have attached thereto additional amino acid residues that result in a sequence that has 100% identity over the entire length of a native sequence.
  • the native sequence can be a sequence such as a tumor-associated antigen from which the epitope is derived.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • 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.
  • MHC Major Histocompatibility Complex
  • HLA human leukocyte antigen
  • a "native” or a “wild type” sequence refers to a sequence found in nature. Such a sequence can comprise a longer sequence in nature.
  • T cell epitope is to be understood as meaning a peptide sequence which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, be recognized and bound by cytotoxic T-lymphocytes or T-helper cells, respectively.
  • a "receptor” is to be understood as meaning 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.
  • the receptor comprises at least one receptor unit, for example, where each receptor unit may consist of a protein molecule.
  • the 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” is to be understood as meaning 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 proteins of MHC class I or MHC class II.
  • 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 of class I or of class II.
  • MHC major histocompatibility complex
  • MHC molecules proteins
  • HLA proteins proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential lymphocyte epitopes, (e.g., T cell epitope and B cell epitope) transporting them to the cell surface and presenting them there to specific cells, in particular cytotoxic T-lymphocytes, T-helper cells, or B cells.
  • 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.
  • the major histocompatibility complex is classified into two gene groups coding for different proteins, namely molecules of MHC class I and molecules of MHC class II. The cellular biology and the expression patterns of the two MHC classes are adapted to these different roles.
  • 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 a-amino and carboxyl groups of adjacent amino acid residues.
  • 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.”
  • a "PanDR binding” peptide, a “PanDR binding epitope” is a member of a family of molecules that binds more than one HLA class II DR molecule.
  • “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 "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, 1 1, 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, 1 1, 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 9, 10, or 1 1 amino acid residues in length.
  • a "supermotif ' is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.
  • a supermotif-bearing peptide described herein is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.
  • Naturally occurring refers to the fact that an object can be found in nature.
  • a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • the term "vaccine” relates to a pharmaceutical preparation
  • a vaccine may be used for the prevention or treatment of a disease.
  • individualized cancer vaccine or “personalized cancer vaccine” concerns a particular cancer patient and means that a cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.
  • a "protective immune response” or “therapeutic immune response” refers to a CTL and/or an HTL response to an antigen derived from an 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.
  • 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.
  • Antigen presenting cells are cells which present peptide fragments of protein antigens in association with MHC molecules on their cell surface. Some APCs may activate antigen specific T cells. Professional antigen-presenting cells are very efficient at internalizing antigen, either by phagocytosis or by receptor-mediated endocytosis, and then displaying a fragment of the antigen, bound to a class II MHC molecule, on their membrane. The T cell recognizes and interacts with the antigen-class II MHC molecule complex on the membrane of the antigen presenting cell. An additional co-stimulatory signal is then produced by the antigen presenting cell, leading to activation of the T cell. The expression of co-stimulatory molecules is a defining feature of professional antigen-presenting cells.
  • dendritic cells which have the broadest range of antigen presentation, and are probably the most important antigen presenting cells, macrophages, B- cells, and certain activated epithelial cells.
  • DCs Dendritic cells
  • Dendritic cells are conveniently categorized as “immature” and “mature” cells, which can be used as a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation.
  • Immature dendritic cells are characterized as antigen presenting cells with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e. g. CD54 and CD1 1) and costimulatory molecules (e. g., CD40, CD80, CD86 and 4-1 BB).
  • 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 nucleic acid (DNA or RNA) that encodes the amino acid or amino acid mimetic.
  • 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.
  • three letter symbols or full names are used without capitals, they can refer to L amino acid residues.
  • 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 Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M, Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S, Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.
  • 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 nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs
  • polynucleotide and nucleic acid can be in vitro transcribed mRNA.
  • the polynucleotide that is administered using the methods of the invention is mRNA.
  • 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,
  • 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 2between.
  • 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 the coding region of a nucleotide sequence.
  • 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).
  • vector means a construct, which is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
  • a polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • an "isolated polynucleotide” encompasses a PCR or quantitative PCR reaction comprising the polynucleotide amplified in the PCR or quantitative PCR reaction.
  • 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.
  • 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.
  • subject and patient are used interchangeably herein in reference to a human subject.
  • the terms "effective amount” or “therapeutically effective amount” or “therapeutic effect” refer to an amount of a therapeutic effective to "treat” a disease or disorder in a subject or mammal.
  • the therapeutically effective amount of a drug has a therapeutic effect and as such can prevent the development of a disease or disorder; slow down the development of a disease or disorder; slow down the progression of a disease or disorder; relieve to some extent one or more of the symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
  • treating or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • Tumor neoantigens which arise as a result of genetic change (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) within malignant cells, represent the most tumor-specific class of antigens.
  • Neoantigens have rarely been used in cancer vaccine or immunogenic compositions due to technical difficulties in identifying them, selecting optimized antigens, and producing neoantigens for use in a vaccine or immunogenic composition.
  • neoplasias/tumors which are present at the DNA level in tumor but not in matched germline samples from a high proportion of subjects having cancer; analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms to generate a plurality of neoantigen T cell epitopes that are expressed within the neoplasia/tumor and that bind to a high proportion of patient HLA alleles; and synthesizing the plurality of neoantigenic peptides selected from the sets of all neoantigen peptides and predicted binding peptides for use in a cancer vaccine or immunogenic composition suitable for treating a high proportion of subjects having cancer.
  • translating peptide sequencing information into a therapeutic vaccine may include prediction of mutated peptides that can bind to HLA molecules of a high proportion of individuals. Efficiently choosing which particular mutations to utilize as immunogen requires the ability to predict which mutated peptides would efficiently bind to a high proportion of patient's HLA alleles.
  • neural network based learning approaches with validated binding and non-binding peptides have advanced the accuracy of prediction algorithms for the major HLA-A and -B alleles.
  • advanced neural network- based algorithms to encode HLA-peptide binding rules several factors limit the power to predict peptides presented on HLA alleles.
  • translating peptide sequencing information into a therapeutic vaccine may include formulating the drug as a multi-epitope vaccine of long peptides.
  • Targeting as many mutated epitopes as practically possible takes advantage of the enormous capacity of the immune system, prevents the opportunity for immunological escape by down-modulation of an immune targeted gene product, and compensates for the known inaccuracy of epitope prediction approaches.
  • Synthetic peptides provide a useful means to prepare multiple immunogens efficiently and to rapidly translate identification of mutant epitopes to an effective vaccine.
  • Peptides can be readily synthesized chemically and easily purified utilizing reagents free of contaminating bacteria or animal substances. The small size allows a clear focus on the mutated region of the protein and also reduces irrelevant antigenic competition from other components (unmutated protein or viral vector antigens).
  • translating peptide sequencing information into a therapeutic vaccine may include a combination with a strong vaccine adjuvant.
  • Effective vaccines may require a strong adjuvant to initiate an immune response.
  • poly-ICLC an agonist of TLR3 and the RNA helicase-domains of MDA5 and RIG3, has shown several desirable properties for a vaccine adjuvant. These properties include the induction of local and systemic activation of immune cells in vivo, production of stimulatory chemokines and cytokines, and stimulation of antigen-presentation by DCs.
  • poly-ICLC can induce durable CD4+ and CD8+ responses in humans.
  • EEF1B2 EEF1DP3, EGFR, EIF2B3, env, EPHB2, ERBB3, ESR1, ESRP1, FAM11 IB, FGFR3, FRG1B,
  • EEF1DP3:FRY fusion polypeptide an EEF1DP3:FRY fusion polypeptide, an EGFR:SEPT14 fusion polypeptide, an EGFRVIII deletion polypeptide, an EML4:ALK fusion polypeptide, an NDRG1 :ERG fusion polypeptide, an ACOl 1997.1 :LRRC69 fusion polypeptide, a
  • RUNXl(ex5)-RUNXlTlfusion polypeptide a TMPRSS2:ERG fusion polypeptide, a NAB: STAT6 fusion polypeptide, a NDRG1 :ERG fusion polypeptide, a PML:RARA fusion polypeptide, a PPP1R1B:STARD3 fusion polypeptide, a MAD1L1 :MAFK fusion polypeptide, a FGFR3:TAC fusion polypeptide, a
  • FGFR3:TACC3 fusion polypeptide a BCR:ABL fusion polypeptide, a CI lorf95:RELA fusion polypeptide, a CBFB:MYH11 fusion polypeptide, a CBFB:MYH11 fusion polypeptide, a CD74:R0S1 fusion polypeptide, a CD74:R0S1 fusion polypeptide, ERVE-4: protease, ERVE-4: reverse transcriptase, ERVE-4: reverse transcriptase, ERVE-4: unknown, ERVH-2 matrix protein, ERVH-2: gag, ERVH-2: retroviral matrix, ERVH48-1 : coat protein, ERVH48-1 : syncytin, ERVI-1 envelope protein, ERVK-5 gag, ERVK-5 env, ERVK-5 pol, EBV A73, EBV BALF3, EBV BALF4, EBV BALF5, EBV BARFO,
  • a neoantigen described herein is due to a mutational event in ⁇ 2 ⁇ , BTK, EGFR, GATA3, KRAS, MLL2, a TMPRSS2:ERG fusion polypeptide, or TP53.
  • the invention provides isolated peptides that comprise a tumor specific mutation from Table 1 or 2. These peptides and polypeptides are referred to herein as "neoantigenic peptides" or
  • peptide is used interchangeably with “mutant peptide” and
  • nucleic acid in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids.
  • polypeptide is used interchangeably with “mutant polypeptide” and “neoantigenic polypeptide” in the present specification to designate a series of residues, e.g., L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids.
  • polypeptides or peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described.
  • sequencing methods are used to identify tumor specific mutations. Any suitable sequencing method can be used according to the invention, for example, Next Generation Sequencing
  • NGS Next Generation Sequencing
  • NGS in the context of the present invention mean all novel high throughput sequencing technologies which, in contrast to the "conventional” sequencing methodology known as Sanger chemistry, read nucleic acid templates randomly in parallel along the entire genome by breaking the entire genome into small pieces.
  • Such NGS technologies also known as massively parallel sequencing technologies
  • Multiple NGS platforms which are commercially available or which are mentioned in the literature can be used in the context of the invention e.g. those described in detail in WO 2012/159643.
  • a neoantigenic peptide described herein molecule can comprise, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 1 10, about 120 or greater amino acid residues, and any range derivable therein.
  • a neoantigenic peptide molecule is equal to or less than 100 amino acids.
  • neoantigenic peptides and polypeptides described herein for MHC Class I are 13 residues or less in length and usually consist of between about 8 and about 1 1 residues, particularly 9 or 10 residues. In some embodiments, neoantigenic peptides and polypeptides described herein for MHC Class II are 9-24 residues in length.
  • a longer neoantigenic peptide can be designed in several ways.
  • a longer neoantigenic peptide could consist of (1) individual binding peptides with extensions of 2-5 amino acids toward the N- and C-terminus of each corresponding gene product; or (2) a concatenation of some or all of the binding peptides with extended sequences for each.
  • sequencing reveals a long (>10 residues) neoepitope sequence present in the tumor (e.g.
  • a longer neoantigenic peptide could consist of the entire stretch of novel tumor-specific amino acids as either a single longer peptide or several overlapping longer peptides.
  • 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.
  • two or more peptides can be used, where the peptides overlap and are tiled over the long neoantigenic peptide.
  • the neoantigenic peptides and polypeptides bind an HLA protein (e.g., HLA class I or HLA class II). In specific embodiments the neoantigenic peptides and polypeptides bind an HLA protein with greater affinity than the corresponding wild-type peptide. In specific embodiments the neoantigenic peptide or polypeptide has an IC 50 of at least less than 5000 nM, at least less than 500 nM, at least less than 100 nM, at least less than 50 nM or less.
  • the neoantigenic peptides can be from about 8 and about 50 amino acid residues in length, or from about 8 and about 30, from about 8 and about 20, from about 8 and about 18, from about 8 and about 15, or from about 8 and about 12 amino acid residues in length.
  • the neoantigenic peptides can be from about 8 and about 500 amino acid residues in length, or from about 8 and about 450, from about 8 and about 400, from about 8 and about 350, from about 8 and about 300, from about 8 and about 250, from about 8 and about 200, from about 8 and about 150, from about 8 and about 100, from about 8 and about 50, or from about 8 and about 30 amino acid residues in length.
  • the neoantigenic peptides can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • the neoantigenic peptides can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more amino acid residues in length.
  • the neoantigenic peptides can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acid residues in length.
  • the neoantigenic peptides can be at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or less amino acid residues in length.
  • the neoantigenic peptides can be at most 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, or less amino acid residues in length.
  • the neoantigenic peptides has a total length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least
  • At least 40 at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.
  • the neoantigenic peptides has a total length of at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.
  • the neoantigenic peptides can have a pi value of about 0.5 and about 12, about 2 and about 10, or about 4 and about 8. In some embodiments, the neoantigenic peptides can have a pi value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the neoantigenic peptides can have a pi value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.
  • the neoantigenic peptides can have an HLA binding affinity of between about lpM and about ImM, about ⁇ and about 500 ⁇ , about 500pM and about 10 ⁇ , about InM and about ⁇ , or about ⁇ and about ⁇ some embodiments, the neoantigenic peptides can have an HLA binding affinity of at least 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, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 ⁇ , or more.
  • the neoantigenic peptides can have an HLA binding affinity of at most 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, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900 ⁇ .
  • a neoantigenic peptide described herein can comprise carriers such as those well known in the art, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acid residues such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.
  • carriers such as those well known in the art, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acid residues such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.
  • a neoantigenic peptide described herein can be modified by terminal-NH 2 acylation, e.g., by alkanoyl (C 1 -C 20 ) or thioglycolyl acetylation, terminal-carboxyl amidation, e.g., ammonia, methylamine, etc. In some embodiments these modifications can provide sites for linking to a support or other molecule.
  • a neoantigenic peptide described herein can contain modifications such as but not limited to glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of a surface active material, e.g. a lipid, or can be chemically modified, e.g., acetylation, etc.
  • bonds in the peptide can be other than peptide bonds, e.g., covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.
  • a neoantigenic peptide described herein can contain substitutions to modify a physical property (e.g., stability or solubility) of the resulting peptide.
  • neoantigenic peptides can be modified by the substitution of a cysteine (C) with a-amino butyric acid ("B"). Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substituting a-amino butyric acid for C not only alleviates this problem, but actually improves binding and crossbinding capability in certain instances.
  • Substitution of cysteine with a-amino butyric acid can occur at any residue of aneoantigenic peptide, e.g., at either anchor or non-anchor positions of an epitope or analog within a peptide, or at other positions of a peptide.
  • a neoantigenic peptide described herein can comprise amino acid mimetics or unnatural amino acid residues, e.g. D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2-thieneylalanine; D- or L-l, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3- pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)- phenylglycine; D-(trifluoro-methyl)-phenylalanine; D-.rho.-fluorophenylalanine; D- or L-.rho.-
  • Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
  • Modified peptides that have various amino acid mimetics or unnatural amino acid residues are particularly useful, as they tend to manifest increased stability in vivo. Such peptides can also possess improved shelf-life or manufacturing properties.
  • Peptide stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef, et al., Eur. J.
  • RPMI-1640 or another suitable tissue culture medium.
  • a small amount of reaction solution is removed and added to either 6% aqueous trichloroacetic acid (TCA) or ethanol.
  • TCA 6% aqueous trichloroacetic acid
  • the cloudy reaction sample is cooled (4°C) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific
  • a neoantigenic peptide described herein can be in solution, lyophylized, or can be in crystal form.
  • a neoantigenic peptide described herein can be prepared synthetically, by recombinant DNA technology or chemical synthesis, or can be isolated from natural sources such as native tumors or pathogenic organisms. Epitopes can be synthesized individually or joined directly or indirectly in a peptide. Although a neoantigenic peptide described herein will be substantially free of other naturally occurring host cell proteins and fragments thereof, in some embodiments the peptide can be synthetically conjugated to be joined to native fragments or particles.
  • a neoantigenic peptide described herein can be prepared in a wide variety of ways.
  • the peptides can be synthesized in solution or on a solid support according to conventional techniques.
  • Various automatic synthesizers are commercially available and can be used according to known protocols. (See, for example, Stewart & Young, SOLID PHASE PEPTIDE SYNTHESIS, 2D. ED., Pierce Chemical Co., 1984).
  • individual peptides can be joined using chemical ligation to produce larger peptides that are still within the bounds of the invention.
  • recombinant DNA technology can be employed wherein a nucleotide sequence which encodes a peptide inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • a nucleotide sequence which encodes a peptide inserted into an expression vector transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • These procedures are generally known in the art, as described generally in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
  • recombinant peptides which comprise or consist of one or more epitopes described herein, can be used to present the appropriate T cell epitope.
  • the invention described herein also provides compositions comprising one, at least two, or more than two neoantigenic peptides.
  • a composition described herein contains at least two distinct peptides.
  • the at least two distinct peptides are derived from the same polypeptide.
  • distinct polypeptides is meant that the peptide vary by length, amino acid sequence or both.
  • the peptides are derived from any polypeptide known to or have been found to contain a tumor specific mutation.
  • the isolated neoantigenic peptide is encoded by a gene with a point mutation resulting in an amino acid substitution of the native peptide.
  • the isolated neoantigenic peptide is encoded by a ABL gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a ALK gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a BRAF gene.
  • a x B y C z is
  • the neoantigenic peptide is not DFGLATEKSR or FGLATEKSRW.
  • the isolated neoantigenic peptide is encoded by a BTK gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a EEF1B2 gene.
  • a x B y C z is
  • the neoantigenic peptide is not EAVSGPPPA, FEAVSGPPP or FEAVSGPPPA.
  • the isolated neoantigenic peptide is encoded by a EGFR gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a ERBB3 gene.
  • a x B y C z is EFSTLPLPNLRMVRGTQVYDGKF.
  • the neoantigenic peptide is not RMVRGTQVY, LPLPNLRMV, LRMVRGTQV, TLPLPNLRMV,
  • the isolated neoantigenic peptide is encoded by a ESR1 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a FGFR3 gene.
  • a x B y C z is
  • the neoantigenic peptide is not DVLERCPHR, LERCPHRPI, CPHRPILQA, or LERCPHRPIL.
  • the isolated neoantigenic peptide is encoded by a FRG1B gene.
  • a x B y C z is
  • the neoantigenic peptide is not SASNSCFIR, LSASNSCFI, ALSASNSCF or FQNGKMALSA.
  • the isolated neoantigenic peptide is encoded by a HER2 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a IDH1 gene.
  • a x B y C z is
  • RVEEFKLKQMWKSPNGTIR ILGG FREAIICKNIPRLVSGWVKPIIIGHHAYGDQYRATDFVVPGP GKVEITTTPSDGTQKVTYLVHNFEEGGGVAMGM
  • the neoantigenic peptide is not PIIIGHHAY, GHHAYGDQY, KPIIIGHHAY, IGHHAYGDQY, PIIIGCHAY, GCHAYGDQY, KPIIIGCHAY, IGCHAYGDQY, PIIIGGHAY, GGHAYGDQY, KPIIIGGHAY, or IGGHAYGDQY.
  • the isolated neoantigenic peptide is encoded by a KIT gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is MEK gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a MYC gene.
  • a x B y C z is
  • MPLNVSFTOR YDLDYDSVQPYFYCDEEENFYQQQQSDLQPPAPSEDIWKKFELLPTPPLSPSR SG LCSPSYVAVTPFSLRGDNDGG,
  • the isolated neoantigenic peptide is encoded by a PDGFRa gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a PIK3CA gene.
  • a x B y C z is
  • the neoantigenic peptide is not ISTRDPLSK, STRDPLSKI, LSKITEQEK, AISTRDPLSK, SKITEQEKDF, SEITKQEKDF, KQEKDFLWSH, FMKQMNDAR, KQMNDARHG, RHGGWTTKM, YFMKQMNDAR, FMKQMNDARH, KQMNDARHGG,
  • the isolated neoantigenic peptide is encoded by a POLE gene.
  • a x B y C z is
  • the neoantigenic peptide is not TTKLPLKFR, RDAETDQIM, KFRDAETDQI, ETTKLPLKFR, or RDAETDQIMM.
  • the isolated neoantigenic peptide is encoded by a PTEN gene.
  • a x B y C z is
  • the neoantigenic peptide is not QTGVMICAY, GKGQTGVMI, GQTGVMICAY, or KAGKGQTGVM.
  • the isolated neoantigenic peptide is encoded by a RACl gene.
  • a x B y C z is
  • the neoantigenic peptide is not TTNAFSGEY, FSGEYIPTV, SGEYIPTVF, YTTNAFSGEY, TTNAFSGEYI, or FSGEYIPTVF.
  • the isolated neoantigenic peptide is encoded by a TP53 gene.
  • a x B y C z is
  • the neoantigenic peptide is not SSCMGSMNR, GSMNRRPIL, MGSMNRRPI, CNSSCMGSM, SMNRRPILTI, SSCMGSMNRR, NSSCMGSMNR, MGSMNRRPIL, MCNSSCMGSM, CMGSMNRRPI, TEVVRHCPH, VVRHCPHHER, SQHMTEVVRH, MNQRPILTI, NQRPILTII, CMGGMNQRPI, GMNQRPILTI, SSCMGGMNQR, NQRPILTIIT, NWRPILTII, SSCMGGMNW, MGGMNWRPI, MNWRPILTI, CMGGMNWRPI,
  • GMNWRPILTI SSCMGGMNWR, MNWRPILTII, NSSCMGGMNW, NSFEVCVCA, EVCVCACPGR, or FEVCVCACPG.
  • the isolated neoantigenic peptide is encoded by a gene with a frameshift mutation
  • the isolated neoantigenic peptide is encoded by a ACVR2A gene.
  • a x B y C z is GVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDI CYDRTDCVEKKRQP or
  • the isolated neoantigenic peptide is C15ORF40 gene. In related embodiments,
  • the isolated neoantigenic peptide is encoded by a CNOT1 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a EIF2B3 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a EPHB2 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a ESRPl gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a FAM1 IB gene.
  • a x B y C z is
  • the neoantigenic peptide is not KLNRMKVPL, PLMKLITRV, RMKVPLMKL, ISKKKHYNR, SKKKHYNRK, KHYNRKISI, HYNRKISIK, YNRKISIKK, KISIKKLNR, SIKKLNRMK, LNRMKVPLM, ISIKKLNRM, MKVPLMKLI, KLNRMKVPLM, RMKVPLMKLI, ISIKKLNRMK, I SKKKHYNRK, KHYNRKISIK, HYNRKISIKK, KISIKKLNRM, SIKKLNRMKV, LNRMKVPLMK, KVPLMKLITR, DISKKKHYNR, KKHYNRKISI
  • the isolated neoantigenic peptide is encoded by a GBP3 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a JAK1 gene.
  • a x B y C z is VNTLKEGKRLPCPPNCPDEVYQLMRKCWEFQPSNRTSFQNLIEGFEALLKTSN or
  • the isolated neoantigenic peptide is encoded by a LMAN1 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a MSH3 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a NDUFC2 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a RBM27 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a RPL22 gene.
  • a x B y C z is MAPVKKLVVKGGKKKEASSEVHS or MAPVKKLVVKGGKKRSKF.
  • the neoantigenic peptide is not VVKGGKKRSK or VKGGKKRSK
  • the isolated neoantigenic peptide is encoded by a SEC31A gene.
  • a x B y C z is MPSHQGAEQQQQQHHVFISQVVTEKEFLSRSDQLQQAVQSQGFINYCQKKN or
  • the isolated neoantigenic peptide is encoded by a SEC63 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a SLC35F5 gene.
  • a x B y C z is NIMEIRQLPSSHALEAKLSRMSYPVKEQESILKTVGKLTATQVAKISFFFALCGFWQICHIKKHFQTHK LL.
  • the isolated neoantigenic peptide is encoded by a SMAP1 gene.
  • a x B y C z is
  • the neoantigenic peptide is not ALKKLRSPL, KISNWSLKK, SLKKVPALK, KLRSPLWIF, KKRKRKREK, RKREKRSQK,
  • HLQLKSCRR WSLKKVPAL, RQNHLQLKS, KKVPALKKL, LKKLRSPLW, KKLRSPLWI,
  • KISNWSLKKV KSRQNHLQLK, SLKKVPALKK, WSLKKVPALK, KRKREKRSQK, RSQKSRQNHL, HLQLKSCRRK, RRKISNWSLK, CRRKISNWSL, NWSLKKVPAL, QKSRQNHLQL, RQNHLQLKSC, LQLKSCRRKI, ALKKLRSPLW, or KKLRSPLWI
  • the isolated neoantigenic peptide is encoded by a TFAM gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a TGFBR2 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a THAP5 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a TTK gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a XPOT gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a APC gene.
  • C z is
  • the neoantigenic peptide is not ADVLLSVHL, ADVLLSVHLI, APFRVNHAV, ARHKAVEFL, CLADVLLSV, CLADVLLSVH, DVLLSVHLI, DVLLSVHLIV, FLQERNLPPK, FRVNHAVEW, HLIVLRVVR, HLIVLRVVRL,
  • HLNTMFRRPH HPKVHLNTM, HPKVHLNTMF, KAVEFLQER, KVHLNTMFR, KVHLNTMFRR, KVVLRHPKV, LLSVHLIVL, LLSVHLIVLR, LPAPFRVNH, LPAPFRVNHA, LQERNLPPKV,
  • LRVVRLPAPF LSVHLIVLR, LSVHLIVLRV, MFRRPHSCL, MFRRPHSCLA, NLPPKVVLR,
  • NTMFRRPHSC QERNLPPKV, RHPKVHLNTM, RNLPPKVVL, RNLPPKVVLR, RPHSCLADV, RPHSCLADVL, RVVRLPAPF, RVVRLPAPFR, S ARHKAVEFL, SVHLIVLRV, SVHLIVLRVV, TMFRRPHSC, TMFRRPHSCL, VEFLQERNL, VLLSVHLIV, VLLSVHLIVL, VLRHPKVHL,
  • VLRVVRLPA VVRLPAPFR
  • VVRLPAPFRV VLRVVRLPA
  • the isolated neoantigenic peptide is encoded by a ARID 1 A gene.
  • C z is
  • the neoantigenic peptide is not
  • AMPILPLPQL APLLAAPSPA, APRTNFHSS, APRTNFHSSL, ASNKLPSLPL, AWRSCIALW,
  • ETVSLHPLA FHSSLAETV
  • GMYSPSRYPR GQWMAHMAL
  • GQWMAHMALL GQWMAHMALL
  • GRCTACHTAL GQWMAHMALL
  • GTAWQLVPL HMALLPSGT
  • HMALLPSGTK HPLAPMPSK
  • HSSLAETVSL HTALGRGSL
  • HTALGRGSL HTALGRGSL
  • IALWCASSV ILATPPSAA, ILATPPSAAW, IPMAISSPP, IPMAISSPPK, ISSPPKAPL, ISSPPKAPLL,
  • KMYTTSMAMP LAAPSPASR, LAAPSPASRL, LAETVSLHPL, LATPPSAAW, LATPPSAAWR, LLAAPSPASR, LLLSADQQAA, LLSADQQAA, LPASNKLPS, LPASNKLPSL, LPLPQLLLS, LPLPQLLLSA, LPSLPLSKM, LPSLPLSKMY, LQCINSNSRI, LQCRRAVSA, LQCRRAVSAT,
  • LSADQQAAPR LSKMYTTSM
  • LSKMYTTSMA LWCASSVTER
  • LWYCWPTWL LWYCWPTWLR
  • MAHMALLPS MALLPSGTK
  • MPILPLPQL MPILPLPQLL
  • MYSPSRYPR MYTTSMAMPI
  • RAVSATSWAS RAVSATSWAS, RCAGRWLWY, RCTACHTAL, RGTAWQLVPL, RLQCINSNSR, RRAVSATSW, RTNFHSSLA, RTNFHSSLAE, RTRCAGRWL, RTRCAGRWLW, RWLWYCWPT, RWLWYCWPTW.
  • SAAWRSCIA SAAWRSCIAL
  • SGQWMAHMAL SKMYTTSMA
  • SKMYTTSMAM SKMYTTSMAM
  • SMAMPILPL SNKLPSLPL
  • SPASRLQCI SPPKAPLLAA
  • SPSLPASNKL SRITSGQWM
  • SSLAETVSL SSLAETVSL
  • SSNDMIPMAI SSSNDMIPM
  • SSSSNDMIPM SSSSSSNDM
  • SSSSSSSNDM TACHTALGR
  • TERTRCAGRW TSMAMPILPL, TTSMAMPIL, TVSLHPLAPM, TWLRGTAWQL, VPLQCRRAV, VSLHPLAPM, VTERTRCAGR, WLRGTAWQL, WLRGTAWQLV, WLWYCWPTW, WLWYCWPTWL, WMAHMALLPS, WPTWLRGTA, WPTWLRGTAW, WYCWPTWLR, YPRSSSSSS, YPRSSSSSSS, YTTSMAMPI, or YTTSMAMPIL.
  • the isolated neoantigenic peptide is encoded by a ⁇ 2 ⁇ gene.
  • C z is RMERELKKWSIQTCLSARTGLSISCTTLNSPPLKKMSMPAV, or
  • the neoantigenic peptide is not ALAVLALLSF, ALLSFWPGGY, DSGLLTSSSR, ELLCVWVSSI, EWKVKFPEL,
  • KVKFPELLCV LAVLALLSF, LAVLALLSFW, LLCVWVSSI, LLCVWVSSIR, LLSFWPGGY,
  • REWKVKFPEL REWKVKFPEL, SFWPGGYPA, S FWPGGYPAY, SSREWKVKF, SSSREWKVK, SSSREWKVKF, TSSSREWKV, TSSSREWKVK, VKFPELLCV, VKFPELLCVW, WKVKFPELL, or YPAYSKDSGL.
  • the isolated neoantigenic peptide is encoded by a CDH1 gene.
  • C z is RSACVTVKGPLASVGRHSLSKQDCKFLPFWGFLEEFLLC,
  • the isolated neoantigenic peptide is encoded by a GATA3 gene.
  • C z is
  • the neoantigenic peptide is not AALSRHNVL, ADAPAIQPV, ADAPAIQPVL, AESKIMFAT, AESKIMFATL, AIQPVLWTT, ALQPLQPHA, APAIQPVLW, ARVPAVPFDL, ATLQRSSLW, AVPFDLHFCR, CSMLTGPPA, CSMLTGPPAR, DLHFCRSSI, DLHFCRSSIM, EPHLALQPL,
  • IMFATLQRSS IMKPKRDGY
  • IMKPKRDGYM KAESKIMFA
  • KIMFATLQR KPKRDGYMF
  • KPKRDGYMFL LALQPLQPH
  • LHFCRSSIM LHFCRSSIMK
  • LKAESKIMF LQHGHRHGL
  • LQPHADHAH LQRSSLWCL, LQRSSLWCLC, LSFGPHHPL, LSFGPHPPL, LSFWTTPPL,
  • LSHISALQPL LSHISALQPL, MFATLQRSSL, MFLKAESKI, MFLKAESKIM, MHPPSSLSFW, MKPKRDGYMF, MLTGPPARV, MSSLSHISA, MSSLSHISAL, NPAALSRHNV, PARVPAVPF, PAVPFDLHF,
  • PEPHLALQPL PEPHLALQPL, PPARVPAVPF, QPVLWTTPPL, RHGLEPCSM, RHNVLPEPHL, RSSIMKPKR, SALQPLQPH, SHISALQPL, S IMKPKRDGY, SLSFGPHHPL, SLSFGPHPPL, SLSFWTTPPL,
  • the isolated neoantigenic peptide is encoded by a MLL2 gene.
  • C z is
  • the neoantigenic peptide is not APGPRGRTC,
  • EVSRLSPCL EVSRLSPCL
  • GLKSPLRSQA GLRNRICPL
  • GLRNRICPLS GLRSHTYLR
  • GLRSHTYLRR GLRSHTYLRR
  • GLRSRTCPPG GLRSRTCPPG
  • HACPPGLRNR HAYALCLRSH
  • HHLRTHLLPH HLGSHPCRL
  • HLLPHHRRTR HLLPHHRRTR
  • HLRLHASPH HLRLHASPH
  • HLRSCPCSL HLRTHLLPH
  • HLRTHLLPHH HLRTPPHPH
  • HLRTPPHPHH HLRYRAYPP
  • HLRYRAYPPC HPCCHYLRSR
  • HPHHLRTHL HPHHLRTHLL
  • LHLRLHASPH LHLRSCPCSL
  • LLPHHRRTR LPCPHRLRSL
  • LPHHRRTRS LPHHRRTRS
  • LPHHRRTRSC LHLRLHASPH
  • LHLRSCPCSL LHLRSCPCSL
  • LLPHHRRTR LPCPHRLRSL
  • LPHHRRTRS LPHHRRTRS
  • LPHHRRTRSC LHLRLHASPH
  • LHLRSCPCSL LHLRSCPCSL
  • LLPHHRRTR LPCPHRLRSL
  • LPHHRRTRS LPHHRRTRS
  • LPHHRRTRSC LPHHRRTRSC
  • LPLGNHPYL LPRPLHLRL, LRLHASPHHL, LRNCTCPPRL, LRNHTCPPSL, LRNRICPLSL,
  • LRSCPCSLPL LRSHACPPGL, LRSHACPPNL, LRSHAYALCL, LRSHTCPPRL, LRSHTCPPSL,
  • LRSHTYLRRL LRSLPRPLHL, LRSQANALHL, LRTPPHPHHL, LRYRAYPPCL, LSLGNHLCPL,
  • LSLRSHPCPL LWCHACLHRL, MSPHLRYRA, MSPHLRYRAY, NLPCPHRLR, NLRNHTCPP, PMSPHLRYR, PPRLRSRTCL, PPSLRSHAY, RAYPPCLWCH, RDHICPLSL, RGRTCHPGL, RGRTCHPGLR, RLHASPHHL, RLHASPHHLR, RLRDHICPL, RLRDHICPLS, RLRNLPCPH,
  • RLRNLPCPHR RLRSHTCPP, RLRSHTCPPS, RLRSLPRPL, RLRSLPRPLH, RLRSRTCLL,
  • RLRSRTCLLC RLSPCLWCHA
  • RNHTCPPSL RNHTCPPSLR
  • RNLPCPHRLR RNRICPLSL
  • RNRICPLSLR RPLHLRLHA, RPLHLRLHAS, RSCPCRWRSH, RSCPCSLPL, RSHACPPGL,
  • RSHACPPGLR RSHACPPNL, RSHACPPNLR, RSHAYALCL, RSHAYALCLR, RSHPCCHYL, RSHPCCHYLR, RSHPCPLGL, RSHPCPLGLK, RSHTCPPRL, RSHTCPPRLR, RSHTCPPSL,
  • RSHTCPPSLR RSHTYLRRL, RSHTYLRRLR, RSLPRPLHL, RSLPRPLHLR, RSQANALHL,
  • RSRTCPPGLR RTCHPGLRSR, RTHLLPHHR, RTHLLPHHRR, RTPPHPHHL, RTPPHPHHLR, RTRSCPCRW, RTRSCPCRWR, RWRSHPCCH, RWRSHPCCHY, RYRAYPPCL, RYRAYPPCLW, SHAYALCLR, SLGNHLCPL, SLPLGNHPY, SLPLGNHPYL, SLPRPLHLR, SLPRPLHLRL,
  • SPHHLRTPP SPHHLRTPPH, SPHLRYRAY, SPLRSQANAL, SPMSPHLRY, SPMSPHLRYR,
  • the isolated neoantigenic peptide is encoded by a PTEN gene.
  • C z is SWKGTNWCNDMCIFITSGQIFKGTRGPRFLWGSKDQRQKGSNYSQSEALCVLL, KRTKCFTFG, PIFIQTLLLWDFLQKDLKAYTGTILMM, QKMILTKQIKTKPTDTFLQILR,
  • the neoantigenic peptide is not KMLKRTKCF, MLKRTKCFT, LKRTKCFTF, MLKRTKCFTF,
  • KQNKMLKRTK KMLKRTKCFT
  • NKMLKRTKCF NKMLKRTKCF
  • the isolated neoantigenic peptide is encoded by a TP53 gene.
  • C z is
  • the neoantigenic peptide is not APASAPWPST, APPWPLHQQL, APWPSTSSH, ASCILGQPSL,
  • PEAAPPWPL PTRAATVSV, QMKLPECQR, QQLLHRRPL, QQLLHRRPLH, QRLLPPWPL,
  • QWFTEDQVQM RAATVSVWA.
  • RLLPPWPLH RMPEAAPPW
  • RPAPASAPW RPAPASAPW
  • SLPRKPTRA SLPRKPTRA
  • TVSVWASCIL TYQGSYGFV, TYQGSYGFVW, TYQGSYVSV, TYQGSYVSVW, VSVWASCIL, WPLHQQLLH, WPSTSSHST, YGFVWASCI, YGFVWASCIL, YQGSYGFVW, YQGSYGFVWA, YQGSYVSVW, YQGSYVSVWA, YVSVWASCI, or YVSVWASCIL.
  • the isolated neoantigenic peptide is encoded by a VHL gene.
  • C z is
  • the neoantigenic peptide is not CHLSMLTDSL, EMQGHTMGF, FLPISHCQC, FLPISHCQCI, FRDAGHTMGF, FWLTKLNYL, GFWLTKLNY, GFWLTKLNYL, GSSEMQGHTM, HLSMLTDSL, HLSMLTDSLF, HSYRGHLGSS, HTMGFWLTK, HTMGFWLTKL, KERCLQLSGA, KLNYLCHLSM, LFRDAGHTM, LNYLCHLSM, LNYLCHLSML, LPISHCQCI, LPISHCQCIL, LSMLTDSLF, LSMLTDSLFL, LTDSLFLPI, LTKLNYLCHL, MGFWLTKLNY, MLTDSLFL
  • WLFRDAGHT WLFRDAGHT
  • WLFRDAGHTM WLFRDAGHTM
  • YLCHLSMLT YRGHLGSSEM.
  • the isolated neoantigenic peptide is encoded by a fusion of a first gene with a second gene. In some embodiments, the isolated neoantigenic peptide is encoded by an in-frame fusion of a first gene with a second gene.
  • the isolated neoantigenic peptide is encoded by a BCR gene and an ABL gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a CI lorf95 gene and a RELA gene.
  • a x B y C z is ISNSWDAHLGLGACGEAEGLGVQGAEEEEEEEEEEEEEGAGVPACPPKGPELFPLIFPAEPAQASGPY VEIIEQPKQPvGMRFRYKCEGRSAGSIPGEPvSTD.
  • the isolated neoantigenic peptide is encoded by a CBFB gene and an MYH11 gene.
  • the isolated neoantigenic peptide is encoded by a CD74 gene and an ROS1 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a EGFR gene and an SEPT 14 gene.
  • a x B y C z is the first native polypeptide is encoded by an gene and the second native polypeptide is encoded by
  • the isolated neoantigenic peptide is encoded by a EGFR gene and an EGFR gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a EML4 gene and an ALK gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a FGFR3 gene and an TACC3 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a NAB gene and an STAT6 gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a NDRG1 gene and an ERG gene.
  • a x B y C z is
  • the isolated neoantigenic peptide is encoded by a TMPRSS2 gene and an ERG gene.
  • a X B Y C Z is
  • the isolated neoantigenic peptide is encoded by a PML gene and an RARA gene.
  • a X B Y C Z is
  • the isolated neoantigenic peptide is encoded by a RU X 1 gene and an CBFA2T1 (RU X1T1) gene.
  • a X B Y C Z is
  • the isolated neoantigenic peptide is encoded by a fusion of a first gene with an exon of a splice variant of the first gene. In some embodiments, the isolated neoantigenic peptide is encoded by a fusion of a first gene with a cryptic exon of the first gene.
  • the isolated neoantigenic peptide is encoded by a AR-v7 gene and a cryptic exon encoded by the AR-v7 gene. In some embodiments, the isolated neoantigenic peptide is encoded by a AR-v7 gene comprising an exon of a splice variant of an AR gene.
  • a X B Y C Z is SCKVFFKRAAEGKQKYLCASRNDCTIDKFRRKNCPSCRLRKCYEAGMTLGEKFRVGNCKHLKMTRP
  • the isolated neoantigenic peptide is encoded by a fusion of a first gene with a second gene, wherein the peptide comprises an amino acid sequence encoded by an out-of frame sequence resulting from the fusion.
  • the isolated neoantigenic peptide is encoded by an AC011997.1 gene and a LRRC69 gene.
  • y is 1.
  • a X B Y C Z is 1.
  • the isolated neoantigenic peptide is encoded by an EEF1DP3 gene and a FRY gene.
  • y is 1.
  • a X B Y C Z is 1.
  • the isolated neoantigenic peptide is encoded by an MAD 1L1 gene and a MAFK gene.
  • y is 0.
  • a X B Y C Z is
  • the isolated neoantigenic peptide is encoded by an PPP1R1B gene and a STARD3 gene.
  • y is 1.
  • a X B Y C Z is 1.
  • the isolated neoantigenic peptide comprises one or more of the peptide sequences depicted in Table 1. In some embodiments, the isolated neoantigenic peptide comprises one or more of the peptide sequences depicted in Table 1A, Table IB, Table 1C, Table ID, Table IE, and/or Table IF. In some embodiments, the isolated neoantigenic peptide does not comprise one or more of the peptide sequences depicted in Table 2. In some embodiments, the isolated neoantigenic peptide does not comprise one or more of the peptide sequences depicted in Table 2A, Table 2B, Table 2C, and/or Table 2D.
  • Nucleic acids encoding peptides can be DNA or RNA, for example, mRNA, or a combination of DNA and RNA.
  • a nucleic acid encoding a peptide is a self-amplifying mRNA. (Brito et al., Adv. Genet. 2015; 89: 179-233). Any suitable polynucleotide that encodes a peptide described herein falls within the scope of this invention.
  • RNA includes and in some embodiments relates to "mRNA".
  • mRNA means "messenger-RNA” and relates to a "transcript” which is generated by using a DNA template and encodes a peptide or polypeptide.
  • 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 may be generated by in vitro transcription from a DNA template.
  • the in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available.
  • RNA may be stabilized and its translation increased by one or more modifications having a stabilizing effects and/or increasing translation efficiency of RNA. Such modifications are described, for example, in
  • RNA used according to the present invention may be modified within the coding region, i.e. the sequence encoding the expressed peptide or protein, without altering the sequence of the expressed peptide or protein, so as to increase the GC -content to increase mRNA stability and to perform a codon optimization and, thus, enhance translation in cells.
  • modified in the context of the RNA used in the present invention includes any modification of an RNA which is not naturally present in said RNA.
  • the RNA used according to the invention does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating RNA with a phosphatase.
  • the RNA according to the invention may have modified ribonucleotides in order to increase its stability and/or decrease cytotoxicity.
  • 5-methylcytidine is substituted partially or completely, for example, completely, for cytidine.
  • pseudouridine is substituted partially or completely, for example, completely, for uridine.
  • the term "modification” relates to providing an RNA with a 5'-cap or 5 '- cap analog.
  • 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 5' triphosphate linkage. In some embodiments, this guanosine is methylated at the 7-position.
  • the term “conventional 5'-cap” refers to a naturally occurring RNA 5'-cap, to the 7-methylguanosine cap (m G).
  • 5'-cap 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.
  • an mRNA encoding a neoantigen peptide of the invention is administered to a subject in need thereof.
  • the invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising a modified nucleoside, gene therapy vectors comprising same, gene therapy methods and gene transcription silencing methods comprising same.
  • the mRNA to be administered comprises at least one modified nucleoside.
  • polynucleotides encoding peptides described herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103 :3185 (1981).
  • Polynucleotides encoding peptides comprising or consisting of an analog can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native epitope.
  • a large number of vectors and host systems suitable for producing and administering a neoantigenic peptide described herein are known to those of skill in the art, and are commercially available.
  • the following vectors are provided by way of example.
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); p75.6 (Valentis); pCEP (Invitrogen); pCEI (Epimmune).
  • any other plasmid or vector can be used as long as it is replicable and viable in the host.
  • bacterial cells such as E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas,
  • Streptomyces, and Staphylococcus fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23 : 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines or Bowes melanoma; plant cells, etc.
  • a compatible vector for example, the C127, 3T3, CHO, HeLa and BHK cell lines or Bowes melanoma
  • plant cells etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention is also directed to vectors, and expression vectors useful for the production and administration of the neoantigenic peptides described herein, and to host cells comprising such vectors.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors which can be, for example, a cloning vector or an expression vector.
  • the vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the polynucleotides.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the coding sequence will be provided operably linked start and stop codons, promoter and terminator regions, and in some embodiments, and a replication system to provide an expression vector for expression in the desired cellular host.
  • promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence.
  • the resulting expression vectors are transformed into suitable bacterial hosts.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3- phosphoglycerate kinase (PGK), acid phosphatase, or heat shock proteins, among others.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and in some embodiments, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Yeast, insect or mammalian cell hosts can also be used, employing suitable vectors and control sequences.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23 : 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences.
  • Such promoters can also be derived from viral sources, such as, e.g., human cytomegalovirus (CMV-IE promoter) or herpes simplex virus type-1 (HSV TK promoter). Nucleic acid sequences derived from the SV40 splice, and polyadenylation sites can be used to provide the required nontranscribed genetic elements.
  • CMV-IE promoter human cytomegalovirus
  • HSV TK promoter herpes simplex virus type-1
  • Polynucleotides encoding neoantigenic peptides described herein can also comprise a ubiquitination signal sequence, and/or a targeting sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.
  • a targeting sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.
  • ER endoplasmic reticulum
  • Polynucleotides described herein can be administered and expressed in human cells (e.g., immune cells, including dendritic cells).
  • a human codon usage table can be used to guide the codon choice for each amino acid.
  • Such polynucleotides comprise spacer amino acid residues between epitopes and/or analogs, such as those described above, or can comprise naturally-occurring flanking sequences adjacent to the epitopes and/or analogs (and/or CTL, HTL, and B cell epitopes).
  • a neoantigenic peptide described herein can also be administered/expressed by viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, such as vaccinia or fowlpox.
  • vaccinia virus is used as a vector to express nucleotide sequences that encode the neoantigenic peptides described herein.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.
  • Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described by Stover et al., Nature 351 :456-460 (1991).
  • the vector is Modified Vaccinia Ankara (VA) (e.g. Bavarian Noridic (MVA-BN)).
  • Standard regulatory sequences well known to those of skill in the art can be included in the vector to ensure expression in the human target cells.
  • a promoter with a downstream cloning site for polynucleotide e.g., minigene insertion
  • a polyadenylation signal for efficient transcription termination e.g., an E. coli origin of replication
  • an E. coli selectable marker e.g. ampicillin or kanamycin resistance
  • Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • the promoter is the CMV-IE promoter.
  • Polynucleotides described herein can comprise one or more synthetic or naturally -occurring introns in the transcribed region.
  • the inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells can also be considered for increasing polynucleotide expression.
  • a polynucleotide described herein can comprise immunostimulatory sequences (ISSs or CpGs). These sequences can be included in the vector, outside the polynucleotide coding sequence to enhance immunogenicity.
  • ISSs or CpGs immunostimulatory sequences
  • the size of at least one antigenic peptide molecule may comprise, but is not limited to, about 8, about 9, about 10, about 1 1, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or greater amino molecule residues, and any range derivable therein.
  • the antigenic peptide molecules are equal to or less than 50 amino acids. In some embodiments, the antigenic peptide molecules are equal to about 20 to about 30 amino acids.
  • a longer peptide may be designed in several ways. For example, when the HLA-binding regions are predicted or known, a longer peptide may consist of either: individual binding peptides with an extension of 0-10 amino acids toward the N- and C-terminus of each corresponding gene product. A longer peptide may also consist of a concatenation of some or all of the binding peptides with extended sequences for each. In another case, when sequencing reveals a long (>10 residues) epitope sequence present in the diseased tissue (e.g.
  • a longer peptide may consist of the entire stretch of novel disease-specific amino acids.
  • use of a longer peptide requires endogenous processing by professional antigen presenting cells such as dendritic cells and may lead to more effective antigen presentation and induction of T cell responses.
  • the extended sequence is altered to improve the biochemical properties of the polypeptide (properties such as solubility or stability) or to improve the likelihood for efficient proteasomal processing of the peptide.
  • the antigenic peptides and polypeptides may bind an HLA protein.
  • the antigenic peptides may bind an HLA protein with greater affinity than a corresponding native / wild-type peptide.
  • the antigenic peptide may have an IC50 of about less than 1000 nM, about less than 500 nM, about less than 250 nM, about less than 200 nM, about less than 150 nM, about less than 100 nM, or about less than 50 nM.
  • the antigenic peptides do not induce an autoimmune response and/or invoke immunological tolerance when administered to a subject.
  • the invention also provides compositions comprising a plurality of antigenic peptides.
  • Reference to antigenic peptides includes any suitable delivery modality that can result in introduction of the peptide into a subject's cell (e.g., nucleic acid).
  • the composition comprises at least 2 or more antigenic peptides.
  • the composition contains at least about 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 distinct peptides.
  • the composition contains at least one peptide from Table 1.
  • the composition contains at least one peptide from Table 1 and at least one other peptide from Table 1.
  • the composition contains at least one peptide from Table 1 and at least one other peptide from Table 2.
  • the composition contains at least 20 distinct peptides. In some embodiments the composition contains at most 20 distinct peptides. According to the invention, 2 or more of the distinct peptides may be derived from the same polypeptide. For example, if an antigenic mutation encodes a polypeptide, two or more of the antigenic peptides may be derived from the polypeptide. In some embodiments, the two or more antigenic peptides derived from the polypeptide may comprise a tiled array that spans the polypeptide (e.g., the antigenic peptides may comprise a series of overlapping antigenic peptides that spans a portion, or all, of the polypeptide). Antigenic peptides can be derived from any protein coding gene. The antigenic peptides can be derived from mutations in human cancer or from an infectious agent or an autoimmune disease.
  • the antigenic peptides, polypeptides, and analogs can be further modified to contain additional chemical moieties not normally part of the protein.
  • Those derivatized moieties can improve the solubility, the biological half-life, absorption of the protein, or binding affinity.
  • the moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).
  • antigenic peptides and polypeptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g.
  • the antigenic peptide and polypeptides may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding.
  • conservative substitutions may encompass replacing an amino acid residue with another amino acid residue that is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the effect of single amino acid substitutions may also be probed using D- amino acids.
  • the antigenic peptide may also be modified by extending or decreasing the compound's amino acid sequence, e.g., by the addition or deletion of amino acids.
  • the antigenic peptides, polypeptides, or analogs can also be modified by altering the order or composition of certain residues. It will be appreciated by the skilled artisan that certain amino acid residues essential for biological activity, e.g., those at critical contact sites or conserved residues, may generally not be altered without an adverse effect on biological activity.
  • non-critical amino acids need not be limited to those naturally occurring in proteins, such as L-a- amino acids, or their D-isomers, but may include non-natural amino acids as well, such as ⁇ - ⁇ - ⁇ - amino acids, as well as many derivatives of L-a-amino acids.
  • An antigen peptide may be optimized by using a series of peptides with single amino acid substitutions to determine the effect of electrostatic charge, hydrophobicity, etc. on MHC binding. For instance, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions may be made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell receptors. In addition, multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be employed. The substitutions may be homo- oligomers or hetero-oligomers.
  • a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions may be made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell receptors.
  • multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues
  • substitutions should employ amino acid residues or other molecular fragments chosen to avoid, for example, steric and charge interference which might disrupt binding.
  • Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final peptide.
  • An antigenic peptide may be modified to provide desired attributes.
  • the ability of the peptides to induce CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.
  • immunogenic peptides/T helper conjugates are linked by a spacer molecule.
  • a spacer comprises relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under
  • Spacers can be selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer.
  • the -antigenic peptide may be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the peptide. The amino terminus of either the antigenic peptide or the T helper peptide may be acylated.
  • T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378- 389.
  • the present invention is based, at least in part, on the ability to present the immune system of the patient with one or more disease-specific antigens.
  • disease specific antigens may be produced either in vitro or in vivo.
  • Disease specific antigens may be produced in vitro as peptides or polypeptides, which may then be formulated into a vaccine or immunogenic composition and administered to a subject.
  • in vitro production may occur by a variety of methods known to one of skill in the art such as, for example, peptide synthesis or expression of a peptide/polypeptide from a DNA or R A molecule in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptide/polypeptide.
  • disease specific antigens may be produced in vivo by introducing molecules (e.g., DNA, RNA, viral expression systems, and the like) that encode disease specific antigens into a subject, whereupon the encoded disease specific antigens are expressed.
  • the methods of in vitro and in vivo production of antigens is also further described herein as it relates to pharmaceutical compositions and methods of delivery of the therapy.
  • the present invention includes modified antigenic peptides.
  • a modification can include a covalent chemical modification that does not alter the primary amino acid sequence of the antigenic peptide itself. Modifications can produce peptides with desired properties, for example, prolonging the in vivo half-life, increasing the stability, reducing the clearance, altering the immunogenicity or allergenicity, enabling the raising of particular antibodies, cellular targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation.
  • Changes to an antigenic peptide include, but are not limited to, conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticulate encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, the addition of a surface active material, the addition of amino acid mimetics, or the addition of unnatural amino acids.
  • PEG conjugated biomolecules have been shown to possess clinically useful properties, including better physical and thermal stability, protection against susceptibility to enzymatic degradation, increased solubility, longer in vivo circulating half-life and decreased clearance, reduced immunogenicity and antigenicity, and reduced toxicity.
  • PEGs suitable for conjugation to a polypeptide sequence are generally soluble in water at room temperature, and have the general formula R(0-CH2-CH2)nO-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons.
  • R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons.
  • the PEG conjugated to the polypeptide sequence can be linear or branched. Branched PEG derivatives, "star-PEGs" and multi-armed PEGs are contemplated by the present disclosure.
  • Such compositions can be produced by reaction conditions and purification methods know in the art. For example, cation exchange
  • chromatography may be used to separate conjugates, and a fraction is then identified which contains the conjugate having, for example, the desired number of PEGs attached, purified free from unmodified protein sequences and from conjugates having other numbers of PEGs attached.
  • PEG may be bound to a polypeptide of the present disclosure via a terminal reactive group (a "spacer").
  • the spacer is, for example, a terminal reactive group which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol.
  • the PEG having the spacer which may be bound to the free amino group includes N-hydroxysuccinylimide polyethylene glycol which may be prepared by activating succinic acid ester of polyethylene glycol with N- hydroxy succinylimide.
  • Another activated polyethylene glycol which may be bound to a free amino group is 2,4-bis(0- methoxypolyethyleneglycol)-6-chloro-s-triazine which may be prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride.
  • the activated polyethylene glycol which is bound to the free carboxyl group includes polyoxyethylenediamine.
  • Conjugation of one or more of the polypeptide sequences of the present disclosure to PEG having a spacer may be carried out by various conventional methods.
  • the conjugation reaction can be carried out in solution at a pH of from 5 to 10, at temperature from 4°C to room temperature, for 30 minutes to
  • reaction conditions may be selected to direct the reaction towards producing predominantly a desired degree of substitution.
  • high temperature, neutral to high pH e.g., pH>7
  • longer reaction time tend to increase the number of PEGs attached.
  • Various means known in the art may be used to terminate the reaction. In some embodiments the reaction is terminated by acidifying the reaction mixture and freezing at, e.g., -20°C.
  • PEG mimetics have been developed that retain the attributes of PEG (e.g., enhanced serum half- life) while conferring several additional advantageous properties.
  • simple polypeptide chains comprising, for example, Ala, Glu, Gly, Pro, Ser and Thr
  • the peptide or protein drug of interest e.g., Amunix' XTEN technology; Mountain View, CA.
  • Amunix' XTEN technology e.g., Amunix' XTEN technology; Mountain View, CA.
  • established molecular biology techniques enable control of the side chain composition of the polypeptide chains, allowing optimization of immunogenicity and manufacturing properties.
  • Glycosylation can affect the physical properties of proteins and can also be important in protein stability, secretion, and subcellular localization. Proper glycosylation can be important for biological activity. In fact, some genes from eukaryotic organisms, when expressed in bacteria (e.g., E. coli) which lack cellular processes for glycosylating proteins, yield proteins that are recovered with little or no activity by virtue of their lack of glycosylation. Addition of glycosylation sites can be accomplished by altering the amino acid sequence.
  • the alteration to the polypeptide may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues (for O-linked glycosylation sites) or asparagine residues (for N- linked glycosylation sites).
  • the structures of N-linked and O- linked oligosaccharides and the sugar residues found in each type may be different.
  • One type of sugar that is commonly found on both is N-acetylneuraminic acid (hereafter referred to as sialic acid).
  • sialic acid is usually the terminal residue of both N-linked and O- linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycoprotein.
  • Embodiments of the present disclosure comprise the generation and use of N-glycosylation variants.
  • polypeptide sequences of the present disclosure may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Removal of carbohydrates may be accomplished chemically or enzymatically, or by substitution of codons encoding amino acid residues that are glycosylated. Chemical deglycosylation techniques are known, and enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases.
  • Additional suitable components and molecules for conjugation include, for example, molecules for targeting to the lymphatic system, thyroglobulin; albumins such as human serum albumin (HAS); tetanus toxoid; Diphtheria toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses; influenza virus hemaglutinin, influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B virus core protein and surface antigen; or any combination of the foregoing.
  • albumins such as human serum albumin (HAS); tetanus toxoid; Diphtheria toxoid
  • polyamino acids such as poly(D-lysine:D-glutamic acid)
  • VP6 polypeptides of rotaviruses influenza virus hemaglutinin, influenza virus nucleoprotein
  • KLH Keyhole
  • Fusion of albumin to one or more polypeptides of the present disclosure can, for example, be achieved by genetic manipulation, such that the DNA coding for HSA, or a fragment thereof, is joined to the DNA coding for the one or more polypeptide sequences. Thereafter, a suitable host can be transformed or transfected with the fused nucleotide sequences in the form of, for example, a suitable plasmid, so as to express a fusion polypeptide. The expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo from, for example, a transgenic organism.
  • the expression of the fusion protein is performed in mammalian cell lines, for example, CHO cell lines. Transformation is used broadly herein to refer to the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells.
  • albumin itself may be modified to extend its circulating half-life. Fusion of the modified albumin to one or more polypeptides can be attained by the genetic manipulation techniques described above or by chemical conjugation; the resulting fusion molecule has a half- life that exceeds that of fusions with non-modified albumin.
  • albumin-binding strategies have been developed as alternatives for direct fusion, including albumin binding through a conjugated fatty acid chain (acylation). Because serum albumin is a transport protein for fatty acids, these natural ligands with albumin - binding activity have been used for half-life extension of small protein therapeutics.
  • insulin detemir an approved product for diabetes, comprises a myristyl chain conjugated to a genetically-modified insulin, resulting in a long- acting insulin analog.
  • Another type of modification is to conjugate (e.g., link) one or more additional components or molecules at the N- and/or C-terminus of a polypeptide sequence, such as another protein (e.g., a protein having an amino acid sequence heterologous to the subject protein), or a carrier molecule.
  • a polypeptide sequence such as another protein (e.g., a protein having an amino acid sequence heterologous to the subject protein), or a carrier molecule.
  • a conjugate modification may result in a polypeptide sequence that retains activity with an additional or complementary function or activity of the second molecule.
  • a polypeptide sequence may be conjugated to a molecule, e.g., to facilitate solubility, storage, in vivo or shelf half-life or stability, reduction in immunogenicity, delayed or controlled release in vivo, etc.
  • Other functions or activities include a conjugate that reduces toxicity relative to an unconjugated polypeptide sequence, a conjugate that targets a type of cell or organ more efficiently than an unconjugated polypeptide sequence, or a drug to further counter the causes or effects associated with a disorder or disease as set forth herein (e.g., diabetes).
  • a polypeptide may also be conjugated to large, slowly metabolized macromolecules such as proteins; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads; polymeric amino acids such as polyglutamic acid, polylysine; amino acid copolymers; inactivated virus particles; inactivated bacterial toxins such as toxoid from diphtheria, tetanus, cholera, leukotoxin molecules; inactivated bacteria; and dendritic cells.
  • Additional candidate components and molecules for conjugation include those suitable for isolation or purification.
  • binding molecules such as biotin (biotin-avidin specific binding pair), an antibody, a receptor, a ligand, a lectin, or molecules that comprise a solid support, including, for example, plastic or polystyrene beads, plates or beads, magnetic beads, test strips, and membranes.
  • cation exchange chromatography may be used to separate conjugates by charge difference, which effectively separates conjugates into their various molecular weights.
  • the content of the fractions obtained by cation exchange chromatography may be identified by molecular weight using conventional methods, for example, mass spectroscopy, SDS-PAGE, or other known methods for separating molecular entities by molecular weight.
  • the amino- or carboxyl- terminus of a polypeptide sequence of the present disclosure can be fused with an immunoglobulin Fc region (e.g., human Fc) to form a fusion conjugate (or fusion molecule).
  • Fc fusion conjugates have been shown to increase the systemic half-life of
  • biopharmaceuticals and thus the biopharmaceutical product may require less frequent administration.
  • Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells that line the blood vessels, and, upon binding, the Fc fusion molecule is protected from degradation and re- released into the circulation, keeping the molecule in circulation longer.
  • This Fc binding is believed to be the mechanism by which endogenous IgG retains its long plasma half-life.
  • More recent Fc-fusion technology links a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical as compared to traditional Fc-fusion conjugates.
  • the present disclosure contemplates the use of other modifications, currently known or developed in the future, of the polypeptides to improve one or more properties.
  • One such method for prolonging the circulation half-life, increasing the stability, reducing the clearance, or altering the immunogenicity or allergenicity of a polypeptide of the present disclosure involves modification of the polypeptide sequences by hesylation, which utilizes hydroxyethyl starch derivatives linked to other molecules in order to modify the molecule's characteristics.
  • hesylation which utilizes hydroxyethyl starch derivatives linked to other molecules in order to modify the molecule's characteristics.
  • Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, in vitro translation, or the chemical synthesis of proteins or peptides.
  • Peptides can be readily synthesized chemically utilizing reagents that are free of contaminating bacterial or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc.85:2149-54, 1963).
  • antigenic peptides are prepared by (1) parallel solid-phase synthesis on multi-channel instruments using uniform synthesis and cleavage conditions; (2) purification over a RP-HPLC column with column stripping; and re-washing, but not replacement, between peptides; followed by (3) analysis with a limited set of the most informative assays.
  • the Good Manufacturing Practices (GMP) footprint can be defined around the set of peptides for an individual patient, thus requiring suite changeover procedures only between syntheses of peptides for different patients.
  • a nucleic acid e.g., a polynucleotide
  • the polynucleotide may be, e.g., DNA, cDNA, PNA, CNA, RNA, either single- and/or double-stranded, or native or stabilized forms of polynucleotides, such as e.g. polynucleotides with a phosphorothiate backbone, or combinations thereof and it may or may not contain introns so long as it codes for the peptide.
  • in vitro translation is used to produce the peptide.
  • An expression vector capable of expressing a polypeptide can also be prepared. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation.
  • the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
  • an expression vector such as a plasmid
  • the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the expression vector.
  • the vector is then introduced into the host bacteria for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
  • Expression vectors comprising the isolated polynucleotides, as well as host cells containing the expression vectors, are also contemplated.
  • the antigenic peptides may be provided in the form of RNA or cDNA molecules encoding the desired antigenic peptides.
  • One or more antigenic peptides of the invention may be encoded by a single expression vector.
  • the polynucleotides may comprise the coding sequence for the disease specific antigenic peptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and/or secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell).
  • a polypeptide having a leader sequence is a preprotein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides can comprise the coding sequence for the disease specific antigenic peptide fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide, which may then be incorporated into a personalized disease vaccine or immunogenic composition.
  • the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.
  • a mammalian host e.g., COS-7 cells
  • Additional tags include, but are not limited to, Calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag, Spy Tag, Biotin Carboxyl Carrier Protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TC tag, Ty tag, and the like.
  • Calmodulin tags include, but are not limited to, Calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag, Spy Tag, Biotin Carboxyl Carrier Protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TC tag, Ty
  • the polynucleotides may comprise the coding sequence for one or more of the disease specific antigenic peptides fused in the same reading frame to create a single concatamerized antigenic peptide construct capable of producing multiple antigenic peptides.
  • isolated nucleic acid molecules having a nucleotide sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least
  • the isolated disease specific antigenic peptides described herein can be produced in vitro (e.g., in the laboratory) by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.
  • sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81 :5662-5066 (1984) and U.S. Pat. No.4,588,585.
  • a DNA sequence encoding a polypeptide of interest would be constructed by chemical synthesis using an oligonucleotide synthesizer.
  • Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest is produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene.
  • a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3 ' overhangs for complementary assembly
  • the polynucleotide sequences encoding a particular isolated polypeptide of interest is inserted into an expression vector and optionally operatively linked to an expression control sequence appropriate for expression of the protein in a desired host.
  • an expression control sequence appropriate for expression of the protein in a desired host.
  • Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host.
  • the gene in order to obtain high expression levels of a transfected gene in a host, the gene can be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
  • Recombinant expression vectors may be used to amplify and express DNA encoding the disease specific antigenic peptides.
  • Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a disease specific antigenic peptide or a bioequivalent analog operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes.
  • a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail herein.
  • Such regulatory elements can include an operator sequence to control transcription.
  • the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated.
  • DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • signal peptide secretory leader
  • a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence
  • a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • operatively linked means contiguous, and in the case of secretory leaders, means contiguous and in reading frame.
  • Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as Ml 3 and filamentous single -stranded DNA phages.
  • Suitable host cells for expression of a polypeptide include prokaryotes, yeast, insect or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli.
  • Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems could also be employed.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known in the art (see Pouwels et al, Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).
  • Suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23 : 175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa and BHK cell lines.
  • Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5 ' or 3 ' flanking nontranscribed sequences, and 5 ' or 3 ' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).
  • the proteins produced by a transformed host can be purified according to any suitable method.
  • Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography, and the like), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence, glutathione-S- transferase, and the like can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.
  • supernatants from systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • a suitable purification matrix for example, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
  • a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be employed for final purification steps.
  • Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • the present invention also contemplates the use of nucleic acid molecules as vehicles for delivering antigenic peptides/polypeptides to the subject in need thereof, in vivo, in the form of, e.g., DNA/RNA vaccines (see, e.g., WO2012/159643, and WO2012/159754, hereby incorporated by reference in their entirety).
  • antigens may be administered to a patient in need thereof by use of a plasmid.
  • plasmids which usually consist of a strong viral promoter to drive the in vivo transcription and translation of the gene (or complementary DNA) of interest (Mor, et al, (1995). The Journal of Immunology
  • Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:
  • Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88)
  • ID intradermally
  • Gene gun delivery the other commonly used method of delivery, ballistically accelerates plasmid DNA (pDNA) that has been adsorbed onto gold or tungsten microparticles into the target cells, using compressed helium as an accelerant (Alarcon et al, (1999). Adv. Parasitol. Advances in Parasitology 42: 343- 410; Lewis et al., ( 1999). Advances in Virus Research (Academic Press) 54: 129-88).
  • pDNA plasmid DNA
  • Alternative delivery methods may include aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88) and topical administration of pDNA to the eye and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54: 129-88).
  • Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors.
  • DNA or RNA may also be delivered to cells following mild mechanical disruption of the cell membrane, temporarily permeabilizing the cells. Such a mild mechanical disruption of the membrane can be accomplished by gently forcing cells through a small aperture (Ex Vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells, Sharei et al, PLOS ONE
  • a disease specific vaccine or immunogenic composition may include separate DNA plasmids encoding, for example, one or more antigenic peptides/polypeptides.
  • the exact choice of expression vectors can depend upon the peptide/polypeptides to be expressed, and is well within the skill of the ordinary artisan.
  • the expected persistence of the DNA constructs is expected to provide an increased duration of protection.
  • One or more antigenic peptides of the invention may be encoded and expressed in vivo using a viral based system (e.g., an adenovirus system, an adeno associated virus (AAV) vector, a poxvirus, or a lentivirus).
  • a viral based system e.g., an adenovirus system, an adeno associated virus (AAV) vector, a poxvirus, or a lentivirus.
  • the disease vaccine or immunogenic composition may include a viral based vector for use in a human patient in need thereof, such as, for example, an adenovirus.
  • a viral based vector for use in a human patient in need thereof, such as, for example, an adenovirus. Plasmids that can be used for adeno associated virus, adenovirus, and lentivirus delivery have been described previously (see e.g., U.S. Patent Nos. 6,955,808 and 6,943,019, and U.S. Patent application No. 20080254008, hereby incorporated by reference).
  • the peptides and polypeptides of the invention can also be expressed by a vector, e.g., a nucleic acid molecule as herein-discussed, e.g., RNA or a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopox virus, avipox virus, or adenovirus, AAV or lentivirus.
  • a vector e.g., a nucleic acid molecule as herein-discussed, e.g., RNA or a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopox virus, avipox virus, or adenovirus, AAV or lentivirus.
  • a vector e.g., a nucleic acid molecule as herein-discussed, e.g., RNA or a DNA plasmid, a viral vector such as a poxvirus,
  • retrovirus gene transfer methods often resulting in long term expression of the inserted transgene.
  • the retrovirus is a lentivirus.
  • high transduction efficiencies have been observed in many different cell types and target tissues.
  • the tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells.
  • a retrovirus can also be engineered to allow for conditional expression of the inserted transgene, such that only certain cell types are infected by the lentivirus.
  • Cell type specific promoters can be used to target expression in specific cell types.
  • Lentiviral vectors are retroviral vectors (and hence both lentiviral and retroviral vectors may be used in the practice of the invention). Moreover, lentiviral vectors are able to transduce or infect non- dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system may therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the desired nucleic acid into the target cell to provide permanent expression.
  • Widely used retroviral vectors that may be used in the practice of the invention include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., ( 1992) J. Virol. 66:2731-2739; Johann et al., (1992) J. Virol.66: 1635- 1640;
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • a minimal non-primate lentiviral vector such as a lentiviral vector based on the equine infectious anemia virus (EIAV).
  • the vectors may have cytomegalovirus (CMV) promoter driving expression of the target gene.
  • CMV cytomegalovirus
  • the invention contemplates amongst vector(s) useful in the practice of the invention: viral vectors, including retroviral vectors and lentiviral vectors.
  • the delivery is via a lentivirus.
  • Dosages e.g., 10 ⁇ of a recombinant lentivirus having a titer of 1 x 10 9 transducing units (TU)/mL, can be adapted or extrapolated to use of a retroviral or lentiviral vector in the present invention.
  • the viral preparation is concentrated by ultracentrifugation. Other methods of concentration such as ultrafiltration or binding to and elution from a matrix may be used.
  • the amount of lentivirus administered may be lxlO 5 or about lxlO 5 plaque forming units (PFU), 5xl0 5 or about 5xl0 5 PFU, lxlO 6 or about LxlO 6 PFU, 5xl0 6 or about 5xl0 6 PFU, lxlO 7 or about lxl07PFU, 5xl0 7 or about 5xl0 7 PFU, lxlO 8 or about lxlO 8 PFU, 5xl0 8 or about 5xl0 8 PFU, lxlO 9 or about lxl0 9 PFU, 5xl0 9 or about 5xl0 9 PFU, lxlO 10 or about lxlO 10 PFU or 5xl0 10 or about 5xl0 10 PFU as total single dosage for an average human of 75 kg or adjusted for the weight and size and species of the subject.
  • PFU plaque forming units
  • an adenovirus vector Also useful in the practice of the invention is an adenovirus vector.
  • One advantage is the ability of recombinant adenoviruses to efficiently transfer and express recombinant genes in a variety of mammalian cells and tissues in vitro and in vivo, resulting in the high expression of the transferred nucleic acids. Further, the ability to productively infect quiescent cells, expands the utility of recombinant adenoviral vectors. In addition, high expression levels ensure that the products of the nucleic acids will be expressed to sufficient levels to generate an immune response (see e.g., U.S. Patent No.7,029,848, hereby incorporated by reference).
  • adenovirus vectors useful in the practice of the invention mention is made of US Patent No.6,955, 808.
  • the adenovirus vector used can be selected from the group consisting of the Ad5, Ad35, Adl 1, C6, and C7 vectors.
  • Ad5 The sequence of the Adenovirus 5 (“Ad5") genome has been published. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285; the contents if which is hereby incorporated by reference).
  • Ad35 vectors are described in U.S. Pat.
  • Adl l vectors are described in U.S. Pat. No. 6,913,922.
  • C6 adenovirus vectors are described in U.S. Pat. Nos. 6,780,407; 6,537,594; 6,309,647; 6,265,189; 6,156,567; 6,090,393; 5,942,235 and 5,833,975.
  • C7 vectors are described in U.S. Pat. No. 6,277,558.
  • Adenovirus vectors that are El-defective or deleted, E3- defective or deleted, and/or E4-defective or deleted may also be used.
  • adenoviruses having mutations in the El region have improved safety margin because El -defective adenovirus mutants are replication-defective in non-permissive cells, or, at the very least, are highly attenuated.
  • Adenoviruses having mutations in the E3 region may have enhanced the immunogenicity by disrupting the mechanism whereby adenovirus down-regulates MHC class I molecules.
  • Adenoviruses having E4 mutations may have reduced immunogenicity of the adenovirus vector because of suppression of late gene expression. Such vectors may be particularly useful when repeated re- vaccination utilizing the same vector is desired.
  • Adenovirus vectors that are deleted or mutated in El, E3, E4, El and E3, and El and E4 can be used in accordance with the present invention.
  • "gutless" adenovirus vectors, in which all viral genes are deleted can also be used in accordance with the present invention.
  • Such vectors require a helper virus for their replication and require a special human 293 cell line expressing both Ela and Cre, a condition that does not exist in natural environment.
  • Such "gutless" vectors are non-immunogenic and thus the vectors may be inoculated multiple times for re -vaccination.
  • the "gutless" adenovirus vectors can be used for insertion of heterologous inserts/genes such as the transgenes of the present invention, and can even be used for co-delivery of a large number of heterologous inserts/genes.
  • the delivery is via an adenovirus, which may be at a single booster dose.
  • the adenovirus is delivered via multiple doses.
  • AAV is advantageous over other viral vectors due to low toxicity and low probability of causing insertional mutagenesis because it doesn't integrate into the host genome.
  • AAV has a packaging limit of 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kb result in significantly reduced virus production.
  • AAV ITR can serve as a promoter and is advantageous for eliminating the need for an additional promoter element.
  • CMV CMV
  • CAG CAG
  • CBh CBh
  • PGK PGK
  • SV40 Ferritin heavy or light chains
  • brain expression the following promoters can be used: Synapsinl for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.
  • Promoters used to drive RNA synthesis can include: Pol III promoters such as U6 or HI .
  • RNA guide RNA
  • AAV vectors useful in the practice of the invention mention is made of US Patent Nos. 5658785, 7115391, 7172893, 6953690, 6936466, 6924128, 6893865, 6793926, 6537540, 6475769 and 6258595, and documents cited therein.
  • the AAV can be AAVl, AAV2, AAV5 or any combination thereof.
  • AAV8 is useful for delivery to the liver. In some embodiments the delivery is via an AAV. The dosage may be adjusted to balance the therapeutic benefit against any side effects.
  • effectively activating a cellular immune response for a disease vaccine or immunogenic composition can be achieved by expressing the relevant antigens in a vaccine or immunogenic composition in a non-pathogenic microorganism.
  • a non-pathogenic microorganism such microorganisms are Mycobacterium bovis BCG, Salmonella and Pseudomona (See, U.S. Patent No.6,991,797, hereby incorporated by reference in its entirety).
  • a Poxvirus is used in the disease vaccine or immunogenic composition.
  • These include orthopoxvirus, avipox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see e.g., Verardiet al., Hum Vaccin Immunother. 2012 Jul;8(7):961-70; and Moss, Vaccine. 2013; 31(39): 4220- 4222).
  • Poxvirus expression vectors were described in 1982 and quickly became widely used for vaccine development as well as research in numerous fields. Advantages of the vectors include simple construction, ability to accommodate large amounts of foreign DNA and high expression levels.
  • poxviruses such as Chordopoxvirinae subfamily poxviruses (poxviruses of vertebrates), for instance, orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC ® VR-1354), Copenhagen Strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox virus (e.g., Wheatley C93 Strain, ALVAC), fowlpox virus (e.g., FP9 Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, inter alia, synthetic or non- naturally occurring recombinants thereof, uses thereof, and methods for making and using such recombinants may be found in scientific and
  • the vaccinia virus is used in the disease vaccine or immunogenic composition to express a antigen.
  • a antigen Rudolph et al., Recombinant viruses as vaccines and immunological tools. Curr Opin Immunol 9:517-524, 1997.
  • the recombinant vaccinia virus is able to replicate within the cytoplasm of the infected host cell and the polypeptide of interest can therefore induce an immune response.
  • Poxviruses have been widely used as vaccine or immunogenic composition vectors because of their ability to target encoded antigens for processing by the major histocompatibility complex class I pathway by directly infecting immune cells, in particular antigen-presenting cells, but also due to their ability to self-adjuvant.
  • ALVAC is used as a vector in a disease vaccine or immunogenic composition.
  • ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens (Horig H, Lee DS, Conkright W, et al. Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human
  • a Modified Vaccinia Ankara (MVA) virus may be used as a viral vector for an antigen vaccine or immunogenic composition.
  • MVA is a member of the Orthopoxvirus family and has been generated by about 570 serial passages on chicken embryo fibroblasts of the Ankara strain of Vaccinia virus (CVA) (for review see Mayr, A., et al., Infection 3, 6- 14, 1975).
  • CVA Ankara strain of Vaccinia virus
  • the resulting MVA virus contains 31 kilobases less genomic information compared to CVA, and is highly hosT cell restricted (Meyer, H. et al, J. Gen. Virol. 72, 1031- 1038, 1991).
  • MVA is characterized by its extreme attenuation, namely, by a diminished virulence or infectious ability, but still holds an excellent
  • MVA-BN ® -HER2 is a candidate immunotherapy designed for the treatment of HER-2 -positive breast cancer and is currently in clinical trials.
  • Methods to make and use recombinant MVA has been described (e.g., see U.S. Patent Nos. 8,309,098 and 5, 185, 146 hereby incorporated in its entirety).
  • recombinant viral particles of the vaccine or immunogenic composition are administered to patients in need thereof.
  • Neoantigen binding peptides are administered to patients in need thereof.
  • the present invention provides a binding protein (e.g., an antibody or antigen- binding fragment thereof), or a T cell receptor (TCR), or a chimeric antigen receptor (CAR) capable of binding with a high affinity to a neoantigen peptide:human leukocyte antigen (HLA) complex.
  • a binding protein e.g., an antibody or antigen- binding fragment thereof
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • HLA human leukocyte antigen
  • the present invention provides a CAR that is capable of binding with a high affinity to a neoantigenic peptide derived from the extracellular domain of a protein.
  • a neoantigen-specific binding protein or TCR or CAR as described herein includes variant polypeptide species that have one or more amino acid substitutions, insertions, or deletions in the native amino acid sequence, provided that the binding protein retains or substantially retains its specific binding function.
  • Conservative substitutions of amino acids are well known and may occur naturally or may be introduced when the binding protein or TCR is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, N Y, 2001).
  • Oligonucleotide- directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired.
  • random or saturation mutagenesis techniques such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to prepare immunogen polypeptide variants (see, e.g., Sambrook et al., supra).
  • amino acid that is substituted at a particular position in a peptide or polypeptide is conservative (or similar).
  • a similar amino acid or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine
  • amino acids with nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • amino acids with beta-branched side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
  • Proline which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine)
  • substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
  • similarity between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide (e.g., using GENEWORKS, Align, the BLAST algorithm, or other algorithms described herein and practiced in the art).
  • a neoantigen specific binding protein, TCR or CAR is capable of (a) specifically binding to a neoantigen: HLA complex on a cell surface independent or in the absence of CD8.
  • a neoantigen specific binding protein is a T cell receptor (TCR), a chimeric antigen receptor or an antigen-binding fragment of a TCR, any of which can be chimeric, humanized or human.
  • an antigen-binding fragment of the TCR comprises a single chain TCR (scTCR).
  • composition comprising a neoantigen-specific binding protein or high affinity recombinant TCR according to any one of the above embodiments and a
  • Methods useful for isolating and purifying recombinantly produced soluble TCR can include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant soluble TCR into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate can be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods can also be employed when isolating an immunogen from its natural environment.
  • Methods for large scale production of one or more of the isolated/recombinant soluble TCR described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR may be performed according to methods described herein and known in the art.
  • the present invention is directed to an immunogenic composition, e.g., a vaccine composition capable of raising a neoantigen-specific response (e.g., a humoral or cell-mediated immune response).
  • the immunogenic composition comprises neoantigen therapeutics (e.g., peptides, polynucleotides, TCR, CAR, cells containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.) described herein corresponding to tumor specific neoantigen identified herein.
  • immunogenic peptides are identified from one or more subjects with a disease or condition. In some embodiments, immunogenic peptides are specific to one or more subjects with a disease or condition. In some embodiments, immunogenic peptides can bind to an HLA that is matched to an HLA haplotype of one or more subjects with a disease or condition.
  • a person skilled in the art will be able to select neoantigenic therapeutics by testing, for example, the generation of T cells in vitro as well as their efficiency and overall presence, the proliferation, affinity and expansion of certain T cells for certain peptides, and the functionality of the T cells, e.g. by analyzing the IFN- ⁇ production or tumor killing by T cells. The most efficient peptides can then combined as an immunogenic composition.
  • the different neoantigenic peptides and/or polypeptides are selected so that one immunogenic composition comprises neoantigenic peptides and/or polypeptides capable of associating with different MHC molecules, such as different MHC class I molecule.
  • an immunogenic composition comprises neoantigenic peptides and/or polypeptides capable of associating with the most frequently occurring MHC class I molecules.
  • immunogenic compositions described herein comprise different peptides capable of associating with at least 2, at least 3, or at least 4 MHC class I or class II molecules.
  • an immunogenic composition described herein is capable of raising a specific cytotoxic T cells response, specific helper T cell response, or a B cell response.
  • an immunogenic composition described herein can further comprise an adjuvant and/or a carrier.
  • an adjuvant and/or a carrier examples of useful adjuvants and carriers are given herein below.
  • Polypeptides and/or polynucleotides in the composition can be associated with a carrier such as e.g. a protein or an antigen- presenting cell such as e.g. a dendritic cell (DC) capable of presenting the peptide to a T cell or a B cell.
  • DC-binding peptides are used as carriers to target the neoantigenic peptides and polynucleotides encoding the neoantigen peptides to dendritic cells (Sioud et al. FASEB J 27: 3272-3283 (2013)).
  • the neoantigenic polypeptides or polynucleotides can be provided as antigen presenting cells (e.g., dendritic cells) containing such polypeptides or polynucleotides.
  • antigen presenting cells e.g., dendritic cells
  • such antigen presenting cells are used to stimulate T cells for use in patients.
  • the antigen presenting cells are dendritic cells.
  • the dendritic cells are autologous dendritic cells that are pulsed with the neoantigenic peptide or nucleic acid.
  • the neoantigenic peptide can be any suitable peptide that gives rise to an appropriate T cell response.
  • T cell therapy using autologous dendritic cells pulsed with peptides from a tumor associated antigen is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278.
  • the T cell is a CTL.
  • the T cell is a HTL.
  • an immunogenic composition containing at least one antigen presenting cell e.g., a dendritic cell
  • at least one antigen presenting cell e.g., a dendritic cell
  • APCs are autologous (e.g., autologous dendritic cells).
  • PBMCs peripheral blood mononuclear cells isolated from a patient can be loaded with neoantigenic peptides or polynucleotides ex vivo.
  • APCs or PBMCs are injected back into the patient.
  • the polynucleotide can be any suitable polynucleotide that is capable of transducing the dendritic cell, thus resulting in the presentation of a neoantigenic peptide and induction of immunity.
  • the polynucleotide can be naked DNA that is taken up by the cells by passive loading.
  • the polynucleotide is part of a delivery vehicle, for example, a liposome, virus like particle, plasmid, or expression vector.
  • the polynucleotide is delivered by a vector-free delivery system, for example, high performance electroporation and high-speed cell deformation).
  • such antigen presenting cells e.g., dendritic cells
  • PBMCs peripheral blood mononuclear cells
  • APCs antigen presenting cells
  • PBMCs peripheral blood mononuclear cells
  • T cell e.g., an autologous T cell
  • the T cell is a CTL.
  • the T cell is an HTL.
  • Such T cells are then injected into the patient.
  • CTL is injected into the patient.
  • HTL is injected into the patient.
  • both CTL and HTL are injected into the patient.
  • Administration of either therapeutic can be performed simultaneously or sequentially and in any order.
  • compositions described herein for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration.
  • the pharmaceutical compositions described herein are administered parenterally, e.g., intravenously,
  • compositions for parenteral administration which comprise a solution of the neoantigenic peptides and immunogenic compositions are dissolved or suspended in an acceptable carrier, for example, an aqueous carrier.
  • an aqueous carrier e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.
  • compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • compositions can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected by fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • neoantigenic peptides and polynucleotides described herein can also be administered via liposomes, which target the peptides to a particular cells tissue, such as lymphoid tissue.
  • Liposomes are also useful in increasing the half-life of the peptides. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the
  • liposomes filled with a desired peptide or polynucleotide described herein can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic polypeptide/polynucleotide compositions.
  • Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, for example, cholesterol.
  • the selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
  • a neoantigen polypeptides or polynucleotides to be incorporated into the liposome for cell surface determinants of the desired immune system cells For targeting to the immune cells, a neoantigen polypeptides or polynucleotides to be incorporated into the liposome for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide can be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the polypeptide or polynucleotide being delivered, and the stage of the disease being treated.
  • neoantigen polypeptides and polynucleotides are targeted to dendritic cells.
  • the neoantigen polypeptides and polynucleotides are target to dendritic cells using the markers DEC205, XCR1, CD197, CD80, CD86, CD 123, CD209, CD273, CD283, CD289, CD184, CD85h, CD85j, CD85k, CD85d, CD85g, CD85a, TSLP receptor, Clec9a or CDla.
  • nontoxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more neoantigenic polypeptides or polynucleotides described herein at a concentration of 25%-75%.
  • the neoantigenic polypeptides or polynucleotides can be supplied in finely divided form along with a surfactant and propellant.
  • a surfactant and propellant are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters, such as mixed or natural glycerides can be employed.
  • the surfactant can constitute 0. l%-20% by weight of the composition, or 0.25-5%.
  • the balance of the composition can be propellant.
  • a carrier can also be included as desired, as with, e.g., lecithin for intranasal delivery.
  • nucleic acid can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et al, Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466.
  • the nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles.
  • mRNA encoding the neoantigenic peptides, or peptide binding agents can also be administered to the patient.
  • the mRNA is self-amplifying RNA.
  • the self-amplifying RNA is a part of a synthetic lipid nanoparticle formulation (Geall et al., Proc Natl Acad Sci U S A. 109: 14604-14609 (2012)).
  • the nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids.
  • cationic compounds such as cationic lipids.
  • Lipid-mediated gene delivery methods are described, for instance, in WO 96/18372, WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).
  • neoantigenic peptides and polypeptides described herein can also be expressed by attenuated viruses, such as vaccinia or fowlpox.
  • vaccinia virus as a vector to express nucleotide sequences that encode the peptide described herein.
  • the recombinant vaccinia virus Upon introduction into an acutely or chronically infected host or into a noninfected host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL response.
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.
  • BCG Bacillus Calmette Guerin
  • BCG vectors are described in Stover et al. (Nature 351 :456-460 (1991)).
  • a wide variety of other vectors useful for therapeutic administration or immunization of the peptides described herein will be apparent to those skilled in the art from the description herein.
  • Adjuvants are any substance whose admixture into the immunogenic composition increases or otherwise modifies the immune response to the therapeutic agent.
  • Carriers are scaffold structures, for example a polypeptide or a polysaccharide, to which a neoantigenic polypeptide or polynucleotide, is capable of being associated.
  • adjuvants are conjugated covalently or non-covalently to the polypeptides or polynucleotides described herein.
  • an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune -mediated reaction, or reduction in disease symptoms.
  • an increase in humoral immunity can be manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T cell activity can be manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion.
  • An adjuvant can also alter an immune response, for example, by changing a primarily humoral or T helper 2 response into a primarily cellular, or T helper 1 response.
  • Suitable adjuvants include, but are not limited to poly(I:C), poly-ICLC, STING agonist, 1018 ISS, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS,
  • PLG microparticles PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Pam3CSK4, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox. Quil or Superfbs. Adjuvants also include incomplete Freund's or GM-CSF.
  • cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, PGE1, PGE2, IL-1, IL-lb, IL-4, IL-6 and CD40L) (U.S. Pat. No. 5,849,589 incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418).
  • CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides act by activating the innate (non- adaptive) immune system via Toll-like receptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell immunogenic pharmaceutical compositions, autologous cellular immunogenic pharmaceutical compositions and polysaccharide conjugates in both prophylactic and therapeutic immunogenic pharmaceutical compositions.
  • TLR Toll-like receptors
  • TH1 bias induced by TLR9 stimulation is maintained even in the presence of adjuvants such as alum or incomplete Freund's adjuvant (IF A) that normally promote a TH2 bias.
  • CpG oligonucleotides show even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nano particles, lipid emulsions or similar formulations, which are especially necessary for inducing a strong response when the antigen is relatively weak.
  • 6,406,705 B l describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and an antigen to induce an antigen-specific immune response.
  • a commercially available CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, GERMANY), which is a component of the pharmaceutical composition described herein.
  • Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 can also be used.
  • CpGs e.g. CpR, Idera
  • non-CpG bacterial DNA or RNA e.g. ssRNA40 for TLR8, as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which can act therapeutically and/or as an adjuvant.
  • CpGs e.g. CpR, Idera
  • non-CpG bacterial DNA or RNA e.g. polyi:
  • adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan without undue experimentation.
  • Additional adjuvants include colony- stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).
  • GM-CSF Granulocyte Macrophage Colony Stimulating Factor
  • an immunogenic composition according to the present invention can comprise more than one different adjuvants.
  • the invention encompasses a therapeutic composition comprising any adjuvant substance including any of the above or combinations thereof.
  • the neoantigenic therapeutic e.g., a humoral or cell-mediated immune response.
  • the immunogenic composition comprises neoantigen therapeutics (e.g., peptides, polynucleotides, TCR, CAR, cells containing TCR or CAR, dendritic cell containing polypeptide, dendritic cell containing polynucleotide, antibody, etc.) and the adjuvant can be administered separately in any appropriate sequence.
  • a carrier can be present independently of an adjuvant.
  • the function of a carrier can for example be to increase the molecular weight of in particular mutant in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life.
  • a carrier can aid presenting peptides to T cells.
  • the carrier can be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell.
  • a carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier comprises a human fibronection type III domain (Koide et al. Methods Enzymol. 2012;503 : 135-56).
  • the carrier must be a physiologically acceptable carrier acceptable to humans and safe.
  • tetanus toxoid and/or diptheria toxoid are suitable carriers
  • the carrier can be dextrans for example sepharose.
  • the polypeptides can be synthesized as multiply linked peptides as an alternative to coupling a polypeptide to a carrier to increase immunogenicity.
  • Such molecules are also known as multiple antigenic peptides (MAPS).
  • Neoantigens that induce an immune response can be used as a composition when combined with an acceptable carrier or excipient. Such compositions are useful for in vitro or in vivo analysis or for
  • compositions can include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.
  • compositions comprising a protein of interest, e.g. , a neoantigen described herein, can be prepared for storage by mixing the neoantigen having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Oslo, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
  • hexamethonium chloride benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e
  • Acceptable carriers are physiologically acceptable to the administered patient and retain the therapeutic properties of the compounds with/in which it is administered. Acceptable carriers and their formulations are generally described in, for example, Remington' pharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA 1990).
  • One exemplary carrier is physiological saline.
  • a pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compounds from the administration site of one organ, or portion of the body, to another organ, or portion of the body, or in an in vitro assay system. Acceptable carriers are compatible with the other ingredients of the formulation and not injurious to a subject to whom it is administered. Nor should an acceptable carrier alter the specific activity of the neoantigens.
  • compositions including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration.
  • compositions or pharmaceutical formulations therefore refer to a composition suitable for pharmaceutical use in a subject.
  • the pharmaceutical compositions and formulations include an amount of a neoantigen (or polynucleotide encoding a neoantigen) and a pharmaceutically or physiologically acceptable carrier.
  • Compositions can be formulated to be compatible with a particular route of administration (i.e., systemic or local).
  • routes of administration i.e., systemic or local.
  • compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • a composition further comprises an acceptable additive in order to improve the stability of the neoantigen in the composition and/or to control the release rate of the composition.
  • Acceptable additives do not alter the specific activity of the neoantigens.
  • Exemplary acceptable additives include, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof.
  • Acceptable additives can be combined with acceptable carriers and/or excipients such as dextrose.
  • exemplary acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to increase stability of the peptide and decrease gelling of the solution.
  • the surfactant can be added to the composition in an amount of 0.01% to 5% of the solution. Addition of such acceptable additives increases the stability and half-life of the composition in storage.
  • compositions for injection include aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal.
  • Isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition.
  • the resulting solutions can be packaged for use as is, or lyophilized; the lyophilized preparation can later be combined with a sterile solution prior to administration.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • Suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as needed.
  • Sterile injectable solutions can be prepared by incorporating an active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • compositions can be conventionally administered intravenously, such as by injection of a unit dose, for example.
  • an active ingredient can be in the form of a parenterally acceptable aqueous solution which is substantially pyrogen-free and has suitable pH, isotonicity and stability.
  • suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be included, as required.
  • compositions can be administered via aerosolization.
  • the composition is lyophilized, for example, to increase shelf-life in storage.
  • the compositions can be substantially free of pyrogens such that the composition will not cause an inflammatory reaction or an unsafe allergic reaction when administered to a human patient.
  • Testing compositions for pyrogens and preparing compositions substantially free of pyrogens are well understood to one or ordinary skill of the art and can be accomplished using commercially available kits.
  • Acceptable carriers can contain a compound that stabilizes, increases or delays absorption, or increases or delays clearance.
  • Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and/or buffers.
  • Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers.
  • the compound can be complexed with a composition to render it resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in an appropriately resistant carrier such as a liposome.
  • compositions can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusions sufficient to maintain concentrations in the blood are contemplated.
  • Peptide-based immunogenic pharmaceutical compositions can be formulated using any of the well- known techniques, carriers, and excipients as suitable and as understood in the art.
  • the polypeptides can be a cocktail of multiple polypeptides containing the same sequence, or a cocktail of multiple copies of different polypeptides.
  • the peptides can be modified, such as for example by lipidation, or attachment to a carrier protein. Lipidation can be the covalent attachment of a lipid group to a polypeptide. Lipidated peptides, or lipidated polypeptides, can stabilize structures and can enhance efficacy of the treatment.
  • Lipidation can be classified into several different types, such as N-myristoylation, palmitoylation, GPI-anchor addition, prenylation, and several additional types of modifications.
  • N-myristoylation is the covalent attachment of myristate, a C14 saturated acid, to a glycine residue.
  • Palmitoylation is thioester linkage of long-chain fatty acids (Cie) to cysteine residues.
  • GPI-anchor addition is glycosyl-phosphatidylinositol (GPI) linkage via amide bond.
  • Prenylation is the thioether linkage of an isoprenoid lipid (e.g.
  • Fatty acids for generating a lipidated peptides can include C 2 to C30 saturated, monounsaturated, or polyunsaturated fatty acyl groups.
  • Exemplary fatty acids can include palmitoyl, myristoyl, stearoyl and decanoyl groups.
  • a lipid moiety that has adjuvant property is attached to a polypeptide of interest to elicit or enhance immunogenicity in the absence of an extrinsic adjuvant.
  • a lipidated peptide or lipopeptide can be referred to as a self-adjuvant lipopeptide.
  • Any of the fatty acids described above and elsewhere herein can elicit or enhance immunogenicity of a polypeptide of interest.
  • a fatty acid that can elicit or enhance immunogenicity can include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, and decanoyl groups.
  • Polypeptides such as naked peptides or lipidated peptides can be incorporated into a liposome.
  • lipidated peptides can be incorporated into a liposome.
  • the lipid portion of the lipidated peptide can spontaneously integrate into the lipid bilayer of a liposome.
  • a lipopeptide can be presented on the "surface" of a liposome.
  • Exemplary liposomes suitable for incorporation in the formulations include, and are not limited to, multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium-sized unilamellar vesicles (MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MVV), single or oligolamellar vesicles made by reverse- phase evaporation method (REV), multilamellar vesicles made by the reverse-phase evaporation method (MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods (VET), vesicles prepared by French press (FPV
  • liposomes can be unilamellar or multilamellar, and can vary in size with diameters ranging from about 0.02 um to greater than about 10 ⁇ . Liposomes can adsorb many types of cells and then release an incorporated agent (e.g., a peptide described herein). In some cases, the liposomes fuse with the target cell, whereby the contents of the liposome then empty into the target cell. A liposome can be endocytosed by cells that are phagocytic. Endocytosis can be followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents.
  • an incorporated agent e.g., a peptide described herein
  • the liposomes provided herein can also comprise carrier lipids.
  • the carrier lipids are phospholipids.
  • Carrier lipids capable of forming liposomes include, but are not limited to dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS).
  • DPPC dipalmitoylphosphatidylcholine
  • PC phosphatidylcholine
  • PG phosphatidylglycerol
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • Suitable phospholipids further include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidyglycerol (DPPG), distearoylphosphatidyglycerol (DSPG),
  • DSPC distearoylphosphatidylcholine
  • DMPC dimyristoylphosphatidylcholine
  • DPPG dipalmitoylphosphatidyglycerol
  • DSPG distearoylphosphatidyglycerol
  • dimyristoylphosphatidylglycerol dipalmitoylphosphatidic acid (DPP A); dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS),
  • DMPS dimyristoylphosphatidylserine
  • DSPS distearoylphosphatidylserine
  • DPPE dipalmitoylphosphatidyethanolamine
  • DMPE dimyristoylphosphatidylethanolamine
  • the liposomes further comprise a sterol (e.g., cholesterol) which modulates liposome formation.
  • a sterol e.g., cholesterol
  • the carrier lipids can be any known non-phosphate polar lipids.
  • a pharmaceutical composition can be encapsulated within liposomes using well-known technology.
  • Biodegradable microspheres can also be employed as carriers for the pharmaceutical compositions of this invention.
  • composition can be administered in liposomes or microspheres (or
  • microparticles Methods for preparing liposomes and microspheres for administration to a patient are well known to those of skill in the art. Essentially, material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.
  • Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the blood stream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time ranging from days to months.
  • a polypeptide can also be attached to a carrier protein for delivery.
  • the carrier protein can be an immunogenic carrier element and can be attached by any recombinant technology.
  • Exemplary carrier proteins include Mariculture keyhole limpet hemocyanin (mcKLH), PEGylated mcKLH, Blue Carrier* Proteins, bovine serum albumin (BSA), cationized BSA, ovalbumin, and bacterial proteins such as tetanus toxoid (TT).
  • mcKLH Mariculture keyhole limpet hemocyanin
  • PEGylated mcKLH Blue Carrier* Proteins
  • BSA bovine serum albumin
  • ovalbumin ovalbumin
  • TT tetanus toxoid
  • a polypeptide can also be prepared as multiple antigenic peptides (MAPs). Peptides may be attached at the N-terminus or the C-terminus to small non-immunogenic cores.
  • the core can be a dendritic core residue or matrix composed of bifunctional units.
  • Suitable core molecules for constructing MAPs can include ammonia, ethylenediamine, aspartic acid, glutamic acid, and lysine.
  • a lysine core molecule can be attached via peptide bonds through each of its amino groups to two additional lysines.
  • a polypeptide can be chemically synthesized, or recombinantly expressed in a cell system or a cell- free system.
  • a peptide can be synthesized, such as by a liquid-phase synthesis, a solid-phase synthesis, or by microwave assisted peptide synthesis.
  • a polypeptide can be modified, such as for example, by acylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation, glycosylation, malonylation, hydroxylation, iodination, nucleotide addition (e.g.
  • ADP-ribosylation oxidation, phosphorylation, adenylylation, propionylation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, gly cation, palmitoylation, myristoylation, isoprenylation or prenylation (e.g. farnesylation or
  • geranylgeranylation glypiation, lipoylation, attachement of flavin moiety (e.g. FMN or FAD), attachment of heme C, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol attachment, hypusine formuation, biotinylation, pegylation, ISGylation, SUMUylation, ubiquitination, Neddylation, Pupylation, citrullination, deamidation, eliminylation, carbamylation, or a combination thereof.
  • flavin moiety e.g. FMN or FAD
  • the polypeptide can be subjected to one or more rounds of purification steps to remove impurities.
  • the purification step can be a chromatographic step utilizing separation methods such as affinity-based, size -exclusion based, ion-exchange based, or the like.
  • the polypeptide is at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities.
  • the polypeptide is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100% pure or without the presence of impurities.
  • a polypeptide can include natural amino acids, unnatural amino acids, or a combination thereof.
  • An amino acid residue can refer to a molecule containing both an amino group and a carboxyl group.
  • Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes.
  • the term amino acid, as used herein, includes, without limitation, a-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.
  • a-amino acid can refer to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the a-carbon.
  • ⁇ -amino acid can refer to a molecule containing both an amino group and a carboxyl group in a ⁇ configuration.
  • Naturally occurring amino acid can refer to any one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
  • a table showing a summary of the properties of natural amino acids can be found, e.g., in U.S. Patent Application Publication No. 20130123169, which is herein incorporated by reference.
  • a peptide provided herein can comprise oneor more hydrophobic, polar, or charged amino acids.
  • Hydrophobic amino acids include small hydrophobic amino acids and large hydrophobic amino acids.
  • Small hydrophobic amino acid can be glycine, alanine, proline, and analogs thereof.
  • Large hydrophobic amino acids can be valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof.
  • Poly amino acids can be serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof.
  • Chargeged amino acids can be lysine, arginine, histidine, aspartate, glutamate, and analogs thereof.
  • a peptide provided herein can comprise oneor more amino acid analogs.
  • An "amino acid analog” can be a molecule which is structurally similar to an amino acid and which can be substituted for an amino acid in the formation of a peptidomimetic macrocycle
  • Amino acid analogs include, without limitation, ⁇ -amino acids and amino acids where the amino or carboxy group is substituted by a similarly reactive group (e.g., substitution of the primary amine with a secondary or tertiary amine, or substitution of the carboxy group with an ester).
  • a peptide provided herein can comprises one or more non-natural amino acids.
  • a "non-natural amino acid” can be an amino acid which is not one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V.
  • Non-natural amino acids or amino acid analogs include structures disclosed, e.g., in U.S. Patent Application Publication No. 20130123169, which is herein incorporated by reference.
  • Amino acid analogs can include ⁇ -amino acid analogs.
  • ⁇ -amino acid analogs and analogs of alanine, valine, glycine, leucine, arginine, lysine, aspartic acids, glutamic acids, cysteine, methionine, phenylalanine, tyrosine, proline, serine, threonine, and tryptophan can include structures disclosed, e.g., in U.S. Patent Application Publication No. 20130123169, which is herein incorporated by reference.
  • Amino acid analogs can be racemic.
  • the D isomer of the amino acid analog is used.
  • the L isomer of the amino acid analog is used.
  • the amino acid analog comprises chiral centers that are in the R or S configuration.
  • the amino group(s) of a ⁇ -amino acid analog is substituted with a protecting group, e.g., tert-butyloxycarbonyl (BOC group), 9- fluorenylmethyloxycarbonyl (FMOC), tosyl, and the like.
  • the carboxylic acid functional group of a ⁇ -amino acid analog is protected, e.g., as its ester derivative.
  • the salt of the amino acid analog is used.
  • a "non-essential” amino acid residue can be a residue that can be altered from the wild-type sequence of a polypeptide without abolishing or substantially altering its essential biological or biochemical activity (e.g., receptor binding or activation).
  • An "essential” amino acid residue can be a residue that, when altered from the wild-type sequence of the polypeptide, results in abolishing or substantially abolishing the polypeptide's essential biological or biochemical activity.
  • a "conservative amino acid substitution” can be one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • These families can include amino acids with basic side chains (e.g., K, R, H), acidic side chains (e.g., D, E), uncharged polar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains (e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V, I) and aromatic side chains (e.g., Y, F, W, H).
  • a predicted nonessential amino acid residue in a polypeptide for example, can be replaced with another amino acid residue from the same side chain family.
  • Other examples of acceptable substitutions can be substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2- thienylalanine for phenylalanine, or 6-Cl-tryptophan for tryptophan).
  • Nucleic acid -based immunogenic pharmaceutical compositions can also be administered to a subject.
  • Nucleic acid -based immunogenic pharmaceutical compositions can be formulated using any of the well- known techniques, carriers, and excipients as suitable and as understood in the art.
  • the nucleic acid can be DNA, genomic DNA or cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.
  • the immunogenic pharmaceutical composition can be a DNA-based immunogenic pharmaceutical composition, an RNA-based immunogenic pharmaceutical composition, a hybrid DNA/RNA based immunogenic pharmaceutical composition, or a hybrid nucleic acid/peptide based immunogenic pharmaceutical composition.
  • the peptide can be a peptide derived from a peptide in Table 1 or 2, a peptide that has a sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in sequence homology to a peptide in Table 1 or 2, or a peptide that has a sequence that is at most 40%, 50%, 60%, 70%, 80%, 90%, 95%, or less in sequence homology to a peptide in Table 1 or 2.
  • a nucleic acid described herein can contain phosphodiester bonds, although in some cases, as outlined below (for example in the construction of primers and probes such as label probes), nucleic acid analogs are included that can have alternate backbones, comprising, for example, phosphoramide, phosphorothioate, O- methylphosphoroamidite linkages, and peptide nucleic acid backbones and linkages.
  • Other analog nucleic acids include those with bicyclic structures including locked nucleic acids, positive backbones and non-ribose backbones. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids.

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RS20250878A RS67172B1 (sr) 2016-03-31 2017-03-31 Neoantigeni i postupci za njihovu upotrebu
KR1020227041564A KR20220163523A (ko) 2016-03-31 2017-03-31 신생항원 및 이것의 사용 방법
CR20180519A CR20180519A (es) 2016-03-31 2017-03-31 Neoantígenos y métodos de su uso
US16/094,851 US20190307868A1 (en) 2016-03-31 2017-03-31 Neoantigens and methods of their use
PL17776811.6T PL3436048T3 (pl) 2016-03-31 2017-03-31 Neoantygeny i sposoby ich zastosowania
EP17776811.6A EP3436048B1 (en) 2016-03-31 2017-03-31 Neoantigens and methods of their use
SG11201808196UA SG11201808196UA (en) 2016-03-31 2017-03-31 Neoantigens and methods of their use
CA3018748A CA3018748A1 (en) 2016-03-31 2017-03-31 Neoantigens and methods of their use
FIEP17776811.6T FI3436048T3 (fi) 2016-03-31 2017-03-31 Neoantigeeneja ja niiden käyttömenetelmiä
RU2018138163A RU2773273C2 (ru) 2016-03-31 2017-03-31 Неоантигены и способы их использования
BR112018070183-1A BR112018070183A2 (pt) 2016-03-31 2017-03-31 neoantígenos e métodos de seu uso
DK17776811.6T DK3436048T3 (da) 2016-03-31 2017-03-31 Neoantigener og fremgangsmåder til anvendelse deraf
ES17776811T ES3036912T3 (en) 2016-03-31 2017-03-31 Neoantigens and methods of their use
CN202211177112.5A CN115558030A (zh) 2016-03-31 2017-03-31 新抗原及其使用方法
CN201780034080.4A CN109310739A (zh) 2016-03-31 2017-03-31 新抗原及其使用方法
JP2018551996A JP2019513373A (ja) 2016-03-31 2017-03-31 ネオ抗原およびその使用方法
SI201731623T SI3436048T1 (sl) 2016-03-31 2017-03-31 Neoantigeni in načini uporabe
AU2017240745A AU2017240745B2 (en) 2016-03-31 2017-03-31 Neoantigens and methods of their use
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IL261880A IL261880B (en) 2016-03-31 2018-09-20 Neoantigens and methods for using them
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CONC2018/0011668A CO2018011668A2 (es) 2016-03-31 2018-10-29 Neoantígenos y métodos de su uso
AU2021269272A AU2021269272B2 (en) 2016-03-31 2021-11-15 Neoantigens and methods of their use
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JP2022088325A JP7491965B2 (ja) 2016-03-31 2022-05-31 ネオ抗原およびその使用方法
IL299926A IL299926B1 (en) 2016-03-31 2023-01-16 Neoantigens and methods for using them
US18/311,158 US20230414735A1 (en) 2016-03-31 2023-05-02 Neoantigens and methods of their use
US18/311,155 US20230405102A1 (en) 2016-03-31 2023-05-02 Neoantigens and methods of their use
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