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WO2024216241A2 - Vaccins hétéroclites à néoépitopes - Google Patents

Vaccins hétéroclites à néoépitopes Download PDF

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Publication number
WO2024216241A2
WO2024216241A2 PCT/US2024/024542 US2024024542W WO2024216241A2 WO 2024216241 A2 WO2024216241 A2 WO 2024216241A2 US 2024024542 W US2024024542 W US 2024024542W WO 2024216241 A2 WO2024216241 A2 WO 2024216241A2
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WIPO (PCT)
Prior art keywords
hla
mer
peptide
seq
amino acid
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PCT/US2024/024542
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WO2024216241A3 (fr
Inventor
Amanda Huff
Elizabeth Jaffee
Neeha ZAIDI
Mark Yarchoan
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Johns Hopkins University
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Johns Hopkins University
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Publication of WO2024216241A3 publication Critical patent/WO2024216241A3/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001164—GTPases, e.g. Ras or Rho
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/20—Supervised data analysis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/852—Pancreas
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
    • G16B35/20—Screening of libraries

Definitions

  • HLA human leukocyte antigen
  • Modifications to the peptide amino acid sequence can improve HLA binding or T cell recognition while conserving reactivity to the target neoepitope, thereby enhancing activation of neoantigen-specific T cells.
  • modified epitopes which improve antigen immunogenicity, are termed heteroclitic epitopes or altered peptide ligands (APLs).
  • APLs altered peptide ligands
  • a method of identifying heteroclitic neoepitopes comprises modeling peptides onto human leukocyte antigens (HLA), wherein the modeling comprises identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes; selecting an HLA-peptide structure having a sequence identity greater than a control HLA-peptide structure; modeling the heteroclitic neoepitopes into an HLA cleft by introducing amino acid rotamers with the lowest energy confirmation at each position; validating the resulting HLA-peptide neoepitope complexes using a root-mean-square deviation analysis (C ⁇ -RMSD); performing a high-resolution Monte Carlo simulation with minimization docking of the peptide to the HLA cleft to identify the lowest full- atom energy conformation; and, identifying heteroclitic neoepitopes.
  • HLA human leukocyte antigens
  • the modeling of the library of peptides onto human leukocyte antigens comprises aligning peptide sequences with a consensus binding motif of HLA molecules.
  • the consensus binding motif for 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17- mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes comprises distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors.
  • the root-mean-square deviation analysis is conducted to identify any unstable structural changes of an HLA cleft.
  • anchor energetic-optimized structures are selected to identify anchor residue modifications.
  • peptides are generated comprising combinations of anchor residue amino acid for each HLA class and subgroups thereof.
  • the HLA-types comprise class I, II and subgroups thereof.
  • the peptides are derived from tumor cells.
  • the peptides comprise tumor antigens.
  • a heteroclitic epitope vaccine candidate is selected based on the assessment of one or more of contacts between the peptide and HLA cleft residues, solvent-assessable surface area (SASA), peptide rigidity as a measure of C ⁇ RMSD of the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, electrostatic potential and/or induction of the greatest T cell expansion and activation as compared to parental peptides.
  • SASA solvent-assessable surface area
  • peptide rigidity as a measure of C ⁇ RMSD of the top 10 predicted structural models for each peptide-HLA complex
  • surface hydrophobicity surface hydrophobicity
  • electrostatic potential and/or induction of the greatest T cell expansion and activation as compared to parental peptides.
  • the method comprises identifying a sequence having high homology to the epitope of a parental sequence. In some embodiments, the method comprises introducing amino acid rotamers with the lowest energy confirmation to the sequence having high homology to the epitope of the parental sequence. In some embodiments, the method further comprises quantifying differences in structural features the parental sequence and the sequence having high homology to the epitope of the parental sequence. In some embodiments, the feature comprises contacts between the peptide and HLA cleft residues, solvent-assessable surface area (SASA), peptide rigidity as a measure of C ⁇ RMSD of the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, and/or electrostatic potential.
  • SASA solvent-assessable surface area
  • the method further comprises in vitro co-culturing of the sequence having high homology with HLA-matched lymphocytes to validate which 2 157361737 heteroclitic peptides induce the greatest T cell expansion and activation as compared to parental peptides, as assessed by IFN-gamma ELISpot assay.
  • the method further comprises selecting/identifying the heteroclitic peptide.
  • the heteroclitic peptide comprises at least a 75%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to any one or more of SEQ ID NOs: 1-838.
  • the heteroclitic peptide comprises at least a 75%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to any one or more of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107- 111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170- 179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254- 260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334- 340, 342-348, 350-356
  • the heteroclitic peptide comprise any one or more of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148- 152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232- 236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310- 316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390- 396, 398
  • the heteroclitic peptide comprises an amino acid sequence having at least 95% sequence identity to 3 157361737 any one or more of SEQ ID NOs: 1-838 having one or more conservative substitutions.
  • the heteroclitic peptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 833, or SEQ ID NO: 834.
  • the peptides comprise peptides isolated from tumor cells. In certain embodiments, the peptides comprise tumor antigens. In certain embodiments, the tumor antigen is wherein the tumor antigen is a Kirsten rat sarcoma viral (KRAS) tumor antigen. In certain embodiments, the KRAS tumor antigen comprises one or more mutations. In certain embodiments, the KRAS peptide sequence is positionally aligned with 8-mer, 9-mer, 11-mer, 12- mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes that bind HLA molecules.
  • KRAS Kirsten rat sarcoma viral
  • the root-mean-square deviation analysis is conducted to identify any unstable structural changes of an HLA cleft.
  • anchor energetic-optimized HLA-KRAS structures are selected to identify anchor residue modifications.
  • a library of KRAS peptides is generated comprising combinations of anchor residue amino acid for each HLA class and subgroups thereof.
  • the HLA-types comprise class I, II and subgroups thereof.
  • the KRAS peptides comprise heteroclitic epitopes.
  • an immunogenic peptide is selected based on the assessment of one or more of contacts between the peptide and HLA cleft residues, solvent-assessable surface area (SASA), peptide rigidity as a measure of C ⁇ RMSD of the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, electrostatic potential and/or induction of the greatest T cell expansion and activation as compared to parental peptides.
  • SASA solvent-assessable surface area
  • the heteroclitic peptide sequence is positionally aligned with epitopes that bind HLA molecules.
  • the epitopes are 8-mer epitopes, 9-mer epitopes, 10-mer epitopes, 11-mer epitopes, 12-mer epitopes, 13-mer epitopes, 14-mer epitopes, 15-mer epitopes, 16-mer epitopes, 17-mer epitopes, 18-mer epitopes, 19-mer epitopes, 20-mer epitopes, 21-mer epitopes, 22-mer epitopes, 23-mer epitopes, 24-mer epitopes, or 25-mer epitopes.
  • a method of treating cancer comprises administering to subject in need thereof, a therapeutically effective amount of one or more peptides comprising one or more heteroclitic epitopes.
  • a cancer vaccine comprises one or more peptides, wherein the peptides comprise at least one heteroclitic neoepitope.
  • the peptide is a tumor antigen.
  • the tumor antigen comprises a Kirsten rat sarcoma viral (KRAS) tumor antigen.
  • the KRAS tumor antigen comprises one or more mutations.
  • a method of treating cancer in a subject diagnosed with cancer comprises isolating cells from a biological sample subject; culturing the isolated cells with one or more peptides generated by the methods embodied herein, isolating T cells, NK cells, and/or antigen presenting cells cultured with the one or more peptides and expanding the T cells, NK cells, and/or antigen presenting cells to produce a therapeutically effective composition of tumor antigen specific T cells, NK cells, and/or antigen presenting cells; adoptively transferring the tumor antigen specific T cells, NK cells, and/or antigen presenting cells into the subject, thereby treating the subject diagnosed with cancer.
  • an immunogenic peptide is selected based on the assessment of one or more of contacts between the peptide and HLA cleft residues, solvent-assessable surface area (SASA), peptide rigidity as a measure of C ⁇ RMSD of the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, electrostatic potential and/or induction of the greatest T cell expansion and activation as compared to parental peptides.
  • SASA solvent-assessable surface area
  • D-Ala the “D” stereoisomer of alanine is represented as D-Ala.
  • Standard nomenclature is used for the bases of DNA, with cytosine, guanosine, adenine, and thymine indicated as “C”, “G”, “A”, and “T”, and codons that encode DNA follow the standard genetic code, for example the amino acid Leu is encoded by TTA, TTG, CTT, CTC, CTA or CTG, and Asp is encoded by GAT or GAC.
  • the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
  • the term can mean within an order of magnitude, within 5-fold, and also within 2-fold, of a value.
  • the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • All 7 157361737 numeric values are herein assumed to be modified by the term “about”, whether or not explicitly indicated.
  • the recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).
  • an “adjuvant” refers to a substance that enhances the body's immune response to an antigen or a vaccine and may be added to the formulation that includes the immunizing agent. Adjuvants provide enhanced immune response even after administration of only a single dose of the vaccine.
  • Adjuvants may include, for example, aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), non-metabolizable oil, mineral and/or plant/vegetable and/or animal oils, polymers, carbomers, surfactants, natural organic compounds, plant extracts, carbohydrates, cholesterol, lipids, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, HRA-3 (acrylic acid saccharide cross-linked polymer), HRA-3 with cottonseed oil (CSO), or an acrylic acid polyol cross-linked polymer.
  • saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), non-metabol
  • the emulsion can be based in particular on light liquid paraffin oil (European Pharmacopeia type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters.
  • light liquid paraffin oil European Pharmacopeia type
  • isoprenoid oil such as squalane or squalene
  • oil resulting from the oligomerization of alkenes in particular of isobutene or decene
  • the oil is used in combination with emulsifiers to form the emulsion.
  • the emulsifiers comprise nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the PLURONIC TM brand products, especially L121.
  • mannide e.g. anhydromannitol oleate
  • glycol of polyglycerol
  • propylene glycol and of oleic isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated
  • polyoxypropylene-polyoxyethylene copolymer blocks in particular the PLURON
  • the adjuvant is at a concentration of about 0.01 to about 50%, at a concentration of about 2% to 30%, at a concentration of about 5% to about 25%, at a concentration of about 7% to about 22%, and at a concentration of about 10% to about 20% by volume of the final product.
  • Adjuvanted refers to a composition that incorporates or is combined with an adjuvant.
  • the term “and/or” may also 8 157361737 occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items.
  • HLA-peptide structure refers to a complex where a peptide is bound to one or a plurality of HLA-haplotypes.
  • amino acid refers to naturally occurring and synthetic ⁇ , ⁇ , ⁇ , and ⁇ amino acids, and includes but is not limited to, amino acids found in proteins, i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine.
  • proteins i.e. glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine.
  • the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, ⁇ -alanyl, ⁇ -valinyl, ⁇ -leucinyl, ⁇ -isoleucinyl, ⁇ -prolinyl, ⁇ - phenylalaninyl, ⁇ -tryptophanyl, ⁇ -methioninyl, ⁇ -glycinyl, ⁇ -serinyl, ⁇ -threoninyl, ⁇ -cysteiny
  • the amino acids can be non-naturally occurring amino acids.
  • non-naturally occurring amino acids include, but are not limited to, D-amino acids (i.e. an amino acid of an opposite chirality to the naturally-occurring form), N- ⁇ -methyl amino acids, C- ⁇ -methyl amino acids, ⁇ -methyl amino acids and D- or L- ⁇ -amino acids.
  • Non-naturally occurring amino acids include, for example, ⁇ -alanine ( ⁇ -Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid ( ⁇ -Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic acid ( ⁇ -Ahx), ornithine (orn), sarcosine, ⁇ -amino isobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3-diaP), D- or L-phenylglycine, D-(trifluoromethyl)-phenylalanine, and D-p- fluorophenylalanine.
  • amino acid When the term amino acid is used, it is considered to be a specific and 9 157361737 independent disclosure of each of the esters of ⁇ , ⁇ , ⁇ , and ⁇ glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine in the D and L- configurations.
  • cancer as used herein is meant, a disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art; including colorectal cancer, as well as, for example, leukemias, e.g., acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS related cancers such as Kaposi’s sarcoma; breast cancers; bone cancers such as Osteosarcoma, Chondrosarcomas, Ewing’s sarcoma, Fibrosarcomas, Giant cell tumors, Adamantinomas, and Chordomas; Brain cancers such as Meningiomas, Glioblastomas, Lower- Grade Astrocytomas, Oligodendrocytomas, Pituitary Tumors, Schwannomas, Primary CNS Lymphoma, and Meta
  • combination therapy refers to those situations in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents.
  • two or more different agents may be administered simultaneously or separately.
  • This administration in combination can include simultaneous administration of the two or more agents in the same dosage 10 157361737 form, simultaneous administration in separate dosage forms, and separate administration. That is, two or more agents can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more agents can be simultaneously administered, wherein the agents are present in separate formulations. In another alternative, a first agent can be administered just followed by one or more additional agents.
  • two or more agents may be administered a few minutes apart, or a few hours apart, or a few days apart.
  • Treatment of a subject also includes a variety of combination therapies with both physical, e.g. surgery, and radiation based treatments.
  • the transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.”
  • the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim.
  • Diagnostic or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The “sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of “true positives”).
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • heteroclitic epitope or “heteroclitic analog” or “heteroclitic neoepitope” or “altered peptide ligand (APL)” are used interchangeably herein, and refers to an altered version of an 11 157361737 endogenous peptide sequence (i.e., an analog) engineered to elicit potent immune reactions.
  • Heteroclitic epitopes have increased stimulatory capacity or potency for a specific T cell, as measured by increased responses to a given dose, or by a requirement of lesser amounts to achieve the same response and therefore provide benefit as vaccine components since these epitopes induce T cell responses stronger than those induced by the native epitope.
  • the potent immune reactions improved HLA binding. In some embodiments, the potent immune reactions comprises improved T cell recognition. In some embodiments, the heteroclitic neoepitopes are derived from tumor antigens or cancer neoantigen.
  • An “immunogenic peptide” or “antigenic peptide” are used interchangeably herein, and refers to a peptide or epitope that can be recognized by the immune system and elicit an immune response. Immunogenic peptides or antigenic peptides may comprise a motif such that the peptide will bind an MHC molecule and induce a T cell response, or can be recognized by the B cell receptor on the B cell to induce antibody production.
  • an “immunogenic epitope” or “antigenic epitope” are used interchangeably herein, and refers to a part of an antigen is recognized by the immune system, e.g., by antibodies, B cells, or T cells.
  • the epitope is the specific piece of the antigen to which an antibody binds.
  • epitopes are usually non-self proteins, sequences derived from the host can, in some instances, be recognized.
  • the term “immune cells” refers to any cells of the immune system that are involved in mediating an immune response.
  • Non-limiting examples of immune cells include a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, neutrophil, or combination thereof.
  • an immune cell expresses CD3.
  • the CD3-expressing immune cells are T cells (e.g., CD4 + T cells or CD8 + T cells).
  • an immune cell that can be targeted with a targeting moiety comprises naive CD4 + T cell.
  • an immune cell comprises a memory CD4 + T cell.
  • an immune cell comprises an effector CD4 + T cell.
  • an immune cell comprises a na ⁇ ve CD8 + T cell.
  • an immune cell comprises a memory CD8 + T cell. In some aspects, an immune cell comprises an effector CD8 + T cell. In some aspects, an immune cell comprises a gamma delta T cell. In some aspects, an immune cell is a dendritic cell. In certain aspects, a dendritic cell comprises a plasmacytoid dendritic cell (pDC), a conventional dendritic 12 157361737 cell 1 (cDC1), a conventional dendritic cell 2 (cDC2), inflammatory monocyte derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof.
  • pDC plasmacytoid dendritic cell
  • cDC1 conventional dendritic 12 157361737 cell 1
  • cDC2 conventional dendritic cell 2
  • inflammatory monocyte derived dendritic cells Langerhans cells
  • dermal dendritic cells
  • a “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell and are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intravitreal (i.v.i.), intra-cisterna magna (i.c.m.), or intrasternal injection, or infusion techniques.
  • patient or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • MHC molecules proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential T-cell epitopes, transporting them to the cell surface and presenting them there to specific cells, in particular cytotoxic T-lymphocytes or T-helper cells.
  • MHC molecules of class I consist of a heavy chain and a light chain and are capable of binding a peptide of about 8 to 13 157361737 11 amino acids, but usually 9 or 10 amino acids, if this peptide has suitable binding motifs, and presenting it to cytotoxic T-lymphocytes.
  • the peptide bound by the MHC molecules of class I originates from an endogenous protein antigen.
  • the heavy chain of the MHC molecules of class I is preferably an HLA-A, HLA-B or HLA-C monomer, and the light chain is ⁇ -2-microglobulin ( ⁇ 2M).
  • ⁇ 2M ⁇ -2-microglobulin
  • sample encompass a variety of sample types obtained from a patient, individual, or subject and can be used in a diagnostic, prognostic and/or monitoring assay.
  • the patient sample may be obtained from a healthy subject, a diseased patient, or a patient with lung cancer.
  • a sample that is “provided” can be obtained by the person (or machine) conducting the assay, or it can have been obtained by another, and transferred to the person (or machine) carrying out the assay.
  • a sample obtained from a patient can be divided and only a portion may be used for diagnosis. Further, the sample, or a portion thereof, can be stored under conditions to maintain sample for later analysis.
  • a sample comprises cerebrospinal fluid.
  • a sample comprises a blood sample.
  • a sample comprises a plasma sample.
  • a serum sample is used.
  • sample also includes samples that have been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations.
  • the terms further encompass a clinical sample, and also include cells in culture, cell supernatants, tissue samples, organs, and the like. Samples may also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by immunohistochemistry.
  • Treat,” “treated,” “treating,” “treatment,” and the like are meant to refer to reducing or ameliorating a disorder and/or symptoms associated therewith (e.g., a neoplasia or tumor).
  • Treating may refer to administration of the therapy to a subject after the onset, or suspected onset, of a cancer.
  • Treating includes the concepts of “alleviating”, which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a cancer and/or the side effects associated with cancer therapy.
  • treating also encompasses the concept of “managing” which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.
  • therapeutic effect refers to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasia or tumor) or its associated pathology.
  • “Therapeutically effective amount” refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. “Therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” (e.g., ED 50 ) of the pharmaceutical composition required.
  • transfected or transformed or transduced means to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the transfected/transformed/transduced cell includes the primary subject cell and its progeny.
  • an “unnatural amino acid,” “non-natural amino acid”, “modified amino acid” or “chemically modified amino acid” refers to any amino acid, modified amino acid, or 15 157361737 amino acid analogue other than the twenty genetically encoded alpha-amino acids.
  • Unnatural amino acids have side chain groups that distinguish them from the natural amino acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha-amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural amino acids may have an extended backbone such as beta-amino acids.
  • Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAc ⁇ -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L- phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine,
  • one or more amino acids of the helicase are substituted with one or more unnatural amino acids and/or one or more natural amino acids.
  • variant refers to an amino acid sequence that is altered by one or more amino acid residues.
  • the variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have “nonconservative” changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable.
  • the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • the genes or gene products disclosed herein which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.
  • the genes, nucleic acid sequences, amino acid sequences, 17 157361737 peptides, polypeptides and proteins are human.
  • the term “gene” is also intended to include variants.
  • the practice of the present disclosure employs, unless otherwise indicated, techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989, Molecular Cloning, 2nd Ed.
  • FIG.1 is a series of Word plots of 9-mer peptide binding consensus sequences from each HLA allele. Data was accessed through HLAthena browser.
  • FIG.2 shows the NetMHC predicted binding affinity for parental KRAS G12D epitope (boxed) and all altered peptide ligand heteroclitic epitopes across the panel of HLAs. Gray asterisks indicate weak binding epitopes, Black asterisks indicate strong binders.
  • FIG. 3 is a series of schematic representations showing the structural modelling of the KRAS G12D epitope GADGVGKSA on HLA-A*02:01 . Rotamer substitution of anchor amino acid residues 9 from alanine to leucine, 2 from alanine to leucine, and 6 from glycine to leucine. Contacts are shown. HLA contact residues are shown. Black arrows indicate interactions with increased contacts.
  • FIG. 4A, FIG. 4B show a heatmap and a table and demonstrating the HLA-specific Mutant KRAS minimal epitope targets validated by mass-spec for heteroclitic peptide design.
  • FIG.4B shows a compiled list of mutant KRAS minimal epitopes and HLA restriction that have been confirmed by mass spectrometry.
  • FIG. 5A-FIG. 5C show a table, a heatmap and a plot demonstrating the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-A*02:01.
  • FIG.5A shows peptide binding consensus sequence for 10-mer (left) and 9-mer (right) epitopes for HLA-A*02:01.
  • FIG.5B shows heat map of predicted binding affinity (nM) of parental G12V 10-mer and 9-mer and APLs to HLA- A*02:01.
  • FIG. 5C shows a compiled list of mutant 10-mer and 9-mer KRAS minimal epitopes restricted to HLA-A*02:01 (parental) and modified altered peptide ligands (APL).
  • FIG. 6A-FIG. 6C show a table, a heatmap and a plot demonstrating the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-A*03:01.
  • FIG.6A shows peptide binding consensus sequence for 10-mer (left) and 9-mer (right) epitopes for HLA-A*03:01. Amino acids are shaded by physiochemical property.
  • FIG.6B shows heat map of predicted binding affinity (nM) of parental 10-mer (left- KRAS G12D, G12V, G12C) and 9- mer (right- KRAS G12R, G12V) and APLs to HLA-A*03:01.
  • X indicates location of KRAS mutation.
  • FIG. 7A- FIG. 7C show a table, a heatmap and a plot demonstrating the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-A*11:01.
  • FIG.7A shows peptide binding consensus sequence for 10-mer (left) and 9-mer (right) epitopes for HLA-A*11:01. Amino acids are shaded by physiochemical property.
  • FIG.7B shows heat map of predicted binding affinity (nM) of parental 10-mer (left- KRAS G12D, G12R, G12V, G12C) and 9-mer (right- KRAS G12D, G12R, G12V) and APLs to HLA-A*11:01.
  • X indicates location of KRAS mutation.
  • FIG.7C shows a compiled list of mutant 10-mer and 9-mer KRAS minimal epitopes restricted to HLA-A*11:01 (parental) and modified altered peptide ligands (APL). X indicates location of KRAS mutation.
  • FIG. 8C show a table, a heatmap and a plot demonstrating the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-A*30:01.
  • FIG.8A shows peptide binding consensus sequence for 10-mer (left)F and 9-mer (right) epitopes for HLA-A*30:01. Amino acids are shaded by physiochemical property.
  • FIG.8B shows heat map of predicted binding affinity (nM) of parental 10-mer (left- KRAS G12R, G12V) and 9-mer (right- KRAS G12R, G12V) and APLs to HLA-A*30:01. X indicates location of KRAS mutation.
  • FIG. 8C shows a table, a heatmap and a plot demonstrating the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-A*30:01.
  • FIG.8A shows peptide binding consensus sequence for 10-mer (left)F and 9-mer (
  • FIG. 9A-FIG. 9C show a table, a heatmap and a plot demonstrating the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-A*68:01.
  • FIG.9A shows peptide binding consensus sequence for 10-mer (left) and 9-mer (right) epitopes for HLA-A*68:01. Amino acids are shaded by physiochemical property.
  • FIG.9B shows heat map of predicted binding affinity (nM) of parental 10-mer (left- KRAS G12D G12C, G12R, G12V) and 9-mer (right- KRAS G12V) and APLs to HLA-A*68:01.
  • X indicates location of KRAS mutation.
  • FIG. 9C shows a compiled list of mutant 10-mer and 9-mer KRAS minimal epitopes restricted to HLA-A*68:01 (parental) and modified altered peptide ligands (APL). X indicates location of KRAS mutation.
  • FIG.10A- FIG.10C show a table, a heatmap and a plot demonstrating the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-B*07:02.
  • FIG. 10A shows peptide binding consensus sequence for 10-mer epitopes for HLA-B*07:02. Amino acids are colored by physiochemical property.
  • FIG. 10B shows heat map of predicted binding affinity (nM) of parental 10-mer KRAS G12D and G12R epitopes and APLs to HLA- B*07:02. X indicates location of KRAS mutation.
  • FIG.10C shows a compiled list of mutant 10- mer KRAS minimal epitopes restricted to HLA-B*07:02 (parental) and modified altered peptide ligands (APL).
  • X indicates location of KRAS mutation.
  • FIG. 11A-FIG. 11C show a table, a heatmap and a plot showing the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-C*01:02.
  • FIG. 11A shows peptide binding consensus sequence for 9-mer epitopes for HLA-C*01:02. Amino acids are colored by physiochemical property.
  • FIG. 11A-FIG. 11C show a table, a heatmap and a plot showing the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-C*01:02.
  • FIG. 11A shows peptide binding consensus sequence for 9-mer epitopes for HLA-C*01:02. Amino acids are colored by
  • FIG. 11B shows heat map of predicted binding affinity (nM) of parental 9-mer KRAS G12V epitopes and APLs to HLA-C*01:02.
  • FIG. 11C shows a compiled list of known mutant 10-mer KRAS minimal epitopes restricted to HLA- C*01:02 (parental) and modified altered peptide ligands (APL).
  • FIG. 12A-FIG. 12C show a table, a heatmap and a plot showing the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-C*08:02.
  • FIG.12A shows peptide binding consensus sequence for 10-mer (left) and 9-mer (right) epitopes for HLA-C*08:02.
  • FIG. 12B shows heat map of predicted binding affinity (nM) of parental 10-mer (left- KRAS G12D) and 9-mer (right- KRAS G12D) and APLs to HLA-C*08:02.
  • FIG.12C shows a compiled list of mutant 10-mer and 9-mer KRAS minimal epitopes restricted to HLA-C*08:02 (parental) and modified altered peptide ligands (APL).
  • FIG.13 show a table, a heatmap and a plot demonstrating the amino acid modifications of mKRAS epitopes predicted to improve HLA binding affinity for HLA-C*03:03.
  • FIG. 12B shows heat map of predicted binding affinity (nM) of parental 10-mer (left- KRAS G12D) and 9-mer (right- KRAS G12D) and APLs to HLA-C*08:02.
  • FIG.12C shows a compiled list of mutant 10-mer and 9-mer KRAS minimal epitopes restricted to HLA-C
  • FIG. 13A shows peptide binding consensus sequence for 10-mer (left) and 9-mer (right) epitopes for HLA- C*03:03. Amino acids are colored by physiochemical property.
  • FIG. 13B shows heat map of predicted binding affinity (nM) of parental 10-mer (left- KRAS G12V) and 9-mer (right- KRAS G12V) and APLs to HLA-C*03:03.
  • FIG.13C shows a compiled list of mutant 10-mer and 9-mer 21 157361737 KRAS minimal epitopes restricted to HLA-C*03:03 (parental) and modified altered peptide ligands (APL). [0064] FIG.
  • FIG.14A shows KRAS altered peptide ligand binding assay to HLA-A*11:01 tetramers - overview of tetramer binding assay.
  • HLA-A*11:01 tetramers were incubated with 10mM KRAS parental epitope or APL, 4hr, RT. Peptide loading was then quantified by flow cytometry analysis with anti-exiting peptide antibody.
  • FIG.14B shows peptide loading calculated as a percentage relative to (+) control peptide and no peptide (-) control.
  • FIG. 14C shows NetMHC predicted binding affinities for HLA-A*11:01- specific KRAS peptides and APLs tested with this assay.
  • FIG.15 show a series of graphs showing the results obtained from a KRAS APL HLA- A*11:01 binding assay. Percent binding of KRAS G12D, G12R, G12V, and G12C 10- and 9-mer parental epitopes and altered peptide ligands. Percent binding of each APL is calculated relative to the respective parental epitope.
  • FIG. 16D show a schematic and a series of graphs demonstrating KRAS G12D altered peptide ligands prime T cell responses against parental antigen in vitro.
  • An in vitro immunogenicity assay was conducted for KRAS G12D altered peptide ligands in an HLA- A*11:01 healthy donor.
  • FIG. 16A shows an overview of in vitro T cell priming and expansion assay. Healthy donor dendritic cells were generated by adherence of monocytes to flasks and differentiated in 800U/mL hGMCSF and 400IU/mL hIL-4 for 7 days. Dendritic cells were then matured in hIL-1B, IL-6, TNF ⁇ , and PGE2 for 2 days.
  • Mature dendritic cells were then collected and pulsed with 40 ⁇ g/mL parental or altered peptide ligand for 2hrs. Pulsed dendritic cells were washed twice with PBS and then co-cultured with autologous CD8 + T cells in the presence of hIL- 15, hIL-6, and hIL-21. 7 days later, more peptide-pulsed dendritic cells were added to each co- culture. The following day, fresh hIL2 and IL7 were added. 7 days post the final dendritic cell culture, T cells were collected and stained with an HLA-A*11:01 tetramer containing the parental epitope sequence.
  • FIG.16B shows flow cytometry gates of HLA-A*11:01 KRAS G12D 10-mer + T cells (gated on HLA-DR-CD45RO+ events).
  • FIG.16C shows quantified populations of HLA- A*11:01 G12D 10-mer tet + T cells in control, parental epitope, or altered peptide ligand 22 157361737 expansions.
  • FIG.16D shows KRAS G12D-HLA-A*11:01 tetramer staining of ATVGADGVGK expanded T cells 7 days after 3 rd peptide boost. No tetramer control was used as negative control. [0067] FIG.
  • FIG. 17 shows a schematic of KRAS G12D parental and altered peptide ligand- HLA contacts and surface structure. Structural modeling of KRAS G12D 10-mer parental epitope (G12D) or altered peptide ligands in HLA-A*11:01. Top row indicates contacts between the 10- mer peptide and HLA binding cleft. Bottom row shows surface area of peptide-HLA complex. Measurements are indicated in FIG 20. [0068]
  • FIG. 18 shows a schematic of KRAS G12D parental and altered peptide ligand- HLA hydrophobicity measurements. Hydrophobicity measurements of KRAS G12D parental and altered peptide ligands- HLA complexes. Surface lipophilicity values are mapped.
  • FIG. 19 shows a schematic of KRAS G12D parental and altered peptide ligand- Electrostatic potential measurements. Electrostatic potential measurements of KRAS G12D parental and altered peptide ligands- HLA complexes are shown. Surface electrostatic potential values are mapped [0070]
  • FIG. 20 shows a table of the structural feature measurements of KRAS G12D 10-mer parental epitope and altered peptide ligands modeled in the context of HLA-A*11:01.
  • FIG.21A- FIG.21F show a series of plots and schematics demonstrating that engineered peptides display enhanced contact points and solvent accessible surface area, and reduced rigidity in a murine PDAC model.
  • FIG. 21A- FIG.21F show a series of plots and schematics demonstrating that engineered peptides display enhanced contact points and solvent accessible surface area, and reduced rigidity in a murine PDAC model.
  • FIG.21A shows that mice were vaccinated twice 7 days apart with PBS, 50 ⁇ g APL, or 5 ⁇ g parental peptide. Seven days after the last vaccine dose, splenocytes were restimulated with APL or parental peptides, overnight, and IFN ⁇ production was measured by ELISpot. Two-way ANOVA followed by Tukey’s multiple comparisons test was performed.
  • FIG.21B shows in silico structural modeling of APL or parental peptide in murine H2-Kb (murine MHC class I molecule H2-Kb, minimal epitope of peptide 44, parental or engineered amino acid residue). Structural features analyzed from parental or APL MHC models. Number of contacts between MHC binding cleft and peptide (FIG.
  • FIG. 21C shows C ⁇ –RMSD measure of peptide rigidity.
  • FIG. 22A-FIG. 22C demonstrate that KRAS G12D APL1–HLA*11:01 binding and immunogenicity of engineered antigens show superior binding and T Cell Responses.
  • FIG.22A shows non–linear regression curves and calculated EC50s for binding between HLA-A*11:01 and a parental KRAS G12D 10–mer peptide and 7 engineered antigens.
  • FIG. 22C shows in vitro immunogenicity testing of HLA- A*11:01 parental epitope and its engineered counterpart (APL1).
  • Human monocyte–derived DCs (moDCs) differentiated from healthy donor PBMCs were pulsed with 100 ⁇ M parental or APL1 plus ⁇ 2–microglobulin (4 hours) at 37°C. After peptide pulsing, moDCs were washed with PBS and co–cultured with 6 ⁇ 10 6 autologous CD8 T cells (plus IL-2, IL-7, IL-15, and IL-21) (7 days). Boosts were performed with peptide–pulsed moDCs on day 7, 14, and 21.
  • FIG.23 shows a series of plots showing that KRAS G12D APL4 demonstrates greater T cell responses compared to parental epitope in a different HLA*11:01 human donor.
  • APL4 Human monocyte–derived DCs (moDCs) differentiated from healthy donor PBMCs were pulsed with 100 ⁇ M parental or APL4 plus ⁇ 2–microglobulin (4 hours) at 37°C.
  • FIG. 24A shows structural models of A11-KRAS G12D parental epitope (left), A11- KRAS G12D APL1, and A11- KRAS G12D APL4 in the context of HLA-A*11:01.
  • FIG. 24B shows structural analysis of peptide neoantigen residue (G12D) in contact with HLA 24 157361737 cleft residues that are associated with stabilization of peptide-HLA interactions (from left to right, Arg114, Gln155, Gln70, TRP147, Thr73).
  • FIG.25A shows binding affinity for KRAS G12D APLs in the context of HLA-A*03:01.
  • FIG.25B shows binding affinity for KRAS G12D APLs in the context of HLA-B*07:02 DETAILED DESCRIPTION
  • the disclosure provides herein KRAS G12D heteroclitic epitope vaccine candidates designed and modelled for diverse HLA subtypes. Additionally, the software described herein is applicable to private patient neoantigen targets where the algorithm can prioritize immunogenic neoantigen targets based on structural features of neoantigen display as well as identify optimal heteroclitic peptides for the patient’s HLA subtypes.
  • a heteroclitic peptide disclosed herein is selected based on the assessment of one or more of contacts between the peptide and HLA cleft residues, solvent-assessable surface area (SASA), peptide rigidity as a measure of C ⁇ RMSD of the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, electrostatic potential and/or induction of the greatest T cell expansion and activation as compared to parental peptides.
  • SASA solvent-assessable surface area
  • peptide rigidity as a measure of C ⁇ RMSD of the top 10 predicted structural models for each peptide-HLA complex
  • surface hydrophobicity surface hydrophobicity
  • electrostatic potential and/or induction of the greatest T cell expansion and activation as compared to parental peptides.
  • the innate immune system is the first line of defense against infections, and most potential pathogens are rapidly neutralized by this system before they can cause, for example, a noticeable infection.
  • the acquired immune system reacts to molecular structures, referred to as antigens, of the intruding organism.
  • antigens molecular structures
  • humoral immune reaction antibodies secreted by B cells into bodily fluids bind to pathogen- derived antigens, leading to the elimination of the pathogen through a variety of mechanisms, e.g. complement-mediated lysis.
  • T-cells capable of destroying other cells are activated.
  • MHC proteins are classified into two types, referred to as MHC class I and MHC class II. The structures of the proteins of the two MHC classes are very similar; however, they have very different functions.
  • MHC class I proteins Proteins of MHC class I are present on the surface of almost all cells of the body, including most tumor cells. MHC class I proteins are loaded with antigens that usually originate from endogenous proteins or from pathogens present inside cells and are then presented to naive or cytotoxic T-lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B-lymphocytes, macrophages and other antigen-presenting cells. They mainly present peptides, which are processed from external antigen sources, i.e. outside of the cells, to T-helper (Th) cells. Most of the peptides bound by the MHC class I proteins originate from cytoplasmic proteins produced in the healthy host cells of an organism itself, and do not normally stimulate an immune reaction.
  • CTLs cytotoxic T-lymphocytes
  • cytotoxic T- lymphocytes that recognize such self-peptide-presenting MHC molecules of class I are deleted in the thymus (central tolerance) or, after their release from the thymus, are deleted or inactivated, i.e. tolerized (peripheral tolerance).
  • MHC molecules are capable of stimulating an immune reaction when they present peptides to non-tolerized T-lymphocytes.
  • Cytotoxic T-lymphocytes have both T-cell receptors (TCR) and CD8 molecules on their surface.
  • T-Cell receptors are capable of recognizing and binding peptides complexed with the molecules of MHC class I.
  • Each cytotoxic T-lymphocyte expresses a unique T-cell receptor which is capable of binding specific MHC/peptide complexes.
  • the peptide antigens attach themselves to the molecules of MHC class I by competitive affinity binding within the endoplasmic reticulum, before they are presented on the cell surface.
  • the affinity of an individual peptide antigen is directly linked to its amino acid sequence and 26 157361737 the presence of specific binding motifs in defined positions within the amino acid sequence. If the sequence of such a peptide is known, it is possible to manipulate the immune system against diseased cells using, for example, peptide vaccines.
  • the human leukocyte antigen (HLA) system is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans.
  • the present disclosure provides methods for predicting peptides capable of binding to HLA alleles.
  • the embodiments comprise a set of candidate peptide sequences and identifying one or more structural features indicative of occupancy of the candidate peptides on the binding pocket or cleft of HLA alleles. This is done, for example, by identifying positional sequence similarity of peptide epitopes that bind to one or a plurality of HLA haplotypes.
  • the HLA-peptide structure having a sequence identity greater than a control HLA-peptide structure is selected and the machine learning algorithm models the heteroclitic neoepitopes into an HLA cleft by introducing amino acid rotamers with the lowest energy confirmation at each position.
  • These structural features are input into a machine learning algorithm model to simulating occupancy of one or more binding peptides and one or more non-binding peptides on the HLA binding pocket in a crystal structure of the HLA allele or the crystal structure of a similar HLA allele.
  • the structural features can be extracted from the output models generated during the simulations.
  • the structural features can also be identified using a machine learning algorithm model that infers occupancy of the one or more candidate peptides on the HLA binding pocket.
  • the inference can be based on the model being trained with simulated models of peptides verified to bind to the HLA allele and verified peptides that do not bind to the HLA allele.
  • Vaccine therapies e.g., cancer and infections
  • Vaccine therapies rely on accurate selection of immunizing peptides to potentiate immune responses (e.g., against tumor-specific neoepitopes or viral epitopes). Given the patient's particular complement of HLA alleles, the ability to predict which epitopes will be presented is a fundamental prerequisite for successful vaccine design.
  • an initial input of a candidate peptide or a set of candidate peptides e.g. KRAS peptides is provided.
  • the candidate peptide or set of candidate peptides may be obtained from a subject or group of subjects in need of an immune 27 157361737 response or modified immune response.
  • candidate peptides can be identified in a peptide sequence database (e.g., derived from sequencing of subjects having a specific condition where an immunogenic composition would be useful).
  • a peptide sequence database includes HLA allele binding and non-binding peptides.
  • Candidate peptides can be isolated and sequenced for each HLA-allele to identify HLA-binding peptides.
  • candidate peptides can be obtained by providing a) a population of cells which expresses a single class I HLA allele or a single pair of class II HLA alleles (one ⁇ -chain and one ⁇ -chain); b) isolating the respective HLA-peptide complexes from said cells; c) isolating peptides from said HLA-peptide complexes; and d) sequencing the peptides.
  • the population of cells may express either a single class I HLA allele, a single pair of class II HLA alleles, or a single class I HLA allele and a single pair of class II HLA alleles.
  • Suitable cell populations include, e.g., class I deficient cells lines in which a single HLA class I allele is expressed, class II deficient cell lines in which a single pair of HLA class II alleles are expressed, or class I and class II deficient cell lines in which a single HLA class I and/or single pair of class II alleles are expressed.
  • the population of cells may be professional antigen presenting cells such as macrophages, B cells and dendritic cells.
  • the cells are B cells or dendritic cells.
  • the cells are tumor cells or cells from a tumor cell line.
  • the cells are cells isolated from a patient.
  • the population of cells are further modified, such as by increasing or decreasing the expression and/or activity of at least one gene.
  • the gene encodes a member of the immunoproteasome.
  • the immunoproteasome is involved in the processing of HLA class I binding peptides and includes the LMP2 ( ⁇ ), MECL- 1 ( ⁇ 2 ⁇ ), and LMP7 ( ⁇ 5 ⁇ ) subunits.
  • the immunoproteasome can also be induced by interferon-gamma.
  • the population of cells may be contacted with one or more cytokines, growth factors, or other proteins.
  • the cells are stimulated with inflammatory cytokines such as interferon-gamma, IL-I ⁇ , IL-6, and/or TNF- ⁇ .
  • the population of cells may also be subjected to various environmental conditions, such as stress (heat stress, oxygen deprivation, glucose starvation, DNA damaging agents, etc.).
  • stress heat stress, oxygen deprivation, glucose starvation, DNA damaging agents, etc.
  • the cells are contacted with one or more of a chemotherapy drug, radiation, targeted therapies or immunotherapy. The methods disclosed herein can therefore be used to study the effect of various genes or conditions on HLA peptide processing and presentation.
  • the conditions used are selected so as to match the condition of the patient for which the population of HLA-peptides is to be identified.
  • Any HLA allele may be expressed in the cell population.
  • the HLA allele is selected so as to correspond to a genotype of interest.
  • the HLA allele is a mutated HLA allele, which may be non-naturally occurring allele or a naturally occurring allele in an afflicted patient.
  • the methods disclosed herein have the further advantage of identifying HLA binding peptides for HLA alleles associated with various disorders as well as alleles which are present at low frequency. Accordingly, in one method the HLA allele is present at a frequency of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% within a population. In some embodiments, the HLA allele is present at a frequency of less than 1% within a population. [0089] In some embodiments, the methods further comprise isolating peptides from HLA- peptide complexes and sequencing the peptides to identify input candidate peptides.
  • the peptides are isolated from the complex by any method known to one of skill in the art, such as acid elution.
  • any suitable sequencing method may be used, including methods employing mass spectrometry, such as liquid chromatography-mass spectrometry (LC-MS or LC-MS/MS, or alternatively HPLC-MS or HPLC-MS/MS).
  • mass spectrometry such as liquid chromatography-mass spectrometry (LC-MS or LC-MS/MS, or alternatively HPLC-MS or HPLC-MS/MS).
  • an HLA-allele specific binding peptide sequence database comprises at least 1000 different binding peptide sequences.
  • the methods disclosed herein may also be used to generate a database comprising the HLA-allele specific binding peptide sequences for more than one HLA-allele, for at least two different HLA-alleles, for at least five, for at least ten, fifteen, twenty, thirty or more different alleles.
  • the present disclosure provides a plurality of HLA-allele specific binding peptides, or the sequences thereof, which peptides correspond to the peptides which are presented by one specific HLA allele. More particularly, in some embodiments, an HLA-allele specific 29 157361737 binding peptide sequence database provided is obtained by carrying out the method described herein.
  • the present disclosure provides methods for identifying HLA-allele specific binding peptides, which method comprises analyzing structural features indicative of occupancy of peptides on the binding pocket of HLA alleles.
  • the structural features are amino acid residues capable of fitting a model of peptide occupancy on the binding pocket of HLA alleles (e.g., enrichment in hydrophobicity, exposed hydrophobic surface and charges determined by the peptide's conformation within the binding pocket, as well as the size and position of the various amino acid side chains).
  • the structural features are energetic features that are encoded not by peptide sequence, but by modeled three-dimensional structures of peptide occupancy on the binding pocket of an HLA allele (e.g., energies of attraction, repulsion, and solvation; energies of side chain and backbone hydrogen bonds; and energies and probabilities of side chain and backbone conformations) (see, e.g., Alford R F, Leaver-Fay A, Jeliazkov J R, O'Meara M J, DiMaio F P, Park H, et al. The Rosetta all-atom energy function for macromolecular modeling and design. J Chem Theory Comput.
  • the binding pocket is determined by a structural analysis.
  • Non- limiting structural analysis methods include X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy (Alberts B, Johnson A, Lewis J, et al. Molecular Biolog y of the Cell. 4th edition. New York: Garland Science; 2002. Analyzing Protein Structure and Function).
  • NMR nuclear magnetic resonance
  • a crystal structure is used to determine the binding pocket. Any HLA-allele crystal structure may be used to simulate occupancy of peptides in the binding pocket.
  • a structure is obtained by generating a crystal structure of an HLA-allele.
  • the crystal structures for HLA molecules are obtained from a database (e.g., the Protein Data Bank (PDB, rcsb.org)).
  • structures of similar HLA alleles are used if a structure for the HLA-allele of interest is unavailable or cannot be generated.
  • similar HLA-alleles include HLA alleles having the highest similarity between the amino acid sequences of the binding pocket.
  • the similarity of the binding 30 157361737 pocket can be computed as the sum of pair-wise residue similarities according to a 20 ⁇ 20 amino acid similarity matrix.
  • similar HLA-alleles include HLA alleles having similarity between the binding motifs (i.e. two alleles are considered similar if they bind similar peptides). In certain embodiments, the HLA alleles bind to two or more of the same peptides. In certain embodiments, similar HLA-alleles include HLA alleles from the same class (e.g., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K and HLA-L). In certain embodiments, similar HLA-alleles include HLA alleles having the highest amino acid sequence identity to the HLA- allele of interest.
  • similar HLA-alleles include HLA alleles having at least 85%, 90% 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the HLA-allele of interest.
  • structural features described herein are identified by simulating models of how the candidate peptides occupy the HLA binding pocket.
  • the method comprises analyzing simulations of the occupancy of peptides on the binding pocket of HLA alleles.
  • how peptides occupy the HLA binding pocket is computationally simulated.
  • the immunogenic peptides comprise from about five amino acids to about 50 amino acids.
  • the immunogenic peptides comprise from about seven amino acids to about 45 amino acids. In certain embodiments, the immunogenic peptides comprise from about eight amino acids to about 40 amino acids. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 35 amino acids. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 30 amino acids. In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 25 amino acids.
  • the immunogenic peptides comprise from about five amino acids to about 50 amino acids include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-838.
  • an immunogenic peptide disclosed herein comprises the amino acid sequence of any one of SEQ ID NOs: 1-838. In some embodiments, an immunogenic peptide disclosed herein comprises the amino acid sequence of any one of SEQ ID NOs: 1-838 having one or more conserved amino acid substitutions. In certain embodiments, the immunogenic peptides comprise an amino acid sequence having at least 95% sequence identity to any one or more of SEQ ID NOs: 1-838 having one or more conservative substitutions. In certain embodiments, the immunogenic peptide comprise SEQ ID NO: 2. In some embodiments, the immunogenic peptide comprises SEQ ID NO: 3. In some embodiments, the immunogenic peptide comprises SEQ ID NO. 833.
  • the immunogenic peptide comprises SEQ ID NO.834 [0099] In certain embodiments, the immunogenic peptides comprise five consecutive amino acids of any one or more of SEQ ID NOs: 1-838. In certain embodiments, the immunogenic peptides comprise five consecutive amino acids of any one or more of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300,
  • the immunogenic peptides comprise six consecutive amino acids of any one or more of SEQ ID NOs: 1-838. In certain embodiments, the immunogenic peptides comprise six consecutive amino acids of any one or more of SEQ ID NOs: 2, 3, 5-12, 14- 21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340,
  • the immunogenic peptides comprise seven consecutive amino acids of any one or more of SEQ ID NOs: 1-838. In certain embodiments, the immunogenic peptides comprise seven consecutive amino acids of any one or more of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340,
  • the immunogenic peptides comprise eight consecutive amino acids of any one or more of SEQ ID NOs: 1-838. In certain embodiments, the immunogenic peptides comprise eight consecutive amino acids of any one or 33 157361737 more of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154- 162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238- 244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318- 324, 32
  • the immunogenic peptides comprise nine consecutive amino acids of any one or more of SEQ ID NOs: 1-838. In certain embodiments, the immunogenic peptides comprise nine consecutive amino acids of any one or more of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148- 152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232- 236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310- 316, 318-324, 326-332, 334
  • the immunogenic peptides comprise from about seven amino acids to about 45 amino acids include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, 34 157361737 at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-838.
  • the immunogenic peptides comprise from about eight amino acids to about 40 amino acids. [00101] In certain embodiments, the immunogenic peptides comprise from about nine amino acids to about 35 amino acids include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
  • the immunogenic peptides comprise from about nine amino acids to about 30 amino acids include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-838.
  • the immunogenic peptides comprise from about nine amino acids to about 25 amino acids include an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-838.
  • mutant KRAS epitopes comprise G12V, G12D, G12C, G12R, G12A, G13D or combinations thereof. 35 157361737 [00105]
  • KRAS variants of the present disclosure comprise an amino acid sequence that has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 9
  • a KRAS variant disclosed herein comprises the amino acid sequence of any one of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390-396,
  • the KRAS variant comprise an amino acid sequence having at least 95% sequence identity to any one or more of SEQ ID NOs: .
  • a KRAS variant disclosed herein comprises the amino acid sequence of any one of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334
  • the KRAS variant comprise SEQ ID NO: 2. In some embodiments, the KRAS variant comprises SEQ ID NO: 3. In some embodiments, the KRAS variant comprises SEQ ID NO.833. In some embodiments, the KRAS variant comprises SEQ ID NO.834. [00106] In certain embodiments, the KRAS and/or mKRAS peptides comprise one or more modified amino acids, unnatural amino acids, substituted amino acids or combinations thereof.
  • the KRAS and/or mKRAS peptides comprising any one of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390-396
  • Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2- naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri- O-acetyl-GlcNAc ⁇ -serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L- phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-
  • Candidate Therapeutic Peptides are used to determine an effective neoantigen vaccine. In this context, it is of interest to determine which neoantigen peptides are likely to bind to a subject's HLA so as to effectively function as immunogenic peptides.
  • subject specific HLA alleles or HLA genotype of a subject may be determined by any method known in the art.
  • One of the barriers to developing curative and tumor-specific immunotherapy is the identification and selection of highly specific and restricted tumor antigens to avoid autoimmunity.
  • 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 neoantigens, and producing neoantigens for use in a vaccine or immunogenic composition.
  • identifying mutations in 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 by the methods embodied herein to generate a 38 157361737 plurality of neoantigen epitopes that are expressed within the neoplasia/tumor and that bind to a high proportion of patient HLA alleles; and synthesizing the plurality of the neoantigenic peptides for use in a cancer vaccine or immunogenic composition suitable for treating a high proportion of subjects having cancer.
  • a therapeutic vaccine disclosed herein may include: (1) identification of mutated peptides that can bind to HLA molecules of a high proportion of individuals and (2) formulating the drug as a multi-epitope vaccine of long peptides.
  • targeting as many mutated epitopes as possible can take advantage of the enormous capacity of the immune system, prevents the opportunity for immunological escape by down- modulation of a particular immune targeted gene product, and compensates for the known inaccuracy of epitope prediction approaches.
  • a synthetic peptide described herein provide a particularly 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).
  • a vaccine disclosed herein can be used in (3)combination with a strong vaccine adjuvant.
  • an effective vaccine can 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.
  • methods of identifying the most suitable peptides for preparing an immunogenic composition for a subject comprises selecting from set given set of peptides the plurality of peptides capable of binding an HLA protein of the subject.
  • provided herein are methods of identifying a plurality of subject-specific peptides for preparing a subject-specific immunogenic composition, wherein the subject has a tumor and the subject- specific peptides are specific to the subject and the subject's tumor.
  • the subject-specific peptides each comprise a different tumor neo-epitope that is an epitope specific 39 157361737 to the tumor of the subject and each binds an HLA protein of the subject, as provided by the methods for identifying HLA binding described herein.
  • the cell used in the method for determining HLA binding as described herein is an antigen-presenting cell.
  • Neoantigens the tumor antigens that bind to HLA alleles are identified by the methods described herein. In certain embodiments, the tumor antigens are neoantigens. In a further aspect, the disclosure provides methods for identifying tumor neoantigen-comprising peptides, wherein the methods comprise identifying for a given HLA allele, the peptides binding an HLA allele in a tumor cell from a tumor of a patient. [00116] In some embodiments, the tumor antigen comprises KRAS, BRAF, FBWX7, FGFR3, IDH1, MUC4, NRAS, PIK3CA, PPP2R1A, PTEN, or TP53.
  • the tumor antigen comprises one or more mutations.
  • the one or more mutations comprises KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y.
  • mutated epitopes are effective in inducing an immune response.
  • spontaneous tumor regression or long term survival correlate with CD8 + T-cell responses to mutated epitopes (Buckwalter and Srivastava P K. “It is the antigen(s), harmless” and other lessons from over a decade of vaccine therapy of human cancer.
  • each tumor contains multiple, patient-specific mutations that alter the protein coding content of a gene.
  • Such mutations create altered proteins, ranging from single amino acid changes (caused by missense mutations) to addition of long regions of novel amino acid sequence due to frame shifts, read-through of termination codons or translation of intron regions (novel open reading frame mutations; neoORFs).
  • these mutated proteins are valuable targets for the host's immune response to the tumor as, unlike native proteins, they are not subject to the immune-dampening effects of self-tolerance.
  • mutated proteins are more likely to be immunogenic and are also more specific for the tumor cells compared to normal cells of the patient.
  • the mutated proteins can be referred to as neoantigens.
  • neoantigen or “neoantigenic” means a class of tumor antigens that arises from a tumor- specific mutation(s) which alters the amino acid sequence of genome encoded proteins.
  • Embodiments disclosed herein provide a method of identifying peptides, e.g., neoantigens, including, but not limited to novel unannotated open reading frames (nuORFs), that are capable of eliciting a cancer specific T-cell response.
  • neoantigens including, but not limited to novel unannotated open reading frames (nuORFs)
  • Genomic aberrations in cancer cells give rise to mutant peptides (neoantigens) displayed on the human leukocyte antigen (HLA) molecules and recognized by T cells, thus triggering an immune response against cancer cells.
  • HLA human leukocyte antigen
  • neoantigen-based peptides can display expanded neoantigen-specific T cells, and is a promising avenue for cancer treatment (Ott et al., An immunogenic personal neoantigen vaccine or patients with melanoma, Nature 2017 Jul. 13; 547(7662):217-221; Sahin et al., 2017 Personalized RNA mutanome vaccines mobilize poly- specific therapeutic immunity against cancer”, Nature, vol. 547, 2017, pages 222-226).
  • neoantigens can be predicted based on mutations detected by whole exome sequencing (WES). In some embodiments, their expression levels are estimated using mRNA sequencing (RNA-seq).
  • Ribosome profiling allows to monitor mRNA translation and has been used to predict a plethora of translated novel unannotated ORFs (nuORFs) (Fields et al., 2015. A Regression-Based Analysis of Ribosome-Profiling Data Reveals a conserveed Complexity to Mammalian Translation, Mol Cell, vol. 60, pages 816-827; Ji et al., 2015. Many IncRNAs, 5′UTRs, and pseudogenes are translated and some are likely to express functional proteins, ELIFE, 41 157361737 vol. 4).
  • Ribo-seq analysis of human fibroblasts infected with HSV-1 and HCMV has identified nuORFs that contribute peptides presented on major histocompatibility complex class I (MHC I) (Erhard et al., 2018. Improved Ribo-seq enables identification of cryptic translation events, Nat. Methods, vol.15, no.5, pages 363-366).
  • MHC I major histocompatibility complex class I
  • the methods disclosed herein can be used to select subject specific peptides that are presented by a tumor for any neoplasia.
  • neoplasia is meant any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both.
  • cancer is an example of a neoplasia.
  • cancers include, without limitation, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphom’ (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angio
  • Vaccines and Immunological Compositions are used in a vaccine or immunological composition to treat any disease or condition described herein (e.g., tumor, autoimmunity, infection, transplant).
  • vaccine or “immunological composition” are used interchangeably and are meant to refer in the present context to a pooled 42 157361737 sample of one or more antigenic peptides, for example at least one, at least two, at least three, at least four, at least five, or more antigenic peptides.
  • a “vaccine” is to be understood as including a protective vaccine, which is a composition for generating immunity for the prophylaxis and/or treatment of diseases (e.g., neoplasia/tumor).
  • a “vaccine” is also to be understood as including a tolerizing vaccine, which is a composition for reducing immunity for the prophylaxis and/or treatment of diseases (e.g., autoimmune disease).
  • a tolerizing vaccine may be formulated with antigenic epitopes specific for an allergen or for an autoimmunity antigen identified according to the present disclosure.
  • a protective vaccine may be formulated with antigenic epitopes specific for a pathogen or for a cancer cell.
  • vaccines can be medicaments which comprise antigens and are intended to be used in humans or animals for generating specific defense and protective substance by vaccination.
  • a “vaccine composition” can include a pharmaceutically acceptable excipient, carrier or diluent.
  • the vaccine may include one or more peptides identified according to the present disclosure. For example, 1 to 10 peptides. Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • “nested sub-ranges” that extend from either end point of the range are specifically contemplated.
  • a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
  • a protective vaccine is used to treat cancer.
  • Additional examples of cancers and cancer conditions that can be treated with the therapy of this document include, but are not limited to a patient in need thereof that has been diagnosed as having cancer, or at risk of developing cancer.
  • the subject may have a solid tumor such as breast, ovarian, prostate, lung, kidney, gastric, colon, testicular, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs and hematological tumors, such as lymphomas and leukemias, including acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, and B cell lymphomas, tumors of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves and other parts of the CNS, such 43 157361737 as glioblastomas or medulla blastomas); head and/or neck cancer, breast tumors, tumors of the circulatory system (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors, and tumor-associated vascular tissue); tumors of the blood and lymphatic system’ (
  • cancers that can be treated using the therapy described herein may include among others cases which are refractory to treatment with other chemotherapeutics.
  • refractory refers to a cancer (and/or metastases thereof), which shows no or only weak antiproliferative response (e.g., no or only weak inhibition of tumor growth) after treatment with another chemotherapeutic agent. These are can be cancers that cannot be treated satisfactorily with other chemotherapeutics.
  • refractory cancers encompass not only (i) cancers where one or more chemotherapeutics have already failed during treatment of a patient, but also (ii) cancers that can be shown to be refractory by other means, e.g., biopsy and culture in the presence of chemotherapeutics.
  • the therapy described herein is also applicable to the treatment of patients in need thereof who have not been previously treated.
  • the therapy described herein is also applicable where the subject has no detectable neoplasia but is at high risk for disease recurrence.
  • the therapy described herein is also applicable where the subject has undergone Autologous Hematopoietic Stem Cell Transplant (AHSCT), and in particular patients who demonstrate residual disease after undergoing AHSCT.
  • AHSCT Autologous Hematopoietic Stem Cell Transplant
  • the post-AHSCT setting is characterized by a low volume of residual disease, the infusion of immune cells to a situation of homeostatic expansion, and the absence of any standard relapse-delaying therapy.
  • Proteins or peptides disclosed herein 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.
  • the nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art.
  • One such database is the National Center for Biotechnology Information's Genbank and GenPept databases located at the National Institutes of Health website.
  • coding regions for known genes may be amplified and/or expressed using the techniques disclosed herein or as would 45 157361737 be known to those of ordinary skill in the art.
  • various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
  • peptides can be readily synthesized chemically utilizing reagents that are free of contaminating bacterial or animal substances (Merrifield R B: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963).
  • neoantigenic 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
  • a nucleic acid encoding a neoantigenic peptide of the disclosure may be used to produce the neoantigenic peptide in vitro.
  • 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. Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, 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.
  • an expression vector such as a plasmid
  • neoantigenic peptides comprising the isolated polynucleotides, as well as host cells containing the expression vectors, are also contemplated.
  • the neoantigenic peptides may be provided in the form of RNA or cDNA molecules encoding the desired neoantigenic peptides.
  • One or more neoantigenic peptides of the disclosure may be encoded by a single expression vector.
  • the polynucleotides may comprise the coding sequence for the tumor specific neoantigenic 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.
  • 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 85% identical, at least 90% identical, at least 95% identical, or at least 96%), 97%, 98% or 99% identical to a polynucleotide encoding a tumor specific neoantigenic peptide of the present disclosure, can be provided.
  • nucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence.
  • These mutations of the reference sequence can occur at the amino- or carboxy- terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, 47 157361737 Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • Bestfit program Wiconsin Sequence Analysis Package, 47 157361737 Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • Recombinant expression vectors may be used to amplify and express DNA encoding the tumor specific neoantigenic peptides.
  • Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a tumor specific neoantigenic 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 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.
  • DNA regions are operatively linked when they are functionally related to each other.
  • DNA for a signal peptide secretory leader
  • a promoter can be operatively linked to a coding sequence if it controls the transcription of the sequence
  • a ribosome binding site can be 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 can 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 especially mammals or humans 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 MI 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). [00141] Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified and completely functional.
  • 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.
  • the proteins produced by a transformed host can be purified according to any suitable method.
  • 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 49 157361737 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.
  • the present disclosure also contemplates the use of nucleic acid molecules as vehicles for delivering neoantigenic peptides/polypeptides to the subject in need thereof, in vivo, in the form of, e.g., DNA/RNA vaccines.
  • antigens may be administered to a patient in need thereof by use of a plasmid.
  • these can be 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), Journal Immunol.155 (4): 2039-2046). Intron A may sometimes be included to improve mRNA stability and hence increase protein expression (Leitner et al. (1997), Journal Immunol. 159 (12): 6112-6119). Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta- globulin polyadenylation sequences Multi cistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.
  • a way of enhancing protein expression is by optimizing the codon usage of pathogenic mRNAs for eukaryotic cells.
  • promoters may be the SV40 promoter or Rous Sarcoma Virus (RSV).
  • Plasmids may be introduced into animal tissues by a number of different methods. The two most used approaches are injection of DNA in saline, using a standard hypodermic needle, and gene gun delivery. In some embodiments, injection in saline can be conducted intramuscularly (EVI) in skeletal muscle, or intradermally (ID), with DNA being delivered to the extracellular spaces.
  • EVI intramuscularly
  • ID intradermally
  • DNA or RNA that encodes a peptide disclosed herein can be administered to a subject. 50 157361737 [00147]
  • Alternative delivery methods may include aerosol instillation of naked DNA on mucosal surfaces, such as the nasal and lung mucosa, and topical administration of pDNA to the eye and vaginal mucosa.
  • 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 DOI: 10.1371/journal.pone.O118803 Apr.13, 2015).
  • a neoplasia vaccine or immunogenic composition may include separate DNA plasmids encoding, for example, one or more neoantigenic peptides/polypeptides as identified in according to the disclosure.
  • 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 disclosure 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 neoplasia 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 (see, e.g., Baden et al. First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). J Infect Dis. 2013 Jan.
  • adeno associated virus 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.
  • this approach can involve the use of a vector to express nucleotide sequences that encode the peptide of the disclosure.
  • the vector Upon introduction into an acutely or chronically infected host or into a noninfected host, the vector can express the immunogenic peptide, and thereby elicits a host CTL response. 51 157361737 [00150]
  • 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 disclosure).
  • lentiviral vectors can be preferred as they are able to transduce or infect non- dividing cells and may 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 disclosure include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.
  • a minimal non-primate lentiviral vector such as a lentiviral vector based on the equine infectious anemia virus (EIAV) (see, e.g., Balagaan, (2006) J Gene Med; 8: 275- 285, Published online 21 Nov.2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.845).
  • the vectors may have cytomegalovirus (CMV) promoter driving expression of the target gene.
  • CMV cytomegalovirus
  • the disclosure contemplates amongst vector(s) useful in the practice of the disclosure: viral vectors, including retroviral vectors and lentiviral vectors.
  • One of skill in the art can determine suitable dosage.
  • Suitable dosages for a virus can be determined empirically.
  • an adenovirus vector is also useful in the practice of the disclosure.
  • 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.
  • high expression levels 52 157361737 ensure that the products of the nucleic acids will be expressed to sufficient levels to generate an immune response (see e.g., U.S. Pat. No.7,029,848).
  • an 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).
  • Ad35 vectors are described in U.S. Pat. Nos.6,974,695, 6,913,922, and 6,869,794.
  • Adenovirus vectors that are E1-defective or deleted, E3-defective or deleted, and/or E4-defective or deleted may also be used. Certain adenoviruses having mutations in the E1 region have improved safety margin because E1-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 E1, E3, E4, El and E3, and El and E4 can be used in accordance with the present disclosure.
  • “gutless” adenovirus vectors, in which all viral genes are deleted can also be used in accordance with the present disclosure.
  • such vectors require a helper virus for their replication and require a special human 293 cell line expressing both E1a 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 viral vector is an adenovirus vector, an adeno-associated viral vector (AAV), or derivatives thereof.
  • AAV adeno-associated viral vector
  • the adeno-associated viral vector comprises AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, DJ/8 or psudotypes thereof.
  • the AAV can be AAV1, AAV2, AAV5 or any combination thereof.
  • AAV8 can be useful for delivery to the liver.
  • effectively activating a cellular immune response for a neoplasia vaccine or immunogenic composition can be achieved by expressing the relevant antigens in a vaccine or immunogenic composition in a non-pathogenic microorganism.
  • a Poxvirus is used in the neoplasia vaccine or immunogenic composition. These include orthopoxvirus, avipox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see e.g., Verardi et al., Hum Vaccin Immunother.2012 July; 8(7):961-70; and Moss, Vaccine.2013; 31(39): 4220-4222).
  • poxviruses that may be used in the practice of the disclosure, 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.
  • Chordopoxvirinae subfamily poxviruses poxviruses of vertebrates
  • orthopoxviruses and avipoxviruses e.g., vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC® VR-1354), Copenhagen Strain, NYVAC, NYVAC.
  • recombinant viral particles of the vaccine or immunogenic composition are administered to patients in need thereof.
  • Dosages of expressed neoantigen can range from a few to a few hundred micrograms, e.g., 5 to 500 ⁇ g.
  • the vaccine or immunogenic composition can be administered in any suitable amount to achieve expression at these dosage levels.
  • the viral particles can be administered to a patient in need thereof or transfected into cells in an amount of about at least 10 3 pfu; thus, the viral particles are preferably administered to a patient in need thereof or infected or transfected into cells in at least about 10 4 pfu to about 10 6 pfu; however, a patient in need thereof can be administered at least about 10 8 pfu or at least about 10 7 pfu to about 10 9 pfu.
  • a pharmaceutical composition comprises an effective amount of one or more antigenic peptides as described herein (including a pharmaceutically acceptable salt, thereof), optionally in combination with a pharmaceutically acceptable carrier, excipient or additive.
  • pharmaceutically acceptable refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S.
  • a “pharmaceutically acceptable excipient, carrier or diluent” refers to an excipient, carrier or diluent that can be administered to a subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent.
  • a “pharmaceutically acceptable salt” of pooled tumor specific neoantigens as recited herein may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication.
  • Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.
  • Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC—(CH 2 ) n
  • pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium.
  • pharmaceutically acceptable salts for the pooled tumor specific neoantigens provided herein, including those listed by Remington's Pharmace ut ical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).
  • a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method.
  • the therapeutic agents for example, the neoantigenic peptides
  • the compositions may be administered once daily, twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once per year.
  • the dosing interval can be adjusted according to the needs of individual patients. For longer intervals of administration, extended release or depot formulations can be used.
  • Combination Therapies [00166] The disclosure also contemplates the combination of the composition of the present disclosure with other drugs and/or in addition to other treatment regimens or modalities such as surgery. When the composition of the present disclosure is used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture. For example, in the case of cancer chemotherapeutic agents may be administered as part of the combination therapy.
  • the KRAS and/or mKRAS peptides are administered in conjunction with a cancer therapy.
  • compositions disclosed herein are administered in conjunction with a cancer therapy.
  • an immunogenic peptide comprising at least 90%, 95%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-838 can be administered in conjunction with a cancer therapy.
  • one or more immunogenic peptides comprise at least a 75% sequence identity to any one or more of SEQ ID NOs: 1-838 can be administered with a cancer therapy.
  • the immunogenic peptides comprise any one or more of SEQ ID NOs: 1-838 can be administered with a cancer therapy.
  • the immunogenic peptides comprises an amino acid sequence having at least 95% sequence identity to any one or more of SEQ ID NOs: 1-838 having one or more conservative substitutions can be administered with a cancer therapy.
  • the immunogenic peptides comprising the amino acid 56 157361737 sequence of any one of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294
  • the immunogenic peptides comprise the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 833, or SEQ ID NO: 834 and can be administered with a cancer therapy.
  • cancer therapy refers to a therapy useful in treating cancer.
  • anti-cancer therapeutic agents include, but are not limited to, e.g., surgery, chemotherapeutic agents, immunotherapy, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti- tubulin agents, and other agents to treat cancer, such as anti-HER-2 antibodies (e.g., HERCEPTIN TM ), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (TARCEVA TM )), platelet derived growth factor inhibitors (e.g., GLEEVEC TM (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, Erb
  • a “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include Erlotinib (TARCEVA TM , Genentech/OSI Pharm.), Bortezomib (VELCADE TM , Millennium Pharm.), Fulvestrant (FASLODEX TM , Astrazeneca), Sutent (SU11248, Pfizer), Letrozole (FEMARA TM , Novartis), Imatinib mesylate (GLEEVEC TM , Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin TM , Sanofi), 5-FU (5- 57 157361737 fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE TM , Wyeth), Lapatinib (GSK572016, GlaxoSmithKline
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN TM doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin
  • chemotherapeutic agent also included in this definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX TM (tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON TM (toremifene); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE TM (megestrol acetate), AROMASIN TM (exemestane), formestanie, fadrozole, RIVISOR TM (vorozole), FEMARA
  • the cancer therapeutic is an immunotherapy selected from the group comprising oncolytic virus, bacteria, oncolytic bacteria or other bacterial compositions, Bacillus Calmette-Guerin (BCG), a microbiome modulator, and/or a toll-like receptor (TLR) agonist.
  • the TLR agonist is a TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and/or TLR13 agonist.
  • the TLR agonist is derived from virus, plants, bacteria and/or made synthetically.
  • the immunotherapy is a is a stimulator of interferon genes (STING) pathway modulator.
  • GM-CSF myeloid derived innate immune system cells
  • PI3K inhibitors e.g., PI3K delta inhibitors
  • 5FU e.g., capecitabine
  • PI3K inhibitors or histone deacetylase inhibitors to remove inhibitory myeloid derived suppressor cells.
  • PI3K inhibitors include, but are not limited to, LY294002, Perifosine, BKM120, Duvelisib, PX-866, BAY 80-6946, BEZ235, SF1126, GDC-0941, XL147, XL765, Palomid 529, GSK1059615, PWT33597, IC87114, TG100-15, CAL263, PI-103, GNE- 477, CUDC-907, and AEZS-136.
  • the PI3K inhibitor is a PI3K delta inhibitor such as, but not limited to, Idelalisib, RP6530, TGR1202, and RP6503.
  • the immunotherapy may also comprise the administration of an interleukin such as IL-2, or an interferon such as INF ⁇ .
  • an interleukin such as IL-2
  • an interferon such as INF ⁇ .
  • the KRAS and/or mKRAS peptides are administered with one or more immune checkpoint modulators.
  • the composition disclosed herein can be administered with an immune checkpoint modulator.
  • an 60 157361737 immunogenic peptide comprising at least 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-838 can be administered in conjunction with a checkpoint modulator.
  • one or more immunogenic peptides comprise at least a 75% sequence identity to any one or more of SEQ ID NOs: 1-838 can be administer with an immune checkpoint modulator. In certain embodiments, the immunogenic peptides comprise any one or more of SEQ ID NOs: 1-838 can be administered with an immune checkpoint modulator.
  • an immunogenic peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148- 152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232- 236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310- 316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390- 396
  • the immunogenic peptides comprises an amino acid sequence having at least 95% sequence identity to any one or more of SEQ ID NOs: 1-838 having one or more conservative substitutions can be administered with an immune checkpoint modulator.
  • the immunogenic peptides comprise the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 833, or SEQ ID NO: 834 can be administered with an immune checkpoint modulator.
  • Immune checkpoints refer to inhibitory pathways of the immune system that are responsible for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses.
  • checkpoint inhibitor examples include, without limitation an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR- ⁇ .
  • the term “checkpoint inhibitor” means a group of molecules on the cell surface of CD4 + and/or CD8 + T cells that fine-tune immune responses by down-modulating or inhibiting an anti- 61 157361737 tumor immune response.
  • Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRP ⁇ (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO 2012/177624).
  • Anti-immune checkpoint inhibitor therapy refers to the use of agents that inhibit immune checkpoint inhibitors.
  • Inhibition of one or more immune checkpoint inhibitors can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer.
  • exemplary agents useful for inhibiting immune checkpoint inhibitors include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint inhibitor nucleic acids, or fragments thereof.
  • Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint inhibitor proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint inhibitor proteins (e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint inhibitor nucleic acid transcription or translation; and the like.
  • a non-activating form of one or more immune checkpoint inhibitor proteins e.g., a dominant negative polypeptide
  • small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and its natural receptor(s)
  • fusion proteins e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin
  • agents can directly block the interaction between the one or more immune checkpoint inhibitors and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response.
  • agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response.
  • a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand.
  • anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA-4 antibodies either alone or used in combination.
  • such therapy involves blockade of programmed cell death 1 (PD- 1).
  • such therapy involves treatment with an agent that interferes with an 62 157361737 interaction involving PD-1 (e.g., with PD-L1).
  • such therapy involves administration of an antibody agent that specifically interacts with PD-1 or with PD-L1.
  • such therapy involves administration of one or more of nivolumab (BMS-936558, MDX-1106, ONO-4538, a fully human Immunoglobulin G4 (IgG4) monoclonal PD-1 antibody), pembrolizumab (MK-3475, a humanized monoclonal IgG4 anti-PD-1 antibody), BMS-936559 (a fully human IgG4 PD-L1 antibody), MPDL3280A (a humanized engineered IgG1 monoclonal PD- L1 antibody) and/or MEDI4736 (a humanized engineered IgG1 monoclonal PD-L1 antibody).
  • BMS-936558 a fully human Immunoglobulin G4 (IgG4) monoclonal PD-1 antibody
  • MK-3475 a humanized monoclonal IgG4 anti-PD-1 antibody
  • BMS-936559 a fully human IgG4 PD-L1 antibody
  • MPDL3280A a humanized engineered
  • EXAMPLE 1 HETEROCLITIC NEOEPITOPE VACCINES DERIVED FROM COMPUTATIONAL STRUCTURAL MODELING
  • a library of KRAS G12D heteroclitic epitopes was generated for structural analysis in a panel of 18 HLAs representing >90% of the global population.
  • the consensus binding motif for epitopes of each HLA showed distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors (FIG. 1).
  • Major anchor residues were predominately at position 2, 3, 5, and 9. Some alleles had additional minor anchor residues at positions 1, 4, 6, and 7.
  • each peptide was first modelled onto HLA-A*02:01 to investigate peptide-HLA interactions (FIG. 3).
  • a Multiple Sequence Comparison by Log-Expectation (MUSCLE) peptide sequence alignment was performed, which takes into consideration positional sequence similarity, of the KRAS G12D epitopes with all epitopes that bind each HLA and have a crystal structure available.
  • MUSCLE Log-Expectation
  • HLA-KRAS neoepitope complexes were structurally validated using the root-mean- square deviation analysis (C ⁇ -RMSD) to ensure no major destabilizing structural changes have occurred to overall HLA cleft.
  • C ⁇ -RMSD root-mean- square deviation analysis
  • High-resolution, Monte Carlo with minimization docking of the peptide to the HLA cleft was performed to identify the lowest full-atom energy conformation.
  • the energetic-optimized structures are used to introduce each anchor residue modification found in the heteroclitic epitope library (FIG.2 and FIG.3).
  • Structural parameters such as contacts between peptide and HLA cleft residues, solvent-assessable surface area (SASA), peptide rigidity as a measure of C ⁇ RMSD of the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, and electrostatic potential. These measurements are compared to modelled parental neoepitopes in each HLA molecule. [00182] Using the neoepitope-HLA structural models, amino acid modifications of mKRAS epitopes with improved HLA binding affinity were predicted for HLA-A*02:01 (FIG.5A- FIG. 5C), HLA-A*03:01 (FIG. 6A-FIG.
  • HLA-A*aa:01 (FIG. 7A- FIG. 7C), HLA-A*30:01 (FIG.8A- FIG.8C), HLA-A*68:01 (FIG.9A- FIG.9C), HLA-B*07:02 (FIG.10A- FIG.10C), 64 157361737 HLA-C*01:02 (FIG.11A- FIG.11C), HLA-C*08:02 (FIG.12A- FIG.12C), and HLA-C*03:03 (FIG.13A- FIG.13C).
  • Predicted binding affinity (nM) of altered peptide ligands (APL) of parental G12V 10- mer and 9-mer and APLs to HLA-A*02:01 was generated (FIG.5B).
  • Predicted binding affinity (nM) of altered peptide ligands (APL) of parental 10-mer (KRAS G12D, G12V, G12C) and 9-mer (KRAS G12R, G12V) and APLs to HLA-A*03:01 was generated (FIG.6B).
  • Predicted binding affinity (nM) of altered peptide ligands (APL) of parental 10-mer (KRAS G12D G12C, G12R, G12V) and 9-mer (KRAS G12V) and APLs to HLA-A*68:01 was generated (FIG.9B).
  • Predicted binding affinity (nM) of altered peptide ligands (APL) of parental 10-mer (KRAS G12D and G12R) and APLs to HLA-B*07:02 was generated (FIG.10B).
  • Predicted binding affinity (nM) of altered peptide ligands (APL) of parental 10-mer (KRAS G12V) and 9-mer (KRAS G12V) and APLs to HLA-C*03:03 was generated (FIG.13B).
  • HLA-A*11:01-KRAS APL binding was tested using MBL tetramer quicks witch assay. HLA-A*11:01 tetramers were incubated with 10mM KRAS parental epitope or APL at room temperature for 4 hours. Peptide loading was then quantified by flow cytometry analysis with anti- exiting peptide antibody.
  • % APL binding relative to parental peptide binding to tetramer was 65 157361737 calculated (FIG. 14A). NetMHC prediction was used to predict binding affinities for HLA- A*11:01-specific KRAS peptides and APLs tested with this assay.
  • Percent binding of KRAS G12D, G12R, G12V, and G12C 10- and 9-mer parental epitopes and altered peptide ligands is shown in FIG.15. APLs with greater binding percentage relative to parental are shaded in gray.
  • the ability of KRAS G12D APLs to prime T cell responses against parental antigen was tested in vitro.
  • Pulsed dendritic cells were washed twice with PBS and then co-cultured with autologous CD8 + T cells in the presence of hIL-15, hIL-6, and hIL-21. 7 days later, more peptide-pulsed dendritic cells were added to each co-culture. The following day, fresh hIL2 and IL7 were added. 7 days post the final dendritic cell culture, T cells were collected and stained with an HLA-A*11:01 tetramer containing the parental epitope sequence (FIG. 16A). Flow cytometry was used to determine T cell expansion (FIG. 16B).
  • KRAS G12D parental and altered peptide ligand- HLA contacts and surface structure was determined (FIG.17). Hydrophobicity of KRAS G12D parental and altered peptide ligand- HLA was measured (FIG.18). Electrostatic potential of KRAS G12D parental and altered peptide ligand was measured.
  • FIG.21A-FIG.21F demonstrate that engineered peptides display enhanced contact points and solvent accessible surface area, and reduced rigidity in a murine PDAC model.
  • Mice were vaccinated twice 7 days apart with PBS , 50 ⁇ g APL, or 5 ⁇ g parental peptide. Seven days after the last vaccine dose, splenocytes were restimulated with APL or parental peptides, overnight, and IFN ⁇ production was measured by ELISpot. Two-way ANOVA followed by Tukey’s multiple comparisons test was performed (FIG 21A).
  • FIGS.21C murine MHC class I molecule H2-Kb, blue, minimal epitope of peptide 44, orange, parental or engineered amino acid residue.
  • FIG. 21E shows C ⁇ –RMSD, measure of peptide rigidity.
  • FIG. 22A-FIG. 22C demonstrate that KRAS G12D APL1– HLA*11:01 binding and immunogenicity of engineered antigens show superior binding and T Cell Responses.
  • FIG. 22A shows non–linear regression curves and calculated EC50s for binding between HLA-A*11:01 and a parental KRAS G12D 10–mer peptide and 7 engineered antigens.
  • FIG.21B 1 ⁇ 10 6 expanded CD8 T cells from no peptide, irrelevant peptide, parental peptide, or APL1 were seeded in an ELISpot capture plate coated with anti-human IFN ⁇ antibody. T cells were stimulated with control or parental antigen overnight and IFN ⁇ capture was read out.
  • FIG. 21C shows in vitro immunogenicity testing of HLA-A*11:01 parental epitope and its engineered counterpart (APL1).
  • Human monocyte–derived DCs (moDCs) differentiated from healthy donor PBMCs were pulsed with 100 ⁇ M parental or APL1 plus ⁇ 2–microglobulin (4 hours) at 37°C.
  • T cells were collected and stained with HLA-A*11:01 tetramer loaded with parental peptide (Left) or subject to IFN ⁇ ELISPOT (Right).
  • APL1 and APL4-pulsed T cells showed increased IFN ⁇ spots as compared to the parental control, indicating superior T cell response.
  • Structural analysis of KRAS G12D HLA-A*11:01 APL1 and APL4 identify peptide rigidity and stabilizing HLA cleft interactions as features associated with enhanced peptide immunogenicity (FIG.24A-FIG.24C).
  • FIG.24B CaRMSD was calculated for the 10-mer peptide in each model compared to all other models for that epitope. Two way ANOVA was performed followed by Tukey’s multiple comparisons test.
  • FIG.24B CaRMSD was calculated for the 10-mer peptide in each model compared to all other models for that epitope.
  • G12D peptide neoantigen residue
  • HLA cleft residues that are associated with stabilization of peptide-HLA interactions (from left to right, Arg114, Gln155, Gln70, TRP147, Thr73).
  • Quantification of G12D residue interactions with each of the HLA-cleft residues across the top 10 structural models for parental G12D, APL1, or APL4 demonstrates APL4 has an altered pattern of HLA cleft binding with a significantly higher proportion of interactions with the Gln70 residue suggested more diverse stabilizing interactions within the HLA cleft.
  • Binding affinity changes for KRAS G12D APLs in the context of HLA-A*03:01 and HLA-B*07:02 (FIG.25A and FIG.25B).
  • Optimized APLs for HLA-A*03:01 and B*07:02 were design and tested for enhanced binding affinity relative to parental neoantigen in K562-TAP1KO- HLA expressing cell lines. Cells were incubated with increasing concentration of peptide overnight at 37C. Cells were then stained for HLA-ABC expression on the surface of cells. Median florescent 68 157361737 intensity of HLA expression was quantified for each peptide at each peptide concentration in duplicates.
  • Control peptide corresponds to a positive control peptide specific to each HLA.
  • Non- linear regression curves were fit to each graph.
  • the peptide sequences that correspond to APL1 and parental are summarized in the tables below. The altered residue is indicated in italics.

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Abstract

Compositions de peptides modifiés pour améliorer la liaison HLA ou la reconnaissance des lymphocytes T tout en conservant la réactivité au néoépitope cible, améliorant l'activation des lymphocytes T spécifiques du néoantigène. Les procédés d'utilisation comprennent le traitement du cancer par l'administration d'un ou de plusieurs de ces peptides générés par modélisation informatique.
PCT/US2024/024542 2023-04-14 2024-04-14 Vaccins hétéroclites à néoépitopes Pending WO2024216241A2 (fr)

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WO1998058951A1 (fr) * 1997-06-23 1998-12-30 Ludwig Institute For Cancer Research Nonapeptides et decapeptides isoles se fixant a des molecules hla, et leur utilisation
US20180068054A1 (en) * 2016-09-06 2018-03-08 University Of Washington Hyperstable Constrained Peptides and Their Design
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